Figure 3
Induction of phosphorylation of Pyk2 and PECAM by cell aggregation. A, Phosphorylation of Pyk2 and PECAM in high versus low cell density cultures. Different concentrations of HBE, SK-LU-1, or H460 cells (H: 5.0 × 105 cells L: 2.5 × 105 cells) were cultured in 60-mm polyHEMA-coated dishes for 0, 6, or 15 h. Cell lysates were resolved by SDS-PAGE and analyzed by immunoblotting with anti-phospho-Pyk2 (Tyr881), or anti-Pyk2 antibody (left). Equal amounts of whole-cell extracts were immunoprecipitated with anti-PECAM antibody followed by immunoblotting with 4G10 antibody (Right). B. Phosphorylation of Pyk2 and PECAM in regular suspension versus methyl-cellulose suspension cultures. H1792 and H460 cells were cultured in polyHEMA-coated dishes (S) or methyl-cellulose dishes (MS) for 6 or 15 h, harvested, and lysed with RIPA buffer. A, Equal amounts of whole-cell extracts were immunoprecipitated with anti-PECAM antibody followed by immunoblotting with anti-phosphotyrosine 4G10 antibody (Upper). Equal amounts of whole-cell extracts were immunoblotted with anti-pPyk2 (Tyr881) or anti-Pyk2 antibody (Lower).