Anti pakt ser473 clone d9e

Cells were lysed in SDS lysis buffer (100 mM Tris рН 8.8, 1% SDS, 5 mM EDTA, 20 mM DTT, and 2 mM AEBSF). Total protein extracts were resolved in SDS polyacrylamide gel and blotted onto a Hybond PVDF membrane (GE Healthcare, Chicago, IL, USA). After being blocked with PBST 5% non-fat dry milk (Bio-Rad), membranes were incubated over night with primary antibodies at +4°C, washed and hybridized for 1 h at room temperature using the appropriate horseradish peroxidase-conjugated secondary antibody (rabbit and mouse, Bio-Rad, Hercules, California, USA). Detection was performed with the ECL chemiluminescence kit (Perkin Elmer, Waltham, Massachusetts, USA). The following antibodies were used: anti-IRS1 (D32G12, Cell Signaling), anti-p63 (clone 4A4, Santa Cruz Biotechnology), anti-p-IRS1 (Ser612, clone C15H5, Cell Signaling), anti-p-AKT (Ser473, clone D9E, Cell Signaling), anti-AKT (clone #9272, Cell Signaling), anti-p-S6 Ribosomal Protein (Ser235/236, clone #2211, Cell Signaling), anti-S6 (Clone 5G10, Cell Signaling), anti-p44/42 MAPK (p-ERK1/2) (Thr202/Tyr204, Clone E10, Cell Signaling), anti-ERK1/2 (Clone 137F5, Cell Signaling).