OGF induced p21 expression. (A) BxPC-3 cells were synchronized by nocodazole (67 ng/ml) for 24 hours, and subsequently treated with 10-6 M OGF or sterile water for 3, 6, 9, 12, 15, 18, and 21 hours. Total proteins were resolved by SDS-PAGE, and blotted with p21 specific antibodies. Densitometric analysis of the Western blots was performed, and p21 expression for OGF-treated cells is expressed relative to controls at each time point. The p21 level was significantly (*p < 0.05) elevated from the control group at 9 hours. (B) To examine whether the OGF-induced downregulation of Cdk2 kinase activity was based on p21/Cdk2 complex formation, homogenates of BxPC-3 cells treated with OGF or sterile water were subjected to Cdk2 immunoprecipitation; the resulting proteins were blotted with antibodies to p21. Densitometric analysis of Western blots of immunoprecipitated p21/Cdk2 complexed protein at 3, 6, 9, 12, 15, 18, and 21 hours revealed an increase at 9, 12, 15, 18, and 21 hours, with significant difference between the OGF and 15-hour control group (*p < 0.05). (C) Opioid receptor mediation of OGF action was evaluated in synchronized BxPC-3 cells treated with 10-6 M OGF, 10-5 M naloxone (NAL), both OGF and NAL, or sterile water (Control) for 9 hours. Protein lysates were resolved on SDS-PAGE, and subjected to Western blot analysis for p21 and actin. Densitometric analysis of p21 expression showed that p21 levels of OGF -treated cells were significantly elevated from controls at *p < 0.05; no change was recorded in the NAL and OGF-NAL groups. Data represent means ± SE for 3 independent experiments.