All lanes : Anti-GEF H1 antibody [EPR17963] - C-terminal (ab201687) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ARHGEF2 knockout HeLa cell lysate
Lane 3 : HEK293 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 111 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?

Lanes 1-3: Merged signal (red and green). Green - ab201687 observed at 75 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab201687 Anti-GEF H1 antibody [EPR17963] - C-terminal was shown to specifically react with GEF H1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265797 (knockout cell lysate ab257223) was used. Wild-type and GEF H1 knockout samples were subjected to SDS-PAGE. ab201687 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution 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 10000 dilution for 1 hour at room temperature before imaging.

 

Homozygous: 1 bp insertion in exon 4.

Representative images of ARHGEF2 knockout HeLa cells, low and high confluency examples (top left and right respectively) and wild-type HeLa cells, low and high confluency (bottom left and right respectively) showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using a EVOS XL Core microscope.