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. 2017 Jul 1;27(10):920–926. doi: 10.1093/glycob/cwx054

Generation and characterization of a monoclonal antibody to the cytoplasmic tail of MUC16

Ilene K Gipson 2,1,†, Ulla Mandel 3,1,†, Balaraj Menon 2, Sandra Michaud 2, Ann Tisdale 2, Diana Campos 4,5, Henrik Clausen 3
PMCID: PMC6283312  PMID: 28673046

Abstract

MUC16 is a large transmembrane mucin expressed on the apical surfaces of the epithelium covering the ocular surface, respiratory system and female reproductive tract. The transmembrane mucin is overexpressed by ovarian carcinomas, it is one of the most frequently used diagnostic markers for the disease and it is considered a promising target for immunotherapeutic intervention. Immunodetection of the mucin has to date been through antibodies that recognize its exceptionally large ectodomain. Similar to other membrane anchored mucins, MUC16 has a short cytoplasmic tail (CT), but studies of the biological relevance of the C-terminal domain of MUC16 has been limited by lack of availability of monoclonal antibodies that recognize the native CT. Here, we report the development of a novel monoclonal antibody to the CT region of the molecule that recognizes native MUC16 and its enzymatically released CT region. The antibody is useful for immunoprecipitation of the released CT domain as demonstrated with the OVCAR3 ovarian cancer cell line and can be used for detailed cytolocalization in cells as well as in frozen sections of ocular surface and uterine epithelium.

Keywords: CA125, MUC16 cytoplasmic tail, MUC16 MAb, transmembrane mucin

Introduction

MUC16 is an exceptionally large transmembrane mucin, originally cloned as the CA125 antigen (O'Brien et al. 2001; Yin and Lloyd 2001), a widely recognized biomarker of ovarian cancer. Much of the interest in MUC16 has been due to its high level of expression in ovarian cancer where it provides a potential target for therapeutic intervention (Das and Batra 2015). The molecule is also highly expressed in native epithelia, specifically at the surface of the corneal and conjunctival epithelia, where it has been demonstrated to provide barrier functions (Gipson et al. 2014), on the female reproductive tract endometrium where it is shed from the surface to allow trophoblast adherence prior to implantation (Gipson et al. 2008), and on the apical surface of the trachea where its function is unknown (Davies et al. 2007). In addition, MUC16 has been reported to be expressed by goblet cells of the respiratory epithelium (Davies et al. 2007; Kesimer et al. 2013) and the conjunctiva where the mucin is associated with the goblet cell mucin granule membrane (Gipson et al. 2015). Its role in the goblet cell mucin granule is unknown.

The MUC16 mucin is a type I transmembrane mucin that like other members of this heavily glycosylated class of glycoproteins expressed by wet-surfaced epithelia, contains a short cytoplasmic tail (CT). Its ectodomain is shed from cell surfaces and can be found in body fluids (Spurr-Michaud et al. 2007; Bottoni and Scatena 2015). Compared to other human transmembrane mucins, MUC16 is the largest at 22,152 amino acids (AA). The molecule has a heavily O-glycosylated terminal region of 12,000 AA without a well-defined structure, a region of 60 tandem repeats of 156 AA each that incorporate 56 SEA modules and both N and O-glycans (Marcos-Silva et al. 2014, 2015), a transmembrane domain and a 32 AA CT (Figure 1) (Hattrup and Gendler 2008; Govindarajan and Gipson 2010; Das and Batra 2015). Upon ectodomain shedding, the C-terminal domain comprised of a short juxtamembrane stalk, the transmembrane helix and the CT is retained. Antibodies generated to MUC16 have been shown to bind the extracellular tandem repeat region of MUC16, although it has been difficult to assign specific peptide sequence epitopes (Nustad et al. 1996, 2002; Marcos-Silva et al. 2014, 2015). A panel of monoclonal antibodies was also generated to the extracellular juxtamembrane region of the MUC16 CT domain retained on the cell surface after shedding of the major part of the ectodomain (Rao et al. 2010), which may have therapeutic value.

Fig. 1.

Fig. 1.

