Phospho total akt

Before gel electrophoresis the cell lysate was supplemented with Bolt reducing agent and Bolt gel loading buffer (both Thermo Fisher Scientific, MA, USA) and incubated at 90 °C for 5 minutes. The samples were then separated by SDS-PAGE (Bolt Gels, Thermo Fisher Scientific, MA, USA), followed by transfer onto nitrocellulose membrane, which were blocked in 3% skimmed milk in PBS + 0.1% Tween (PBS-T) for 1 hour at room temperature. Immunoblotting was performed at 4 °C overnight using the following primary antibodies diluted in blocking buffer: phospho/total p44/42 MAPK, phospho/total Akt, phospho/total PLCgamma1 (all at 1:1000 and all from Cell Signaling Technology, USA), or anti-Myc-tag (1:1000; Merck Millipore, MA, USA). The following day the membrane was washed three times in PBS-T and then incubated with horseradish peroxidase-conjugated secondary antibodies (anti-rabbit IgG or anti-mouse IgG at 1:5000, from Agilent Technologies, CA, USA) diluted in blocking buffer for 4 hours at 4 °C. This step was followed by another 3 washes in PBS-T and finally chemiluminescent development using the ECL Western Blotting Substrate (Promega, WS, USA). The signal was captured with the BioRad ChemiDoc Imager (Bio-Rad Laboratories, CA, USA) and bands quantified using ImageJ (https://imagej.nih.gov/ij/).