Fig. 3. Status of downstream signaling intermediates in HDFs expressing the different E5 proteins. (A, upper panel of B) Whole-cell extracts of proliferating HDFs expressing the indicated E5 protein, v-Sis or no viral oncoprotein (control) were subjected to immunoblotting. In A, blots were probed with antibodies specific for phosphorylated (p) forms of JNK, AKT, ERK1/2 or c-Jun, and then stripped and reprobed with pan-specific antibodies detecting the total amount of each protein. JNK isoforms 1 and 2 are indicated on the right. In B (upper panel) the blot was probed with a cyclin D3-specific antibody and then reprobed with actin antibodies. In B (lower panel) E5-expressing HDFs were either untreated (–) or treated with of 20 µM LY294002 (LY) or 310 nM wortmannin (W) for 24 hours. Whole-cell lysates were prepared and immunoblotted for cyclin D3 and β-tubulin.