FIG. 3.

Cancer cells have elevated PKA activity levels. Whole-cell lysates were prepared from various normal or immortalized or cancer cell lines, and an in vitro PKA enzymatic assay was performed with the cAMP-dependent PKA Signatect Assay kit (A to D, left). The cell lines were transiently transfected with vector, GFP-SAC, or GFP-SAC/155A constructs and the transfectants were visualized by confocal microscopy for GFP fluorescence of Par-4 or mutant constructs and DAPI staining for apoptosis (A to D, right). To determine if Par-4 was preferentially phosphorylated in cancer cells compared to normal cells, whole-cell lysates from prostate cancer cell lines LNCaP, LNCaP/IGFBP5, and PC-3 and immortalized prostate epithelial cell line PZ were subjected to SDS-PAGE, transferred to PVDF membranes, and probed with phospho-T155 antibody, Par-4 antibody, and actin antibody as a loading control (E).