“…Git1 Phosphorylation-BHK-21 cells (2 ϫ 10 5 ), which lack RPTP but contain its substrate Git1, were cultured on Matrigel-coated plates for 24 h in DMEM containing 10% FCS and then transfected with RPTP mutant expression plasmids (9). After cultivation for 24 h, cells were incubated with or without VacA for 1 h at 37°C and then washed twice with ice-cold PBS; this was followed by treatment with N-lysis buffer (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.2% deoxycholic acid, 1 mM sodium orthovanadate, 1 mM NaF, and protease inhibitor) for 30 min on ice.…”