All lanes : Anti-PD-L1 antibody [EPR19759] (ab213524) at 1/1000 dilution

Lane 1 : Wild-type A549 treated with 100 ng/ml IFN gamma for 48 h cell lysate
Lane 2 : CD274 knockout A549 treated with 100 ng/ml IFN gamma for 48 h cell lysate
Lane 3 : U-87 MG cell lysate
Lane 4 : MCF7 cell lysate
Lane 5 : Wild-type A549 untreated cell lysate
Lane 6 : CD274 knockout A549 untreated cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 33 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

Lanes 1 - 6: Merged signal (red and green). Green - ab213524 observed at 50 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab213524 was shown to 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 lines ab267055 (treated and untreated knockout cell lysates ab256866) were used. Wild-type A549 treated with 100 ng/ml IFN gamma for 48 h and CD274 knockout A549 treated with 100 ng/ml IFN gamma for 48 h cell lysates 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 a 1 in 1000 dilution and a 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.

Homozygous: 1 bp insertion in exon4

Representative images of CD274 knockout A549 cells, low and high confluency examples (top left and right respectively) and wild-type A549 cells, low and high confluency (bottom left and right respectively) showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using an EVOS M5000 microscope.