Anti p akt ser473

Protein lysates were separated by 11% SDS-PAGE and blotted on Immobilon-P PVDF membranes (Sigma-Aldrich), following the manufacturer’s instructions and using TE 22 Mini Tank transfer unit (GE Healthcare, Waukesha, WI, USA). Membranes were incubated overnight with the following primary antibodies: CK2α/CK2α′ (MCA3031Z; Bio-Rad, Hercules, CA, USA); anti-CK2β (catalog ab76025) from Abcam (Cambridge, UK); anti-p-Akt (Ser473) (catalog #4060), anti-p-PRAS40 (Thr246) (catalog #13175), anti-p-GSK3β (Ser9) (catalog #5558), anti-GSK3β (catalog #9336), anti-p-ERK1/2 (Thr202/Tyr204) (catalog #4370) and anti-ERK1/2 (catalog #4695) from Cell Signaling Technology (Danvers, MA, USA); anti-Akt1/2/3 (catalog sc-8312) from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and anti-β-actin (catalog A5441) from Sigma-Aldrich (Burlington, MA, USA). After removal of primary antibodies and washing with Tris-buffered saline (TBS), membranes were incubated with the secondary antibodies towards rabbit and mouse IgG, conjugated to horseradish peroxidase (PerkinElmer). A signal was developed using an enhanced chemiluminescent detection system ECL (Amersham Biosciences, Little Chalfont, UK). Immunostained bands were quantified by means of a Kodak Image Station 4000MM-PRO and analyzed with Carestream Molecular Imaging software (New Haven, CT, USA).

Proteins were separated by 8% SDS-PAGE and transferred to nitrocellulose transfer membranes. The blots were blocked with freshly prepared 5% nonfat milk in TBST for 1 h at room temperature. Then the blots were incubated at 4 °C overnight with primary antibodies. After washing with TBST, the blots were incubated with horseradish peroxidase-conjugated (HRP-conjugated) donkey anti-rabbit IgG or sheep anti-mouse IgG (Invitrogen, China) at room temperature for 1 h. ECL substrate (CLiNX, Shanghai, China) and ChemiScope Touch (CLiNX, Shanghai, China) were used for detecting HRP-conjugated antibodies. Primary antibodies including anti-P-AMPK (phospho S496), anti-P-S6K (phospho S424), and anti-P-AKT (Ser473) were brought from Abcam (China). Primary antibodies including anti-LC3, anti-P-ERK(Thr202/Tyr204), anti-PARP1, anti-vimentin, anti-SQSTM1/p62, anti-GAPDH, and anti-IGF2R(M6PR) were brought from Proteintech (China).