Anti phosphorylated akt thr308

The following primary antibodies were used: anti-FLAG (mouse, M2; Sigma-Aldrich, St Louis, MO; dilution used in immunocytochemistry: 1:2000), anti-human influenza hemagglutinin (rabbit, Y-11; Santa Cruz Biotechnology, Dallas, TX; dilution used in Western blotting: 1:3000, ICC: 1:1000), anti-SMYD2 (rabbit, D14H7; Cell Signaling Technology, Danvers, MA; dilution used in WB: 1:1000), anti-phosphorylated AKT (Thr 308) (rabbit, D25E6; Cell Signaling Technology; dilution used in WB: 1:1000), anti-AKT (rabbit, C67E7; Cell Signaling Technology; dilution used in WB: 1:1000), anti–α-tubulin (mouse, DM1A; Calbiochem, Billerica, MA; dilution used in WB: 1:1000), and anti–phospho-PTEN (Ser 380) (rabbit, Cell Signaling Technology, dilution used in WB: 1:1000). An anti-K313 dimethylated PTEN antibody (Sigma-Aldrich; dilution used in WB: 1:1000) was produced in rabbit immunized with a synthetic peptide.

Cells were lysed in cell lysis buffer (50 mM Tris‐HCl pH 7.5, 125 mM NaCl, 5 mM EDTA and 0.1% Triton X‐100) containing both 1% protease inhibitor and 1% phosphatase inhibitor cocktail (Sigma‐Aldrich, St Louis, MO, USA). Equal amounts of protein were separated on 10% SDS‐PAGE and then transferred to polyvinylidene fluoride membranes with an iBlot^®2 Gel Transfer Stack (Thermo Fisher Scientific). The membranes were incubated with primary antibodies at 4°C overnight. The primary rabbit antibodies used were anti‐ANXA10 antibody (1:500, NBP1‐90156SS, Novus Biologicals), anti‐phosphorylated Akt (Ser473) (/1:500, #4060, Cell Signaling Technology, Beverly, MA), anti‐phosphorylated Akt (Thr308) (1:500, #2965, Cell Signaling), anti‐Akt (1:1000, #9272, Cell Signaling), anti‐phosphorylated Erk1/2 antibody (Thr202/Tyr204) (1:500, #9101, Cell Signaling), anti‐Erk1/2 antibody (1:1000, #9102, Cell Signaling), anti‐β‐actin antibody (1:1000, #4970, Cell Signaling). The membranes were then probed with secondary antibodies for 90 min at room temperature. The secondary antibody was horseradish peroxidase‐conjugated donkey anti‐rabbit IgG (1:1000, NA934V, GE Healthcare, Little Chalfont, Buckinghamshire, UK).