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The left panel presents a schematic depiction of the structure of MUC16 with designations of the cytoplasmic tail (CT), the transmembrane (TM) region, the ectodomain with its tandem repeats and SEA modules (after Govindarajan and Gipson 2010). The CT region has a polybasic ERM binding region (Blalock et al. 2007) and a known phosphorylation site is indicated by *P below the amino acid sequence of the CT domain. The plasmid map of the expression vector used in production of the recombinant triple repeat protein of the CT of MUC16 used as antigen to make MAb MUC16CT2C6 is shown on the right. Three identical sequences of MUC16CT with BamHI–EcoRI, EcoRI–XbaI and XbaI–HindIII flanks were amplified by PCR from cDNA of human corneal epithelial cells cultured to express MUC16. The amplified product was gel purified and cloned into the pPROEX-HTb expression vector. The pPROEX-HTb -MUC16 rCT (3X) was ~5000 bp. f1, origin of replication; lacI, lactose operon repressor; AmpR, ampicillin resistance selection marker; MCS, multiple cloning site; His6, 6X histidine tag; arrow represents direction of transcription/translation. This figure is available in black and white in print and in color at Glycobiology online.

Data suggest that MUC16 is a multifunctional molecule with its extracellular domain providing a barrier against pathogen invasion and cell adhesion (Gipson et al. 2008, 2014), which can be facilitated by association with galectin-3 (Argueso et al. 2009). Additionally, data suggest that its CT domain, after ectodomain shedding and release can induce signaling, influencing cancer cell growth on soft agar as well as invasive properties of cancer cells (Rao et al. 2015). The CT domain has been reported to associate with members of the Ezrin, Radixin, Moesin family (Blalock et al. 2007), with JAK2 (Lakshmanan et al. 2012), with beta catenin (Liu et al. 2016) and with SRC and SRC family tyrosine kinase YES (Akita et al. 2013). Ectodomain cleavage of MUC16 has been generally thought to occur extracellularly however a recent study suggests that the initial cleavage of the membrane proximal ectodomain may occur within the Golgi (Das et al. 2015). In this study, it was also shown that the CT domain including a segment of the cell surface juxtamembrane region carrying a reporter tag was detected in both cytosolic and nuclear fractions, suggesting that the entire CT domain can translocate to the nucleus to effect cellular functions (Das et al. 2015). Studies of function of the MUC16 CT domain have been carried out with either synthetic peptides of the MUC16 CT or with recombinant proteins of the C-terminal region including a portion of the ectodomain of the molecule that carry reporter tags. Verification of data from these studies of partial MUC16 constructs in cells and tissues would be facilitated by availability of a specific CT domain antibody that recognizes native protein allowing isolation, characterization and localization of the MUC16 CT.

We report here the development of a monoclonal antibody to the 32 AA CT of MUC16, which was generated by immunization with a recombinant expressed triple repeat of the MUC16 CT (MUC16 rCT(3X)) (Figure 1). The antibody recognizes full length and cellular MUC16 CT by Western blot analysis, and can immunopreciptate from OVCAR3 cell lysates the MUC16 CT released from full length MUC16 on the cells using the MUC16 sheddase ZmpC (Govindarajan et al. 2012). In addition, the antibody allows histochemical localization of the MUC16 CT on OVCAR3 cells and frozen tissue.

Results

Design and production of a MUC16 rCT(3X)

Attempts were made to produce MUC16 CT antibodies using a KLH conjugated synthetic peptide corresponding to the amino acid sequence of the 32 AA CT region. Clones were obtained that recognized the synthetic peptide but they did not recognize native MUC16 in cells or tissues (data not shown). We therefore designed a recombinant protein comprised of three repeated 32 AA sequences with a HIS6 tag introduced at the N-terminus to increase immunogenicity (Figure 1). The expression construct was verified by sequencing (data not shown) and expressed in Escherichia coli. The correct size (approx. 17 kDa) of the resulting affinity purified 135 AA recombinant protein was verified by SDS-PAGE analysis (Figure 2).

Fig. 2.

Fig. 2.

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SDS-PAGE Coomassie analysis of purification of recombinant HIS-tagged MUC16 rCT(3X). Fractions eluted from nickel affinity chromatography were used and the fraction designated #4 (*) containing the approximately 17 kDa HIS6 tagged protein were pooled from multiple batches. A total of 23 μg was obtained and concentrated for antibody production.

