Anti phospho akt s473 d9e xp

293T cells with or without forced expression of EXOSC3 and CNOT4 were lysed with lysis buffer (10 mmol/L Tris‐HCl [pH 7.4], 5 mmol/L EDTA, 150 mmol/L NaCl, 10% glycerol, 1% Triton X‐100, 1% sodium deoxycholate, 0.1% SDS, 50 mmol/L NaF, 1 mmol/L phenylmethylsulfonyl fluoride [PMSF], and 1 mmol/L sodium orthovanadate [Na₃VO₄]) and a protease inhibitor mixture (complete [EDTA‐free] protease inhibitor [Roche]) for 20 min on ice and centrifuged at 15,106 g for 15 min at 4°C. Supernatants were subjected to SDS‐PAGE, and the separated proteins were transferred to a polyvinylidene difluoride membrane (Millipore). The membranes were incubated with primary antibodies at 4°C overnight and then incubated with horseradish peroxidase‐labeled secondary antibodies. The signals were developed using ECL™ Western Blotting Detection Reagents (GE Healthcare) and visualized with an LAS 4000 mini system (GE Healthcare). The antibodies used in this experiment were as follows: anti‐FLAG^® M2‐peroxidase (HRP) (Sigma), anti‐phospho‐p44/42 MAPK (T202/Y204) (D13.14.4E) XP™, anti‐phospho‐JNK, anti‐phospho‐STAT3 (Y705), anti‐phospho‐AKT (S473) (D9E) XP™ (Cell Signaling Technologies), anti‐MYD88 (Santa Cruz Biotechnology), and anti‐actin (clone C4) (Millipore).