RPS6KA3 Antibody / Ribosomal protein S6 kinase alpha-3 (FY12518)

Immunofluorescent staining of RPS6KA3 using anti-RPS6KA3 antibody (green) and anti-Beta Tubulin antibody (red). RPS6KA3 was detected in immunocytochemical section of SiHa cell. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 ug/ml rabbit anti-RPS6KA3 antibody and mouse anti-Beta Tubulin antibody overnight at 4oC. DyLight 488 Conjugated Goat Anti-Rabbit IgG and DyLight 594 Conjugated Goat Anti-Mouse IgG were used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37oC. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Western blot analysis of RPS6KA3 using anti-RPS6KA3 antibody. Lane 1: human CACO-2 whole cell lysates, Lane 2: human SH-SY5Y whole cell lysates, Lane 3: human 293T whole cell lysates, Lane 4: human whole cell lysates, Lane 5: rat skeletal muscle tissue lysates, Lane 6: rat thymus tissue lysates, Lane 7: mouse skeletal muscle tissue lysates, Lane 8: mouse thymus tissue lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-RPS6KA3 antibody at 0.25 ug/ml overnight at 4oC, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal was developed using enhanced chemiluminescent. RPS6KA3/RSK2 (~90 kDa) was detected as a doublet in human samples; the upper, slower-migrating band is consistent with phosphorylation-dependent mobility shifts associated with ERK- and PDK1-mediated activation and Ser386 autophosphorylation.

Immunoprecipitation of RPS6KA3 in whole cell lysate. Western blot analysis of RPS6KA3 using anti-RPS6KA3 antibody. Lane 1: whole cell lysates (30ug) Lane 2: Rabbit control IgG instead of anti-RPS6KA3 antibody in whole cell lysate. Lane 3: anti-RPS6KA3 antibody (2ug) + whole cell lysate (500ug) After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-RPS6KA3 antibody at a dilution of 0.5 ug/ml and probed with a goat anti-rabbit IgG-HRP secondary antibody. The signal is developed using ECL Plus Western Blotting Substrate. The expected molecular weight of RPS6KA3 is ~84 kDa.

Flow Cytometry analysis of 293T cells using anti-RPS6KA3 antibody. Overlay histogram showing 293T cells stained with (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-RPS6KA3 antibody (1 ug/million cells) for 30 min at 20oC. DyLight 488 conjugated goat anti-rabbit IgG (5-10 ug/million cells) was used as secondary antibody for 30 minutes at 20oC. Isotype control antibody (Green line) was rabbit IgG (1 ug/million cells) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.