Key resources table.
| Reagent type (species) or resource |
Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (mouse) |
Frmd8-/-: C57BL/6- Frmd8tm1(KOMP)Vlcg |
This paper | N/A | FRMD8 KO mice generated as described in materials and methods |
| Strain, strain background (mouse) |
WT control: C57BL/6 | This paper | N/A | WT control for FRMD8 KO mice |
| Strain, strain background (mouse) |
Rhbdf2-/-: C57BL/6 -Rhbdl6A22 |
(Adrain et al., 2012) | N/A | |
| Cell line (human) | HEK293T cells | Freeman lab | RRID:CVCL_0063 | |
| Cell line (human) | HEK293T human iRhom1WT |
This paper | N/A | HEK293T cells transduced with pLEX.puro-human iRhom1WT-3xHA |
| Cell line (human) | HEK293T human iRhom2WT |
This paper | N/A | HEK293T cells transduced with pLEX.puro-human iRhom2WT-3xHA |
| Cell line (human) | HEK293T human UNC93B1 |
This paper | N/A | HEK293T cells transduced with pLEX.puro-human UNC93B1-3xHA |
| Cell line (human) | HEK293T FRMD8 KO | This paper | N/A | CRISPR/Cas9-mediated KO cell line as described in materials and methods |
| Cell line (human) | HEK293T FRMD8 KO + human iRhom2WT |
This paper | N/A | FRMD8 KO cells transduced with pLEX.puro-human iRhom2WT-3xHA |
| Cell line (human) | HEK293T endogenous iRhom2-3xHA |
This paper | N/A | CRISPR/Cas9-mediated knock-in cell line as described in materials and methods |
| Cell line (human) | HEK293T iRhom1/iRhom2 double-knockout (DKO) |
This paper | N/A | CRISPR/Cas9-mediated KO cell line as described in materials and methods |
| Cell line (human) | HEK293T iRhom1/iRhom2 DKO + human iRhom2WT |
This paper | N/A | DKO cells transduced with pLEX.puro-human iRhom2WT-3xHA |
| Cell line (human) | HEK293T iRhom1/iRhom2 DKO + human iRhom2Δ300 |
This paper | N/A | DKO cells transduced with pLEX.puro-human iRhom2Δ300-3xHA |
| Cell line (human) | HEK293T iRhom1/iRhom2 DKO + human FRMD8- iRhom2Δ300 |
This paper | N/A | DKO cells transduced with pLEX.puro-human FRMD8- iRhom2Δ300-3xHA |
| Cell line (human) | HEK293T iRhom1/iRhom2 DKO + human iRhom2Δ 201–300 |
This paper | N/A | DKO cells transduced with pLEX.puro-human iRhom2Δ201-300-3xHA |
| Cell line (human) | hiPSC line AH017-13 | (Fernandes et al., 2016) | James Martin Stem Cell Facility |
|
| Cell line (human) | hiPSC line AH017-13 FRMD8 KO clone 1 (clone F1) |
This paper | N/A | CRISPR/Cas9-mediated KO cell line as described in materials and methods |
| Cell line (human) | hiPSC line AH017-13 FRMD8 KO clone 2 (clone G6) |
This paper | N/A | CRISPR/Cas9-mediated KO cell line as described in materials and methods |
| Cell line (human) | hiPSC line AH017-13 WT (clone E4) |
This paper | N/A | unedited WT control for FRMD8 KO iPSCs clones |
| Cell line (mouse) | Frmd8-/- ES cells: C57BL/ 6NTac-Frmd8tm1(KOMP)Vlcg (clone 17364AC3) |
KOMP | RRID:IMSR_KOMP:
VG17364-1-Vlcg |
|
| Antibody | anti-β-actin-HRP, mouse monoclonal (clone AC-15) |
Sigma-Aldrich | Cat#A3854; RRID:AB_262011 |
dilution is described in materials and methods |
