All lanes : Anti-Hsp27 (phospho S85) antibody (ab5594) at 2 µg/ml

Lane 1 : Hela cell lysate - untreated
Lane 2 : Hela cell lysate - treated with 10uM Anisomycin for 30 minutes

Lysates/proteins at 50 µg per lane.

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

Immunocytochemistry/Immunofluorescence analysis of Hsp27 (phospho S85) (green) in HeLa cells either left untreated (left panel) or treated with 10uM Anisomysin (right panel) 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 probed with ab5594 at 20ug/ml for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat anti-rabbit IgG secondary antibody at 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.

Immunoprecipitation of Hsp27 (phospho S85) was performed on HeLa cells treated with 10uM Anisomysin for 30 minutes. Antigen-antibody complexes were formed by incubating 500µg of whole cell lysate with 3µg of ab5594 overnight on a rocking platform at 4°C. The immune complexes were captured on 50µl Protein A/G Agarose, washed extensively, and eluted with Lane Marker Reducing Sample Buffer. HeLa cell lysate (50ug) was loaded as a positive control (left lane). Samples were 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 ab5594 at 2ug/ml overnight rotating at 4°C, washed in TBST, and probed with Clean-Blot IP Detection Reagent at a dilution of 1:1000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura.

Western blot analysis of Hsp27 (phospho S85) was performed by loading rat skeletal muscle tissue extracts on a SDS-PAGE gel. Proteins were transferred to a membrane probed with a Hsp27 (phospho S85) polyclonal antibody (ab5594) at a concentration of 0.25ug/ml followed by an anti-rabbit IgG-HRP secondary antibody. Detection was performed using a chemiluminescent substrate.