Figure 1

Identification of the CAIX Interactome using the BioID proximal ligation strategy and validation of high confidence proximal CAIX-associating proteins by co-immunoprecipitation. (a) Schematic showing the domain structure of the CAIX-BirA* fusion protein used for the BioID studies. SP, signal peptide; PG, proteoglycan-like domain; CA, catalytic domain; TM, transmembrane domain; IC, intracellular domain. (b) FACS analysis showing levels of constitutive expression of the CAIX-BirA* fusion protein expressed by MDA-MB-231 cells (MDA-MB-231-CAIX-BirA*) cultured in normoxia compared to levels of endogenous CAIX expression by WT MDA-MB-231 cells cultured in either normoxia or hypoxia. The filled (gray) histograms indicate specific CAIX staining whereas the dotted lined histograms indicate staining by isotype-specific control IgG. (c) Image showing exogenous CAIX (red) localized at the cell surface of MDA-MB-231-CAIX-BirA* cells grown in normoxia. Scale bar, 10 μM. (d–g) Co-IP of high confidence proximal CAIX-associating proteins (d) ITGB1, (e) ITGA2 (f) CD98hc and (g) MMP14 with exogenous CAIX expressed by MDA-MB-231-CAIX-BirA* cells cultured in normoxia. (h–j) Co-IP of (h) ITGB1, (i) ITGA2 and (j) MMP14 with endogenous CAIX expressed by WT MDA-MB-231 cells cultured in either normoxia (N) or hypoxia (H). Isotype-specific IgG was used as a control for co-IPs and data in (d) to (j) are representative of at least two independent experiments. IgG, immunoglobulin G.