P akt1 2 3 ser473

Total, cytosolic and nuclear proteins were extracted and counted as previously described [23 (link)]. Lysates were separated on SDS-PAGE gel transferred to nitrocellulose membranes and proteins were detected with specific polyclonal (p) or monoclonal (m) antibodies (Abs), recognized by IRDye secondary Abs (LI-COR Corporate, Milan, Italy). The Abs employed were anti-FoxO3a (75D8, #2497), p-FoxO3a (Ser253; #13129), p-FoxO3a (Ser294; #5538), p44/42 MAPK (#9102), Phospho-p44/42 MAPK (Thr202/Tyr204) (#9101) (all from Cell Signaling Technology Europe, B.V., Leiden, The Netherlands). MDM2 (IF2; #33-7100) (ThermoFisher Scientific), AKT 1/2/3 (H136; sc-8312), p-AKT 1/2/3 (Ser473) (sc-7985), p21 (sc-71811), p27 (sc-53871), p53 (sc-126), CD1 (sc-718), β-Actin (AC-15; sc-69879), GAPDH (FL-335; sc-25778) and Lamin B (C-20; sc-6216) all from Santa Cruz Biotechnology, Inc., Heidelberg, Germany. Images were acquired with the Odyssey FC Imaging System (LI-COR Corporate). For each Western blot figure, the whole blots showing all the bands with all molecular weight markers, as well as the densitometry readings/intensity ratio of each band (analyzed using Image J software, NIH, USA) are collected in the Supplemental Materials as a separate file named “Original blots and densitometry”.

Equal amounts of total tissue lysates were separated on either 4-20%, 30 μl Mini-PROTEAN TGX™ gels (Bio-Rad Laboratories, USA) or 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membranes were then blocked with a solution containing 10 mM Tris-buffered saline, 0.05% Tween 20, and 5% nonfat dry milk or 5% bovine serum albumin (BSA) (Sigma-Aldrich) and then incubated with primary antibodies including PGC-1α (Santa Cruz, sc-13067, dilution 1 : 500), Akt 1/2/3, (Santa Cruz, sc-8312, dilution 1 : 500), P-Akt 1/2/3 (Ser⁴⁷³) (Santa Cruz, sc-7985-R, dilution 1 : 500), FoxO3a (Cell Signaling, 2497, dilution 1 : 500), P-FoxO3a (Abcam, ab154786, dilution 1 : 500), Fbx32 (Abcam, ab168372, dilution 1 : 1000), and β-tubulin (Cell Signaling, 2146, dilution 1 : 500) over night at 4°C. The membranes were treated with secondary anti-rabbit and anti-mouse antibody (dilution 1 : 20000) for 1 h at room temperature. Following treatment with the appropriate secondary antibody, the bands were visualized using ImageQuant LAS 500 (GE Healthcare). The changes in the protein level were quantified by a densitometric method using the LASImage software. β-Tubulin was used as a lane loading control. The immunoblotting was performed at least two times.

Fetal bovine serum (FBS) and horse serum (HS) were purchased from BioWest (Nuaillé, France) and Invitrogen (Carlsbad, CA, USA), respectively. Dulbecco’s modified Eagle’s medium (DMEM, low glucose), minimum essential medium (MEM) Vitamin mix, Akti and dimethyl sulfoxide (DMSO) were obtained from Sigma (St. Louis, MO, USA). HEPES was obtained from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). LC3B antibody, AMPKα rabbit mAb, phospho-AMPKα (Thr172) rabbit mAb and AICAR were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). 4E-BP1(R-113), Akt1 (B-1) and p-Akt 1/2/3 (Ser 473) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). HRP-conjugated goat anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG were obtained from Kirkegaard & Perry Laboratories, Inc. (Gaithersburg, MD, USA). Other chemicals were obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan).