Figure 5.
Effect of sst2 expression on E-cadherin protein expression and tyrosine dephosphorylation in pancreatic cancer cells. (A) Cells were cultured in 10-cm diameter dishes in medium containing 5% FCS. After a 12-h attachment phase, cells were cultured in serum-free medium for 24 h. Soluble proteins were subjected to SDS/polyacrylamide gel electrophoresis and immunoblotted (blot E-cadherin) with anti-E-cadherin Abs. Lane 1, PC1.0/neo cells; lane 2, PC-1.0 cells; and lane 3, PC1.0/sst2 cells. The arrow indicates the position of E-cadherin (120 kDa). Results are representative of three independent experiments. (B and C) PC-1.0 and PC-1.0/sst2 cells transiently transfected or not with the pcDNA3/C453S-SHP-1 vector (dominant negative SHP-1 mutant) were cultured in 100-mm dishes (7 × 105) cells/dish in RPMI medium 1640 supplemented with 5% FCS for 18 h. Cells were thereafter solubilized and immunoprecipitated or not with E-cadherin Abs. Solubilized proteins were resolved through 7.5% SDS/polyacrylamide gels and immunoblotted with anti-SHP-1 Abs (B, blot SHP-1). Immunoprecipitated proteins (ip: E-cadherin) were immunoblotted with anti-phosphotyrosine (C, blot: p-Tyr) or anti-E-cadherin (C, blot: E-cadherin, reblotting experiment). The arrow indicates the position of E-cadherin phosphorylation (120 kDa). Lanes 1, PC-1.0 cells; lanes 2, PC-1.0-SHP-1/C453S cells; lanes 3, PC-1.0/sst2 cells; lanes 4, PC-1.0/sst2-SHP-1/C453S cells. The figure is representative of three independent experiments.