FIGURE 3.
Effect of cPAcP expression on tyrosine phosphorylation of ErbB-2 in LNCaP C-81 cells. A, LNCaP C-81 cells were transiently transfected with WT PAcP, mutant PAcP H12A cDNAs, or vector alone. Cells were harvested after 48 and 72 h, respectively, and an equal amount of cellular lysates was separated by SDS-PAGE for Western blot analyses. cPAcP, the total and the specific tyrosine phosphorylation (p) level of ErbB-2, and phosphorylation of p52Shc and PCNA were detected by the corresponding Ab. The protein levels of ErbB-2, Shc, and β-actin were examined with the respective Ab after the membranes were stripped. B, cPAcP, ErbB-2, and total tyrosine phosphorylation of ErbB-2 protein in WT PAcP cDNA-transfected LNCaP-28 and LNCaP-40 stable sublines were detected with anti-PAcP, anti-ErbB-2, and 4G10 anti-Tyr-phosphorylation Abs, respectively. For cell growth kinetics, cPAcP-28, cPAcP-40, and LNCaP C-81 cells were seeded at a density of 5 × 104 cells/well in 6-well plates in regular medium for 3 days. One set of attached cells was harvested and counted as day 0. The remaining cells were fed with regular medium. Cells were harvested on days 2, 4, and 7 for counting. Similar results were obtained in two sets of independent experiments in duplicate wells. *, p < 0.05 versus LNCaP C-81 cells; **, p < 0.01 versus LNCaP C-81 cells.