| Antibody | anti-ADAM10, mouse monoclonal (clone SHM14) |
BioLegend | Cat#352702; RRID:AB_10897813 |
dilution is described in materials and methods |
| Antibody | anti-ADAM10, rabbit polyclonal |
Cell Signaling Technology |
Cat#14194 | dilution is described in materials and methods |
| Antibody | anti-ADAM17, mouse monoclonal (clone A300E) |
(Yamamoto et al., 2012); received from Stefan Düsterhöft |
N/A | dilution is described in materials and methods |
| Antibody | anti-ADAM17, rabbit polyclonal |
Abcam | Cat#ab39162; RRID:AB_722565 |
dilution is described in materials and methods |
| Antibody | anti-calnexin, rabbit polyclonal |
Santa Cruz | Cat#sc-11397; RRID:AB_2243890 |
dilution is described in materials and methods |
| Antibody | anti-FRMD8, rabbit polyclonal |
Abcam | Cat#ab169933 | dilution is described in materials and methods |
| Antibody | anti-HA, rabbit polyclonal | Santa Cruz | Cat#sc-805; RRID:AB_631618 |
dilution is described in materials and methods |
| Antibody | anti-HA, rat monoclonal (clone 3F10) |
Roche | Cat#11867423001; RRID:AB_10094468 |
dilution is described in materials and methods |
| Antibody | anti-HA tag, rabbit monoclonal (C29F4) |
CST | Cat#3724; RRID:AB_1549585 |
dilution is described in materials and methods, used at 1:1000 for IF |
| Antibody | anti-HA-HRP, rat monoclonal (clone 3F10) |
Roche | Cat#12013819001; RRID:AB_390917 |
dilution is described in materials and methods |
| Antibody | anti-iRhom2, rabbit polyclonal |
(Adrain et al., 2012) | N/A | dilution is described in materials and methods |
| Antibody | ant-LAMP1, mouse monoclonal (H4A3) |
Santa Cruz | Cat#sc-20011; RRID:AB_626853 |
dilution is described in materials and methods, used at 1:250 for IF |
| Antibody | anti-transferrin receptor 1, mouse monoclonal (clone H68.4) |
Thermos Fisher Scientific |
Cat#13–6800; RRID:AB_86623 |
dilution is described in materials and methods |
| Antibody | anti-V5, goat polyclonal | Santa Cruz | Cat#sc-83849; RRID:AB_2019670 |
dilution is described in materials and methods, used at 1:1000 for IF |
| Antibody | anti-goat-HRP, mouse monoclonal |
Santa Cruz | Cat#sc-2354; RRID:AB_628490 |
dilution is described in materials and methods |
| Antibody | anti-mouse-HRP, goat polyclonal |
Santa Cruz | Cat#sc-2055; RRID:AB_631738 |
dilution is described in materials and methods |
| Antibody | anti-rabbit-HRP, goat polyclonal |
Sigma-Aldrich | Cat#A9169; RRID:AB_258434 |
dilution is described in materials and methods |
| Antibody | anti-mouse Alexa Fluor 488, donkey polyclonal |
Thermos Fisher Scientific |
Cat#A-21202; RRID:AB_141607 |
dilution is described in materials and methods |
| Antibody | anti-rabbit Alexa Fluor 488, donkey polyclonal |
Thermos Fisher Scientific |
Cat#A-21206; RRID:AB_2535792 |
dilution is described in materials and methods |
| Antibody | anti-rabbit Alexa Fluor 647, donkey polyclonal |
Thermos Fisher Scientific |
Cat#A-31573; RRID:AB_2536183 |
dilution is described in materials and methods |
