All lanes : Anti-GCN2 antibody [EPR5970(2)] (ab134053) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : EIF2AK4 knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 187 kDa
Observed band size: 187 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab134053 observed at 187 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

 ab134053 was shown to react with GCN2 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab267246 (knockout cell lysate ab256902) was used. Wild-type HEK-293T and EIF2AK4 knockout HEK-293T cell lysates were subjected to SDS-PAGE. ab134053 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) 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.

Allele-1: 16 bp deletion in exon9

 

Allele-2: 1 bp insertion in exon 9.

 

Representative images of EIF2AK4 knockout HEK293T cells, low and high confluency examples (top left and right respectively) and wild-type HEK293T 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.