Generation and characterization of MAb MUC16CT2C6

The unconjugated HIS-tagged MUC16-rCT(3X) protein was purified by affinity chromatography to apparent homogeneity (Figure 2) and used for immunization. Hybridoma supernatants were screened by binding to a synthetic 32 AA MUC16 CT peptide with ELISA. As a negative control we used a purified recombinant HIS6-tagged MUC16 protein (MUC16-1.2TR) derived from the tandem repeat region of the ectodomain, which was produced in CHO SimpleCells with truncated O-glycans (Marcos-Silva et al. 2015).

Further characterization of candidate clones was done by Western blot analysis of OVCAR3 cell lysates with comparison of binding to a classical MUC16 antibody, MAb, M11, recognizing the ectodomain tandem repeats of MUC16 (Figure 3). We also compared binding of candidate MAbs to M11 and a similar clone, 5B9 (Marcos-Silva et al. 2015) using immunocytology with human cancer cell lines OVCAR3, HeLa, K562 (Figure 4 and Supplementary Figure 1) as well as by immunohistochemistry of frozen tissue sections of corneal epithelium known to express MUC16 in the apical cell layers (M11 only) (Figure 5). Based on these positive comparisons, clone MUC16CT2C6 was selected.

Fig. 3.

Fig. 3.

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SDS-PAGE Western blot analysis with MAb MUC16CT2C6. (A) OVCAR3 cell lysate blot (50 μg) probed with both MAbs M11 to the MUC16 ectodomain and the MUC16CT2C6 to the cytoplasmic tail. The blot was first probed with the MUC16CT2C6 antibody, then reprobed with the M11 antibody to confirm detection of MUC16. Note that the CT antibody weakly recognized the full length MUC16 and that M11 does not recognize cleaved MUC16 CT. (B) Western blot of MAb MUC16CT2C6 immunoprecipitates from OVCAR3 cell lysates after preincubation of the cells with (+) or without (–) rZmpC to remove the MUC16 ectodomain. After immunoprecipitation, MAb MUC16CT2C6 detected the CT domain in samples pretreated with rZmpC. We note variation in apparent contaminating high molecular weight bands in the IP most notably in the 4 H 60 min experiment with ZmpC, but the banding patterns are similar in all ZmpC treated samples at 30 and 60 min with increased exposure. The lack of detection of the full length MUC16 is likely due to its large size and potential removal from shearing during immunoprecipitation or lack of access of the mAb to the CT from the large MUC16 ectodomain. Without ZmpC treatment (–), the CT was weakly detected, due perhaps to lower protein loading (approximately 20 μg) than in A (50 μg) or to steric hindrance of access of the MAb MUC16CT2C6 to the CT domain by the massive full length MUC16 molecule.

Fig. 4.

Fig. 4.

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Immunocytology with MAb MUC16CT2C6 showing strong cell surface immunoflourescense (A) similar to traditional MUC16 MAb 5B9 (B). MAb 2D10 (GalNAc-T3) showed a different Golgi-like staining pattern (C) and served as isotope control. This figure is available in black and white in print and in color at Glycobiology online.

Fig. 5.

Fig. 5.

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Immunhistology with MAb MUC16CT2C6 (A, C) and control MAb M11 (B, D). Binding of both MAbs on frozen sections of human corneal (A, B) and human uterine (C, D) epithelia has similar apical surface patterns. (E) The secondary antibody only control on corneal epithelium—the same secondary antibody was used for localization of both MAbs MUC16CT2C6 and M11. Bar = 20 μm. This figure is available in black and white in print and in color at Glycobiology online.

