Immobilon p membrane

Immunoblot analyses were performed using a range between 20 μg and 100 μg of lysates depending on the abundance of the protein of interest. The material was electrophoretically resolved on denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels ranging from 6 % to 16 % acrylamide and was transferred to nitrocellulose Immobilon-P membranes (Invitrogen) using the iBlot™ Dry Blotting System (Invitrogen). Membranes were then blocked in 5 % w/v Marvel™ milk or 5 % BSA (Sigma-Aldrich) w/v in 1× TBS-0.1 % Tween for 30 min at RT and were immunoblotted with β-Actin (Abcam), β-Tubulin (Abcam), Bak (Cell Signaling), Bax (Cell Signaling), Bid (Cell Signaling), CREB1 (Cell Signaling), pCREB1 (Cell Signaling), Cyclin D1 (Cell Signaling), Histone H3 (Abcam), p21 (Cell Signaling), p27 (Cell Signaling), PARP (Cell Signaling), SIK2 (BioLegend, Sigma-Aldrich or Cell Signaling), TORC1 (Cell Signaling), TORC2 (Cell Signaling) and TORC3 (Cell Signaling) primary antibodies overnight at 4°C. Membranes were then washed 5 times in 1× TBS-0.1 % Tween and were incubated with an appropriated HRP-conjugated secondary antibody (Dako) for 1 h at RT. Membranes were then washed 5 times in 1× TBS-0.1 % Tween and proteins were visualised using ECL Plus Western Blotting Detection System (GE Healthcare).