| Recombinant DNA reagent |
cDNA: human iRhom2 (NM_024599.2) |
Origene | Cat#SC122961 | |
| Recombinant DNA reagent |
cDNA: human FRMD8 (NM_031904) |
Origene | Cat#SC107202 | |
| Recombinant DNA reagent |
cDNA: human UNC93B1 | (Brinkmann et al., 2007) | N/A | |
| Recombinant DNA reagent |
Plasmid: pLEX.puro | Thermo Fisher Scientific |
Cat#OHS4735 | |
| Recombinant DNA reagent |
Plasmid: pcDNA3.1(+) | Thermo Fisher Scientific |
Cat#V790-20 | |
| Recombinant DNA reagent |
Plasmid: entiviral packaging plasmid |
(Adrain et al., 2012) | N/A | |
| Recombinant DNA reagent |
Plasmid: entiviral envelope plasmid |
(Adrain et al., 2012) | N/A | |
| Recombinant DNA reagent |
Plasmid: pSpCas9(BB) −2A-Puro (pX459) |
(Ran et al., 2013) | Addgene plasmid #48139 |
|
| Recombinant DNA reagent |
Plasmid: epX459(1.1) | received from Joey Riepsaame |
N/A | |
| Recombinant DNA reagent |
Plasmid: pSpCas9(BB) −2A-Puro V2.0 (pX462 V2.0) |
(Ran et al., 2013) | Addgene plasmid #62987 |
|
| Transfected construct (human) |
Plasmid: pLEX.puro-human iRhom1WT-3xHA |
(Christova et al., 2013) | N/A | |
| Transfected construct (human) |
Plasmid: pLEX.puro-human iRhom2WT-3xHA |
This paper | N/A | cloned from human iRhom2 (NM_024599.2) |
| Transfected construct (human) |
Plasmid: pLEX.puro-human iRhom2Δ100-3xHA |
This paper | N/A | human iRhom2 lacking amino acids 1–100 |
| Transfected construct (human) |
Plasmid: pLEX.puro-human iRhom2Δ200-3xHA |
This paper | N/A | human iRhom2 lacking amino acids 1–200 |
| Transfected construct (human) |
Plasmid: pLEX.puro-human iRhom2Δ300-3xHA |
This paper | N/A | human iRhom2 lacking amino acids 1–300 |
| Transfected construct (human) |
Plasmid: pLEX.puro-human iRhom2Δ201-300-3xHA |
This paper | N/A | human iRhom2 lacking amino acids 1–382 |
| Transfected construct (human) |
Plasmid: pLEX.puro-human iRhom2Δ382-3xHA |
This paper | N/A | human iRhom2 lacking amino acids 201–300 |
| Transfected construct (human) |
Plasmid: pLEX.puro-human FRMD8-iRhom2Δ300-3xHA |
This paper | N/A | human FRMD8 fused to human iRhom2Δ300 via a flexible linker (GSGSGS) |
| Transfected construct (mouse) |
Plasmid: pM6P.blast-mouse iRhom2WT-3xHA |
(Grieve et al., 2017) | N/A | |
| Transfected construct (mouse) |
Plasmid: pM6P.blast-mouse iRhom2cub-3xHA |
(Grieve et al., 2017) | N/A | |
| Transfected construct (mouse) |
Plasmid: pM6P.blast-mouse iRhom2pDEAD-3xHA |
(Grieve et al., 2017) | N/A | |
| Sequence-based reagent |
gRNA targeting exon 7 of human FRMD8 (ACCC ATAAAACGGCAGCTCG) |
This paper | N/A | gRNA targeting exon 7 of human FRMD8 |
| Sequence-based reagent |
gRNA targeting exon 19 of human RHBDF2 (AG CGGTCAGTGCAGCACCT) |
This paper | N/A | gRNA targeting exon 19 of human RHBDF2 |
| Sequence-based reagent |
gRNA targeting exon 3 of human RHBDF1 (GGAACC ATGAGTGAGGCCCC) |
This paper | N/A | gRNA targeting exon 3 of human RHBDF1 |
| Sequence-based reagent |
gRNA targeting exon 3 of human RHBDF1 (GGGTGG CTTCTTGCGCTGCC) |
