Figure 5. PKA activity is proportional to phosphorylation of EMSY and HDR activity in EMSY-overexpressing cells.

(A) EMSY-overexpressed 293T cells were treated with DMSO, the PKA activator forskolin, or the PKA inhibitor H-89. The left panels show the input lysates. The right panels show V5-immunoprecipitaions (V5-IP) and detection of EMSY phosphorylation with the PKA-phosphosubstrate (PKA-p-sub) antibody. Overexpressed EMSY is detected with the V5 (V5^EMSY) antibody. Ponceau staining and anti-β-Actin served as loading controls. (B) DR-GFP assay measuring HDR activity in OVCAR8^DR-GFP overexpressing wild type EMSY. The cells are treated with DMSO, forskolin, or H-89. HDR activity was measured 24 h post-transfection and post-treatment. All data are normalized to DMSO-treated mock control (vector only) cells.