Lane 1 : Wild-type A549 cell lysateLane 2 : NFE2L2 knockout A549 cell lysateLane 3 : HepG2 cell lysateLane 4 : Huh7 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Observed band size: 85 kDa why is the actual band size different from the predicted?

Western blot: Anti-NFE2L2 antibody (ab137550) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab137550 was shown to bind specifically to NFE2L2. A band was observed at 85 kDa in wild-type A549 cell lysates with no signal observed at this size in NFE2L2 knockout cell line ab285359 (knockout cell lysate ab289682). To generate this image, wild-type and NFE2L2 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.