αpt308akt

αGenAKT, αpERK1/2(pTEY-ERK), αGenERK1/2 and αGenMEK1/2 Abs were from Sigma-Aldrich (Rehovot, Israel). αMEK1(H8), and αMEK1(C18) Abs were from Santa Cruz Biotechnology (CA, USA). αpT308AKT, αpS473AKT, αpS218/222MEK, αGenMEK2, αpS256FoxO1 and αGenFoxO1 Abs were from Cell Signaling Technology (Boston, MA). αpS298MEK1 Ab was from AbCam (Cambridge, England). αGFP Ab was from Roche Diagnostics GmbH (Mannheim, Germany). αpS306MEK2 Phosphosite-specific Abs for the S306-phosphorylated form of MEK2 were prepared by the Antibody Unit of the Weizmann Institute of Science. The rabbit Abs were generated against a sequence corresponding to residues 298–311 of the human MEK2 that contained a phosphorylated Ser residue: C-PRPPGRPV(pS)GHGMD. Rabbit serum Abs were affinity purified against their immunizing peptide as well as against the non-phosphorylated peptide using the SulfoLink kit (Thermo Scientific, Rockford, IL).

For lysates, cells were serum deprived for 2 h and stimulated or not with 30 ng/ml VEGF-A for 15 min. When used, inhibitors were added to cells for 1 h prior to growth factor stimulation. Total proteins were extracted in Laemmli buffer (2.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol), quantified and equal amounts of each sample were resolved by SDS-PAGE and transferred to PVDF membrane. After blocking with TBS/0.1% Tween 20/5% BSA, membranes were incubated with primary antibody overnight at 4° C. The following primary antibodies were used: rabbit α-pY1175 VEGFR2, α-VEGFR2, α-pS473 Akt, α-pT308 Akt, α-Akt, α-pS235/236 S6, α-pS65 and pT37/46 4EBP1 (all from Cell Signaling Technology), α-β actin (Santa Cruz Biotecnology), α-p110 α (ThermoFisher Scientific). Immunoreactive proteins were identified with secondary antibody coupled to HRP antibody and visualized by ECL. Quantification of the bands was done by NIH ImageJ on representative western blots; band intensities of phosphorylated Akt were normalized on total Akt, and analogously those for phosphorylated VEGFR2 were normalized on total VEGFR2, whereas band intensities of phosphorylated S6 and 4EBP1 were normalized on β actin.