This paper | N/A | gRNA targeting exon 3 of human RHBDF1 |
| Sequence-based reagent |
gRNA targeting exon 10 of human RHBDF1 (AGCCGT GTGCATCTATGGCC) |
This paper | N/A | gRNA targeting exon 10 of human RHBDF1 |
| Sequence-based reagent |
gRNA targeting exon 10 of human RHBDF1 (CCGTCTC ATGCTGCGAGAAC) |
This paper | N/A | gRNA targeting exon 10 of human RHBDF1 |
| Sequence-based reagent |
gRNA targeting exon 2 of human RHBDF2 (GCAGAG CCGGAAGCCACCCC) |
This paper | N/A | gRNA targeting exon 2 of human RHBDF2 |
| Sequence-based reagent |
gRNA targeting exon 2 of human RHBDF2 (GGGTCT CTTTCTCGGGTGGC) |
This paper | N/A | gRNA targeting exon 2 of human RHBDF2 |
| Sequence-based reagent |
gRNA targeting exon 9 of human RHBDF2 (AAACTC GTCCATGTCATCATCACC) |
This paper | N/A | gRNA targeting exon 9 of human RHBDF2 |
| Sequence-based reagent |
gRNA targeting exon 9 of human RHBDF2 (ACGGG TGCGATGCCATACGC) |
This paper | N/A | gRNA targeting exon 9 of human RHBDF2 |
| Sequence-based reagent |
non-targeting siGENOME control pool |
Dharmacon | D-001206-13-50 | |
| Sequence-based reagent |
siGENOME SMARTpool for human FRMD8 |
Dharmacon | M-018955-01-0010 | |
| Sequence-based reagent |
siRNA targeting human RHBDF2 (HSS128594) |
Thermo Fisher Scientific |
Cat#1299001 | |
| Sequence-based reagent |
siRNA targeting human RHBDF2 (HSS128595) |
Thermo Fisher Scientific |
Cat#1299001 | |
| Sequence-based reagent |
Human ACTB
(Hs99999903_m1) |
Thermo Fisher Scientific |
Cat#4331182 | |
| Sequence-based reagent |
Human ADAM17
(Hs01041915_m1) |
Thermo Fisher Scientific |
Cat#4331182 | |
| Sequence-based reagent |
Human FRMD8
(Hs00607699_mH) |
Thermo Fisher Scientific |
Cat#4331182 | |
| Sequence-based reagent |
Human RHBDF2
(Hs00226277_m1) |
Thermo Fisher Scientific |
Cat#4331182 | |
| Sequence-based reagent |
Human TNF
(Hs00174128_m1) |
Thermo Fisher Scientific |
Cat#4331182 | |
| Commercial assay or kit |
BCA Protein Assay Kit | Thermo Fisher Scientific |
Cat# 23225 | |
| Commercial assay or kit |
Human TNF alpha ELISA Kit |
Thermo Fisher Scientific |
Cat#88-7346-86 | |
| Commercial assay or kit |
SuperScript VILO cDNA synthesis kit |
Thermo Fisher Scientific |
Cat#11754050 | |
| Chemical compound, drug |
1,10-Phenanthroline | Sigma-Aldrich | Cat#131377–5G | |
| Chemical compound, drug |
DSP (dithiobis(succinimidyl propionate)) |
Thermos Fisher Scientific |
Cat#22585 | |
| Chemical compound, drug |
EDTA-free protease inhibitor mix |
Roche | Cat#11873580001 | |
| Chemical compound, drug |
GW280264X (GW) | (Lorenzen et al., 2016); received from Stefan Düsterhöft |
N/A | |
| Chemical compound, drug |
GI254023X (GI) | (Lorenzen et al., 2016); received from Stefan Düsterhöft |
N/A | |
| Chemical compound, drug |
LPS | Sigma-Aldrich | Cat#L5668-2ML | |
| Chemical compound, drug |
nocodazole | Sigma-Aldrich | Cat#M1404 | |
| Chemical compound, drug |
PNPP substrate | Thermos Fisher Scientific |
Cat#34045 | |
| Chemical compound, drug |
