All lanes : Anti-HuR / ELAVL1 antibody [4C8] (ab136542) at 1 µg/ml

Lane 1 : Wild-type SW480 cell lysate at 40 µg
Lane 2 : ELAVL1 knockout SW480 cell lysate at 40 µg
Lane 3 : HEK293 cell lysate at 20 µg
Lane 4 : HCT116 cell lysate at 20 µg

Performed under reducing conditions.

Predicted band size: 36 kDa
Observed band size: 36 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab136542 observed at 36 kDa. Red - loading control, ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) observed at 55kDa.

ab136542 was shown to react with ELAVL1 in wild-type SW480 cells in western blot. Loss of signal was observed when ELAVL1 knockout sample was used. Wild-type and ELAVL1 knockout SW480 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBS-T (0.1% Tween^®) before incubation with ab136542 and ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) overnight at 4°C at 1 µg/ml and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 hour at room temperature before imaging.

Overlay histogram showing MCF7 cells stained with ab136542 (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 (ab136542, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150113) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

Electrophoretic Mobility Shift Assay of HuR/mRNA complex (Lane 1). The complex is significanlty supershifted by the addition of 10ng of ab136542 (Lane 2).