Binding of MAb MUC16CT2C6 to full length MUC16 and to MUC16CT released by rZmpC

To ascertain that MAb MUC16CT2C6 bound to native MUC16 and the MUC16 CT after MUC16 ectodomain release, and to determine if the antibody could be used to immunoprecipitate the MUC16 CT, OVCAR3 cells as well as OVCAR3 cells exposed to recombinant ZmpC, a metalloproteinase expressed by Streptococcus pneumoniae and known to induce MUC16 ectodomain release (Govindarajan et al. 2012) was used. MAb MUC16CT2C6 recognized both full length MUC16 as well as a >20 kDa low molecular weight protein comparable in size to the MUC16 CT in OVCAR cell lysates (Figure 3A). In OVCAR3 cell lysates from cells pretreated with ZmpC for 30 or 60 min to remove the MUC16 ectodomain, the MAb MUC16CT2C6 immunoprecipitated an approximately 22–25 kDA protein comparable in size to the predicted mass of the MUC16 CT with inclusion of the transmembrane and immediate juxtamembrane region of the ectodomain retained after shedding (Figure 3B). The CT size is to be expected since ZmpC is predicted to cleave the extracellular domain more N-terminal than the endogenous cleavage site (Govindarajan et al. 2012).

Immunohistochemistry with MAb MUC16CT2C6

By immunocytology MAb MUC16CT2C6 showed strong binding to the ovarian cancer cell line OVCAR3, extremely weak binding to the cervix cancer cell line HeLa and no reactivity with the lymphoblast cell line K562 cell (Figure 4 and Supplementry Figure 1). The expression of MUC16 in these cell lines was monitored by MAb 5B9, and this correlated well including the extremely weak expression of MUC16 in HeLa.

By immunohistology MAb MUC16CT2C6 labeled the apical surface of both corneal epithelium as well as uterine epithelium (Figure 5). The binding was similar to that of MAbs M11 and 5B9 (not shown) directed to the ectodomain of MUC16, albeit less intense and not as discretely localized at the cell membrane. Since the latter MAbs recognize repeated epitopes in the SEA domains it may be expected to enhance the immunofluorescence signal (Marcos-Silva et al. 2014). We noted that MAb MUC16CT2C6 did not bind formalin fixed, paraffin embedded tissue, thus frozen sections must be used. Different fixation protocols were tested on OVCAR3 cells including paraffin embedding of cell pellets. In brief, acetone and paraformaldehyde were tolerated well on cells attached to coverslips, but paraffin embedding of cell pellets rendered cells unreactive even after antigen retrieval by microwave (pH 6.0). Also we noted that overnight incubation with the antibody enhanced immunofluorescence signal.

Discussion

We report here generation and characterization of the first MAb to MUC16 that detects the CT domain of the large cell membrane mucin. The antibody will be a useful reagent for studies of the fate and biology of the retained intracellular domain of MUC16 retained after shedding of the ectodomain, and it will facilitate studies to determine whether the native CT is phosphorylated and has binding partners that impact function. We were only successful in stimulating appropriate immunity to make the MAb with a unique antigen design comprised of a recombinantly derived repeated CT peptide. The resultant MAb designated MUC16CT2C6 was reactive in both Western blot analysis and immunohistochemistry with fresh frozen cells and tissues. Importantly, the antibody immunoprecipitated MUC16 CT released from the ectodomain by bacterial ZmpC.

As an illustrative example of usefulness of mucin CT antibodies, a monoclonal antibody generated to a synthetic peptide to the CT domain of the mucin MUC1, designated MUC1 CT2, has been widely used (Pemberton et al. 1992), and as a result the role of the MUC1 CT in signaling evaluated [for review see (Hanson and Hollingsworth 2016; Cascio and Finn 2016)]. In addition the MUC1 CT2 antibody as well as other antibodies to the MUC1 CT have been used to follow the movement of the CT and its associated proteins from the cell membrane to the nucleus after ectodomain release where the assemblages are involved in gene regulation (Wei et al. 2005; Singh et al. 2008; Liu et al. 2014).

Compared to the current knowledge of the role of the MUC1 CT in signaling, much less is known regarding the MUC16 CT. The MUC1 CT has 74 AA compared to MUC16's 32, and it has 22 potential phosphorylation sites, approximately half of which have been characterized (see Hanson and Hollingsworth 2016). MUC16 has several potential phosphorylation sites, although only one of these has been validated (for review see Hanson and Hollingsworth 2016). Availability of MAb MUC16CT2C6 may facilitate characterization of the additional potential phosphorylation sites.

