Lane 1: Wild-type A549 treated with 100 ng/mL IFN gamma (ab259377) for 48 h cell lysate (20 µg)

Lane 2: CD274 knockout A549 treated with 100 ng/mL IFN gamma (ab259377) for 48 h cell lysate (20 µg)

Lane 3: U-87 MG cell lysate (20 µg)

Lane 4: MCF7 cell lysate (20 µg)

Lane 5: Wild-type A549 untreated cell lysate (20 µg)

Lane 6: CD274 knockout A549 untreated cell lysate (20 µg)

Lanes 1 - 6: Merged signal (red and green). Green - ab213524 observed at 50 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab213524 Recombinant Anti-PD-L1 antibody [EPR19759] was shown to specifically react with PD-L1 in wild-type A549 treated with 100 ng/mL IFN gamma for 48 h cells in western blot. Loss of signal was observed when both treated and untreated knockout cell line ab267054 (treated and untreated knockout cell lysates ab256831) were used. Wild-type and PD-L1 knockout samples were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab213524 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Allele-1: 7 bp deletion in exon4

Allele-2: 2 bp deletion in exon4

Allele-3: 1 bp insertion in exon4