Figure 3. Verification of the specificity of the anti-CERKL antibody RA. A: Western blot analysis of mouse retinal extract with the affinity-purified RA antibody. The antibody detects a main specific band, which corresponds to the expected size of the primary and most abundant CERKL isoform in the adult mouse retina (isoform a’, 58 kDa). Two additional fainter bands slightly higher than 51 kDa, corresponding to isoforms b’ and c’ (55 and 53 kDa, respectively), can also be observed. All three bands are completely absent following pre-absorption of the RA antibody with the recombinant CERKL protein (right panel), but not with a non-specific protein (BSA; left panel). B: Western blot analysis of extracts from bacteria transformed with mouse Cerkl retinal isoforms a’ (58 kDa), b’ (55 kDa), and d’ (12 kDa). The RA antibody detects proteins of the expected sizes in IPTG-induced, but not in un-induced bacterial extracts. C: Western blot analysis of protein extracts from the ARPE-19 and 661W cell lines. In the mouse-derived cell line, 661W, a major band of approximately 58 kDa, which corresponds to mouse CERKL isoform a’ is detected. In the human-derived cell line, ARPE-19, two bands corresponding to human CERKL isoforms c and d (46 and 51 kDa, respectively) are detected. D: Immunostaining of ARPE-19 cells is omitted following pre-absorption of the RA antibody (green) with the recombinant CERKL protein (right panel), but not with a non-specific protein (BSA; left panel). Nuclei are stained with TO-PRO-3 (blue). Scale bar, 20 µm. E: Immunostaining of a mouse retina section is omitted following pre-absorption of the RA antibody (green) with the recombinant CERKL protein (right panel), but not with a non-specific protein (BSA; left panel). Nuclei are stained with TO-PRO-3 (blue). Scale bar, 20 µm.