Several lines of data suggest that the MUC16 CT has binding partners and signaling capabilities. Constructs of a 114 AA C-terminal domain inserted into 3T3 fibroblasts induced increased growth on and invasion into matrigel and increased tumor growth in mice, suggesting a role for the C-terminal region in signaling (Rao et al. 2015). Data also suggest that the MUC16 CT associates with the Janus associated kinase JAK2 as JAK2 antibodies bind full length MUC16 by western blot (Lakshmanan et al. 2012). The association of MUC16 CT to JAK2 is hypothesized to be through association of JAK2's ERM-like domain. It has been shown previously that ERMs bind to the RRRKK sequence in synthetic peptides that mimic the MUC16 CT (Blalock et al. 2007). Expression of the 114 AA MUC16 C-terminal construct in pancreatic cancer cells induced enhanced nuclear localization of JAK2 as well as nuclear localization of a the tagged114 AA MUC16 CT construct, suggesting that the MUC16 CT terminal region can be transported to the nucleus similar to what has been shown for the MUC1 CT (Das et al. 2015). It would be of interest to determine if native epithelia as well as cancer cells show similar binding partners and signaling patterns; use of MAb MUC16CT2C6 could facilitate such studies.

A recent report demonstrated that a tagged C-terminal construct of MUC16 expressed in colon cancer cells and immunoprecipitated from the cells showed bound SRC as well as the tyrosine-kinase YES a member of the SRC family of kinases (Akita et al. 2013). The binding of SRC to the MUC16 construct resulted in phosphorylation of a tyrosine (corresponding to residue 22,141 in the full coding MUC16 gene) in the MUC16 CT construct that induced shedding of the ectodomain of the construct. It would be of interest to determine if the MUC16 CT moved to the nucleus after the SRC interaction, and the use of MAb MUC16CT2C6 would facilitate such studies.

In native epithelia including that of the ocular surface, the female reproductive tract and the respiratory system, MUC16 ectodomain emanates from the apical surface of these polarized epithelia. Knockdown of MUC16 in an in vitro model of polarized corneal epithelia resulted in loss of tight junctions and increased cell surface size (Gipson et al. 2014), leading to the hypothesis that MUC16 through its polybasic sequence binds members of the ezrin, radixin, moesin family and through this binding the actin cytoskeletal cortical web that plays a role in tight junction formation and protrusion of surface microplicae and microvilli. High-resolution imaging facilitated through use of MAb MUC16CT2C6 may yield further information to characterize the barrier functions of MUC16 in native epithelia.

In summary, MUC16 has been described as being a multifunctional molecule; it serves as a barrier to pathogen penetrance at apical surfaces of native epithelia, it associates with the actin cytoskeleton and its knockdown abrogates tight junction formation and alters cell shape. In cancer cells it appears to be involved in control of cell cycle and cell–cell adhesion. Its extracellular domain, shed from surfaces of cancer cells appears to induce signaling that influences cell proliferation. Many questions remain regarding the function of the MUC16 CT; availability of MAb MUC16CT2C6 will hopefully facilitate future studies of this large transmembrane mucin.

Materials and methods

Design and expression of a MUC16 rCT(3X)

