Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: NAK knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: HepG2 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab12116 observed at 90 kDa. Red - loading control, ab181602, observed at 37 kDa.
ab12116 was shown to recognize NAK when NAK knockout samples were used, along with additional cross-reactive bands. Wild-type and NAK knockout samples were subjected to SDS-PAGE. ab12116 and ab181602 (loading control to GAPDH) were diluted at 1 μg/ml and 1/10 000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

All lanes : Anti-NAK/TBK1 antibody [108A429] (ab12116) at 2 µg/ml

Lane 1 : 293 cells
Lane 2 : 293 cells transfected with Human NAK cDNA

Predicted band size: 87.5 kDa

Overlay histogram showing HeLa cells stained with ab12116 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab12116, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells ) used under the same conditions. Acquisition of >5,000 events was performed.