Figure 7

mPARM-1 and hPARM-1 proteins activate ERK1/2, PI3K/AKT, and STAT3 signaling pathways in NIH/3T3 cells. NIH/3T3 cells were either untransfected or transfected with empty vector, hParm-1-pcDNA3.1A/Myc-His, mParm-1-pcDNA3.1A, respectively. After 48 h, cell lysates were extracted with specific buffer containing proteinase and phosphatase inhibitors and 30 μg of proteins were resolved by 12% SDS-PAGE. Following protein transfer, PVDF membranes were probed with (a) anti-phospho-ERK1/2 (Thr202/Tyr204), (b) anti-phospho-AKT or (c) anti-phospho-STAT3 (All from cell Signaling; 1:1000) antibodies. Loading of equal amount of protein was verified using anti-anti-p44/42 MAPkinase (ERK), anti-AKT, anti-STAT3 (All from cell Signaling; 1:1000) and anti-β-actin antibodies. Expression of murine and human PARM-1 proteins was verified using an anti-Myc antibody (exemplified with only one membrane). Experiments were performed in triplicate.