Anti pi3 kinase p85

The protein extraction methods used in this study were adopted from our previous protocol [11 (link)]. The protein concentration in the cell extract was determined using a Bio-Rad protein assay dye reagent (Richmond, VA, USA). Aliquots of the supernatant containing 50 μg protein were separated through standard SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were incubated with anti-insulin receptor β (IRβ; 1 : 1000; Cell Signaling Technology, Beverly, MA, USA), anti-PI3-kinase p85 (1 : 1000; Cell Signaling Technology), anti-phospho-Akt (Ser473) (1 : 1000; Cell Signaling Technology), anti-Akt (1 : 500; Cell Signaling Technology, Danvers, MA, USA), antiglucose transporter (GLUT)2 (1 : 500; Millipore, