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. 2019 Dec 9;295(4):926–939. doi: 10.1074/jbc.RA119.010472

Figure 4.

Figure 4.

Anti-MRP1 antibodies were tested against HAP1 WT and HAP1 ABCC1 KO samples in SDS-PAGE. 20 μg of HAP1 (WT) and HAP1 MRP1 (ABCC1) KO were loaded into single 3–8% Tris-acetate gels and run under the Tris-acetate buffer system. The protein gel was transferred onto a single nitrocellulose membrane. Membrane was blocked in 3% milk (TBS + 0.1% Tween) solution before being spliced into two individual strips (A and B). A, membrane was incubated with mouse anti-MRP1 antibody (ab24102) and rabbit anti-α-tubulin antibody (ab52866) at a 1:20 dilution and 1:20,000 dilution, respectively. Antibody binding was detected using goat anti-mouse IgG H&L (IRDye® 800CW) preadsorbed and goat anti-rabbit IgG H&L (IRDye® 680RD) preadsorbed secondary antibodies at 1:20,000 dilution. ab24102 clearly displays a single band at 170 kDa in the 800-nm channel (green) that shows reduced signal in the HAP1 MRP-1 (ABCC1) knockout lysate. This confirms that ab24102 identifies MRP-1 as well as other off-target proteins. ab52866 staining α-tubulin at 50 kDa in the 680-nm channel (red) confirms equal protein loading across all lanes. B, membrane was incubated with anti-MRP1 rabbit antibody (ab233383) and mouse anti-vinculin antibody (ab130007) at a 1:1000 dilution and 1:20,000 dilution, respectively. Antibody binding was detected using goat anti-rabbit IgG H&L (IRDye® 800CW) preadsorbed and goat anti-mouse IgG H&L (IRDye® 680RD) preadsorbed secondary antibodies at 1:20,000 dilution. ab233383 displays the expected glycosylated smear for MRP1 between 150 and 200 kDa in the 800-nm channel (green) that is absent in the HAP1 MRP1 (ABCC1) KO lysate. This demonstrates that in HAP1 cells ab233383 reacts only with MRP1 (ABCC1). Ab130007 staining of vinculin at 125 kDa in the 700-nm channel (red) confirms equal protein loading across all lysates. Membranes were visualized using the Odyssey CLx imager with auto-intensity and 169-μm resolution. Separate images were required to visualize HiMarkTM pre-stained protein standard. White dotted line and scissor symbol denote splicing.