All lanes : Anti-Hsp27 (phospho S15) antibody (ab5581) at 1/500 dilution

Lane 1 : HeLa cell lysate - untreated
Lane 2 : HeLa cell lysate - treated with 10nM Anisomycin

Lysates/proteins at 50 µg per lane.

Secondary
All lanes : HRP-conjugated goat anti-rabbit IgG at 1/20000 dilution

Predicted band size: 23 kDa

Immunoprecipitation of Hsp27 (phospho S15) was performed on HeLa cells treated with 10uM Anisomysin for 30 minutes. Antigen-antibody complexes were formed by incubating 500ug of whole cell lysate with 3ug of ab5581 overnight on a rocking platform at 4°C. The immune complexes were captured on 50ul Protein A/G Agarose, washed extensively, and eluted with Lane Marker Reducing Sample Buffer. Samples were then resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with ab5581 at 1:500 overnight rotating at 4°C, washed with TBST, and probed with Clean-Blot IP Detection Reagent at 1/1000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura.

Immunocytochemistry/Immunofluorescence analysis of Hsp27 (phospho S15) (green) in HeLa cells either untreated (left) or treated with 10uM Anisomysin (right) for 30 minutes. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% blocker BSA for 15 minutes at room temperature. Cells were incubated with ab5581 at 1:50 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat anti-rabbit IgG secondary antibody (1:400) for 30 minutes at room temperature. F-Actin (red) was stained with DyLight 554 Phalloidin and nuclei (blue) were stained with Hoechst 33342 dye. 20X magnification.