pERK levels are affected by post-treatment trypsinization at different temperatures. PaSMC cultures were stimulated with PMA 50 nM for 5 minutes or 20% FBS for 15 minutes at 37°C, 5% CO2 and prepared for phosphorylation-specific flow cytometry analysis by trypsinization at 37°C or 0°C. Cells were pretreated with the MEK1 inhibitor PD98059 at 30 μM for 3 hours to inhibit pERK activation. A: Flow cytometry analysis of pERK levels in PMA- and serum-treated PaSMC. Rabbit anti-pERK/goat-anti-rabbit-Alexa Fluor 647 secondary antibody was quantified (10,000 events) and plotted as overlaid histograms of fluorescence versus number of cells. B: Median fluorescence intensity (MFI) measured for PMA- and serum-induced pERK levels following post-treatment trypsinization at 37°C or 0°C. Trypsinization at 37°C both increased the basal pERK levels and attenuated the activation of pERK by PMA. Control: Goat-anti-rabbit Alexa Fluor 647 secondary only. (Representative of three independent experiments).
Abrahamsen and Lorens BMC Cell Biology 2013 14:36 doi:10.1186/1471-2121-14-36