To generate the triple repeat, three identical sequences of MUC16CT with BamHI-EcoRI, EcoRI-XbaI and XbaI-HindIII flanks were amplified by PCR from cDNA of Human Corneal Limbal Epithelial (HCLE) cells grown for optimal mucin expression (Gipson et al. 2003). The amplified product was gel purified and cloned into the BamHI and HindIII restriction sites of the pPROEX-HTb expression vector (gift from Dr. Gordon Cannon at the University of Southern Mississippi). Oligonucleotide sequences used for amplifying the individual MUC16CT sequences were 5′-CGCGGATCCGTGACCACCCGCCGGCGG-3′ and 5′-GGAATTCTTGCAGATCCTCCAGGTC-3′ for sequence 1, 5′-GGAATTCGTGACCACCCGCCGGCGG-3′ and 5′-GCTCTAGATTGCAGATCCTCCAGGTC-3′ for sequence 2, and 5′-GCTCTAGAGTGACCACCCGCCGGCGG-3′ and 5′-CCCAAGCTTTTGCAGATCCTCCAGGTC-3′ for sequence 3. Underlined sequences represent BamHI, EcoRI, XbaI and HindIII restriction sites. DNA sequencing at the University of Maine DNA Sequencing Facility confirmed the construct sequence. Recombinant protein expression was induced by the addition of 0.5 mM IPTG (Promega) to E. coli BL21 cells (New England Biolabs) at OD600 of ~0.4. Expression of the resulting construct with an N-terminal His6 tag in E. coli BL21 cells yielded a triple repeat of MUC16CT (~17 kDa) (Figure 2) with each repeat corresponding to the amino acid sequence, VTTRRRKKEGEYNVQQQCPGYYQSHLDLEDLQ. Purification of the triple repeat of MUC16CT was performed under denaturing conditions using Ni-NTA affinity chromatography as described previously (Menon and Govindarajan 2013). Eluates containing purified protein were passed through a 50-kDa cut off Millipore centrifugal filtration device. Methanol precipitation was used to recover the protein in the flow through fraction. Precipitated protein was resuspended in sterile PBS, the concentration was determined using MicroBCA protein assay (Pierce) and aliquots were run reduced on 4% stacking, 12% separating SDS-PAGE and the gel stained with GelCode Blue (Thermo Scientific) to confirm correct molecular weight. Protein from multiple batches of purification (23 μg in total) was pooled, divided into four equal aliquots in low protein binding tubes (Fisher) and concentrated at room temperature at 30 bars Hg in a LABCONCO Centrivap concentrator for 12 hours. The concentrated samples were then divided into 4 × 100 μL aliquots for antibody production.

Antibodies

Two mouse monoclonal antibodies against the ectodomain of MUC16 were used in this study. MAb M11 (Thermo Fisher Scientific) was generated by immunizing mice with partially purified CA125 antigen derived from ascetic fluid (O'Brien et al., 1991). M11 recognizes the tandem repeated SEA domains in the ectodomain of MUC16 (Nustad et al. 2002; Marcos-Silva et al. 2015). Mab 5B9 was obtained by immunizing mice with a purified recombinant HIS-tagged MUC16 tandem repeat construct (MUC16-1.2TR) produced in CHO Simple Cells with truncated-O-glycosylation. Mab 5B9 has similar specificity as M11 (Marcos-Silva et al. 2015).

Generation of MAb MUC16CT2C6

Two female Balb/c mice were immunized with a single subcutaneous injection of 6 μg of unconjugated pure MUC16 rCT(3X) protein (Figure 1) in a total volume of 200 μL (1:1 mix with Freunds adjuvant) three times and finally an intravenous boost without adjuvant. Three days after the fourth immunization, splenocytes from one mouse were fused with NS1 myeloma cells as described previously (Sorensen et al. 2006). Animal experiments were approved by the Danish authorities (license number 2015-15-0201-00625). Hybridoma supernatants were screened by ELISA and immunocytochemistry after 10–12 days, and selected clones subjected to at least two limiting dilutions. One clone designated MUC16CT2C6 was selected and determined by immunocytology to secrete IgG1.

Cell culture and protein isolation

OVCAR3 cells (ATCC) were plated on plastic and grown to confluence plus 7 days in RPMI-1640 Medium (ATCC) supplemented with 0.01 mg/mL bovine insulin and fetal bovine serum to a final concentration of 20% at 37°C in a 5% carbon dioxide atmosphere to achieve optimal MUC16 expression. Cells were switched to unsupplemented DMEM/F12 for 24 hours prior to treatment with 200 pmol of recombinant ZmpC (rZmpC) (Menon and Govindarajan 2013) diluted in unsupplemented DMEM/F12 or with vehicle control (unsupplemented DMEM/F12) for 30 min (n = 2 each), 60 min (n = 2 each) or 4 h (rZmpC treatment only; n = 2). Cells were then washed once with Modified Dulbecco's PBS and lysed with ice-cold Pierce Co-Immunoprecipitation lysis buffer (0.025 M Tris, 0.15 M NaCl, 0.001 M EDTA, 1% NP-40, 5% glycerol; pH 7.4) plus Halt protease and phosphatase inhibitor cocktail (Thermo Scientific) and incubated on ice for 5 min with periodic mixing. The lysate was cleared of cell debris by centrifugation at 13,000 × g for 10 min. A BCA protein assay (Pierce) was performed on the cleared cell lysate.

