SSH1 Antibody / Slingshot protein phosphatase 1 (FY13149)

Immunofluorescent staining of SSH1 using anti-SSH1 antibody (red). SSH1 was detected in an immunocytochemical section of U2OS cells. 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-SSH1 antibody overnight at 4oC. Cy3 Conjugated Goat Anti-Rabbit IgG was used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37oC. The section was counterstained with DAPI nuclear stain (blue). Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Western blot analysis of SSH1 using anti-SSH1 antibody. Lane 1: human Hela whole cell lysates, Lane 2: human 293T whole cell lysates, Lane 3: human Jurkat whole cell lysates, Lane 4: human Raji whole cell 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-SSH1 antibody at 0.5 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. SSH1 antibody detects a doublet at ~110-120 kDa with an additional slower band at ~150 kDa across the indicated lysates. Although the predicted mass is ~116 kDa, Slingshot-1 typically shows phosphorylation-dependent mobility shifts, with hyperphosphorylated/14-3-3-bound forms migrating more slowly.

Flow Cytometry analysis of 293T cells using anti-SSH1 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-SSH1 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 (Red line) was also used as a control.