Rho kinase inhibitor Y-27632 |
Abcam | Cat#ab120129 | |
| Peptide, recombinant protein |
Q5 High-Fidelity DNA polymerase |
New England Biolabs | Cat#M0491S | |
| Peptide, recombinant protein |
Sequencing Grade Trypsin |
Promega | Cat#V5111 | |
| Peptide, recombinant protein |
HA peptide | Roche | Cat#I2149-.5MG | |
| Peptide, recombinant protein |
M-CSF | Gibco | Cat#PHC9501 | |
| Peptide, recombinant protein |
IL-3 | Gibco | Cat#PHC0033 | |
| Peptide, recombinant protein |
BMP-4 | Invitrogen | Cat#PHC9534 | |
| Peptide, recombinant protein |
VEGF | PeproTech | Cat#100–20 | |
| Peptide, recombinant protein |
SCF | Miltenyi | Cat#130-094-303 | |
| Peptide, recombinant protein |
RhFGF (bFGF) | R and D | Cat#4114-TC | |
| Other | anti-HA magnetic beads | Thermo Fisher Scientific |
Cat#88837 | |
| Other | anti-V5 magnetic beads | MBL International | Cat#M167-11 | |
| Other | concanavalin A sepharose | Sigma-Aldrich | Cat#C9017-25ML | |
| Other | C18 spin columns | Thermo Fisher Scientific |
Cat#89873 | |
| Other | vivaspin concentrator 500 (10,000 kDa MWCO) |
Sartorius | Cat#VS0102 | |
| Other | mouse IgG agarose | Sigma-Aldrich | Cat#A0919-5ML | |
| Other | DMEM | Thermo Fischer Scientific |
Cat#41965039 | |
| Other | Fetal bovine serum | Thermo Fischer Scientific |
Cat#10500064 | |
| Other | Fish skin gelatin | Sigma-Aldrich | Cat#G7765 | |
| Other | KnockOut -DMEM | Thermo Fischer Scientific |
Cat#10829 | |
| Other | KnockOut - serum replacement |
Thermo Fischer Scientific |
Cat#10828 | |
| Other | MEM Non-Essential Amino Acids (100x) |
Thermo Fischer Scientific |
Cat#11140–035 | |
| Other | GlutaMAX (100x) | Thermo Fischer Scientific |
Cat#35050–038 | |
| Other | 2-Mercaptoethanol (1000x) | Thermo Fischer Scientific |
Cat#31350–010 | |
| Other | Penicillin-Streptomycin (P/S 100x) |
Thermo Fischer Scientific |
Cat#15140–122 | |
| Other | 6-well ultra-low attachment plates |
Corning | Cat#3471 | |
| Other | X-VIVO 15 | Lonza | Cat#BE04-418 | |
| Other | mTeSR1 | Stemcell Technologies | Cat#12491 | |
| Other | Paraformaldehyde 16% | Electron Microscopy Sciences |
Cat#15710 | |
| Other | hESC-qualified Geltrex | Thermo Fischer Scientific |
Cat#A1413302 | |
| Other | ProLong Gold antifade reagent with DAPI |
Molecular Probes | Cat#P36935 | |
| Software, algorithm | FlowJo (version X 10.0.7r2) | FlowJo, LLC |
https://www.flowjo.com/
solutions/flowjo |
|
| Software, algorithm | Prism (version 7) | GraphPad |
https://www.graphpad.com/
scientific-software/prism/ |
|
| Software, algorithm | MaxQuant (version 1.5.0.35) | (Cox and Mann, 2008) |
http://www.coxdocs.org/
doku.php?id=maxquant:start |
|
| Software, algorithm | Perseus (version 1.5.5.3) | (Tyanova et al., 2016) |
http://www.coxdocs.org/
doku.php?id=perseus:start |
|
| Software, algorithm | Fiji (version 2.0.0-rc- 43/1.52a) |
(Schindelin et al., 2012) | https://fiji.sc/ | |
| Software, algorithm | Clustal Omega | EMBL-EBI |
https://www.ebi.ac.uk/
Tools/msa/clustalo/ |