Immunoprecipitation and western blot

Immunoprecipitation (IP) of MUC16 protein from the OVCAR3 cell lysates using the MAb MUC16CT2C6 was performed using the Pierce Co-Immunoprecipitation Kit (Thermo Fisher) according to manufacturer's recommendations. Briefly, 75 μg of MAb MUC16CT2C6 diluted in 1× coupling buffer was immobilized to AminoLink Plus Coupling Resin (Thermo Fisher) in spin columns. The immobilized antibody was incubated with 100 μg of OVCAR3 cell lysate overnight at 4°C on a rocker. The flow through was collected for analysis, the beads were washed 6 times with IP Lysis/Wash Buffer plus Halt Protease and Phosphatase Inhibitors (Thermo Scientific) and bound protein was eluted with Pierce Elution Buffer (Thermo Scientific). The specificity of the IP was confirmed by incubating 100 μg of cell lysate with the AminoLink Plus Coupling resin without immobilized antibody. A 25 μg of OVCAR3 total cell lysate (pre-IP) and 20 μL of each eluate from the IP spin columns were run reduced on 4% stacker, 10% separating SDS-PAGE, transferred to nitrocellulose for 2 h at 500 mA constant current using a MiniBlot Transfer Module (BioRad). The membranes were blocked with 5% BLOTTO in TBST for 1 h at room temperature and transferred proteins were probed with MAb MUC16CT2C6 diluted 1:500 in 5% BLOTTO in TBST overnight at 4°C. The membrane was washed 6 times with TBST, incubated with HRP-conjugated goat antimouse IgG1 (Santa Cruz) for 1 h at room temperature, washed 6 times with TBST and incubated with Pierce SuperSignal West Femto substrate (diluted 1:3 with dH2O) for 5 min and exposed to Hyblot film (Denville Scientific).

Immunocytology

OVCAR3 cells were cultured in RPMI supplemented with 2% glucose, 1% l-glutamine and 15% FBS, and HeLa and K562 cells in DMEM with 10% FBS and 1% l-glutamine. OVCAR3 cells were seeded on sterile coverslips coated with type I collagen (0.4 mg/ml) and cultivated for 2–3 days. The cells were then washed in cold PBS and fixed in cold PFA for 10 min and washed again. OVCAR3, HeLa and K562 cells were also trypsinized and seeded on slides, airdried at RT overnight and fixed for 5 min in ice-cold acetone. Slides were incubated with MAbs as undiluted culture supernatants overnight at 4°C, followed by 1 h at RT with FITC-conjugated goat anti-mouse IgG (γ-chain specific (Southern Biotech) and washing and mounting in Vectashield with DAPI (Vector Laboratories, Inc.). Images were acquired using a Zeiss Axioscope 2 plus with an AxioCam MRc (40x).

Immunohistology

Acquisition of human tissues was done in compliance with Institutional Review Board regulations, informed consent regulations, and the tenets of the Declaration of Helsinki. Human corneal and human uterine tissues, epithelia of which are known to express MUC16 at their apical epithelial surfaces, were used and were those obtained for previous studies (Gipson et al. 2008, 2014). Methods to immunolocalize binding of the MAb MUC16CT2C6 to frozen sections of the two tissues were as previously described (Gipson et al. 2008, 2014). As control for presence of MUC16, MAb M11 (Thermo Fisher Scientific), which recognizes a SEA Module domain in the ectodomain of MUC16 was used, and as secondary antibody, FITC-conjugated donkey antimouse IgG (Jackson Laboratories).

Supplementary Material

Supplementary Data
Click here for additional data file. (2.7MB, pdf)

Supplementary data

Supplementary data is available at Glycobiology online.

Conflict of interest statement

None declared.

Funding

This work was supported by the National Institute of Health grant (NEI, R01 EY03306 to IKG) and the Danish Research Council and the Danish National Research Foundation (DNRF107).

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