Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: FTO knockout HAP1 whole cell lysate (20 µg)
Lane 3: HEK293 whole cell lysate (20 µg)
Lane 4: MOLT4 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab92821 observed at 58 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab92821 was shown to specifically recognize FTO in wild-type HAP1 cells along with additional cross reactive bands. No band was observed when FTO knockout samples were examined. Wild-type and FTO knockout samples were subjected to SDS-PAGE. ab92821 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at a 1/1000 dilution and 1/10,000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

Overlay histogram showing SH-SY5Y cells stained with ab92821 (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 followed by the antibody (ab92821, 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 IgG2a [ICIGG2A] (ab91361, 1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in SH-SY5Y cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

ICC/IF image of ab92821 stained PC12 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab92821, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96879, DyLight® 488 goat anti-mouse IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Anti-FTO antibody [5-2H10] (ab92821) at 1/1000 dilution + Rat testes lysate

Predicted band size: 58 kDa
Observed band size: 58 kDa