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. 2018 Jan 10;9(17):13206–13221. doi: 10.18632/oncotarget.24152

Figure 2. Identification of annexin A2 as the antigen target of 2448.

Figure 2

Immunoprecipitation (IP) of lysate from IGROV1 ovarian cancer cells was done with 2448-coupled Protein G—beads. IP with 2448 enriched for unique antigen bands around 39 kDa (←) on Western blot. The corresponding bands on parallel silver stained gels were excised and sent for liquid chromatography-tandem mass spectrometry (LC-MS/MS). A search against protein databases revealed Annexin A2 as a top match for multiple rounds of IP. (C) Peptide coverage (as highlighted) was found to span across the amino acid sequence of ANXA2. (D) Antigen recognition of 2448 was abolished after cells were transfected with validated ANXA2-specific siRNA (versus scramble siRNA as a control). Loss of binding corresponded to a decrease in ANXA2 expression using a commercial anti-ANXA2 mAb. Results were normalised against beta-actin expression. (E) Antibody 2448 targeted a glycosylated epitope. A mild periodate treatment abolished binding of 2448 on Western blot. No loss of binding was observed using a commercial anti-ANXA2 mAb targeting ANXA2 peptide. Abbreviations: Ab = antibody AA = amino acid, ANXA2 = annexin A2, MW = molecular weight.