All lanes : Anti-HuR / ELAVL1 antibody [19F12AE12] (ab170193) at 2 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 - ab170193 observed at 36 kDa. Red - loading control, ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) observed at 55kDa.

ab170193 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 ab170193 and ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) overnight at 4°C at 2 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.

Immunocytochemistry on HeLa cells.

Cells were paraformaldehyde fixed (4%, 20 minutes) and Triton X-100 permeabilized (0.1%, 15minutes). The cells were then incubated with the anti-HuR antibody (5µg/mL) for 2 hours at room temperature or over night at 4°C. The secondary antibody used was (red) Alexa Fluor® 594 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1 hour. A cytoplasmic antibody (ab75834) was used as counter stain (green). 10% goat serum was used as a blocking agent for all blocking steps.

The target protein localizes mainly to the nucleus.

All lanes : Anti-HuR / ELAVL1 antibody [19F12AE12] (ab170193) at 1 µg/ml

Lane 1 : Human Liver Homogenate
Lane 2 : Human Heart Homogenate
Lane 3 : HepG2 Cell Lysate
Lane 4 : HeLa Cell Lysate
Lane 5 : SH-SY5Y Cell Lysate
Lane 6 : Jurkat Cell Lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-mouse IR800 at 1/5000 dilution

Developed using the ECL technique.

Predicted band size: 36 kDa

All lanes : Anti-HuR / ELAVL1 antibody [19F12AE12] (ab170193) at 1 µg/ml

Lane 1 : H9C2 Rat Cell Lysate
Lane 2 : H4IIE Rat Cell Lysate
Lane 3 : MEF Mouse Cell Lysate
Lane 4 : 3T3 Mouse Cell Lysate
Lane 5 : SH-SY5Y Cell Lysate
Lane 6 : HepG2 Cell Lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-mouse AP at 1/5000 dilution

Developed using the ECL technique.

Predicted band size: 36 kDa

Immunocytochemistry on SH-SY5Y cells (Human neuroblastoma cell line).

Cells cells were paraformaldehyde fixed (4%, 20 minutes) and Triton X-100 permeabilized (0.1%, 15 minutes). The cells were then incubated with the anti-HuR antibody (5µg/mL) for 2 hours at room temperature or overnight at 4°C. The secondary antibody was (red) Alexa Fluor® 594 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. A cytoplasmic antibody (ab75834) was used as counter stain (Green). 10% Goat serum was used as a blocking agent for all blocking steps.

The target protein localizes mainly to the nucleus.  

Flow cytometric analysis of methanol-fixed HeLa cells labeling HuR / ELAVL1 with ab170193 at 1µg/mL (blue) or an equal amount of an isotype control antibody (red).