Serum starved HeLa cells were incubated at 37°C for 60 minutes with vehicle control (0 µM) and different concentrations of phorbol 12-myristate 13-acetate (PMA) (ab120297) in DMSO. Increased expression of PKC mu/PKD (phospho S916) (ab81218) correlates with an increase in phorbol 12-myristate 13-acetate (PMA) concentration, as described in literature.

Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 20µg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 3% milk before being incubated with ab81218 at 1 µg/ml and ab8227 at 1 µg /ml overnight at 4°C. Antibody binding was detected using an anti-rabbit HRP secondary antibody (ab97051) at 1/10000 dilution and visualised using ECL development solution.

All lanes : Anti-PKC mu/PKD (phospho S916) antibody [EP1494Y] (ab81218) at 1/100000 dilution

Lane 1 : lysate from untreated HeLa cells
Lane 2 : lyaste from HeLa cells treated with TPA

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled Goat anti-Rabbit at 1/2000 dilution

Predicted band size: 102 kDa

Immunohistochemistry analysis of paraffin-embedded Human placenta tissue using 1/100 ab81218.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.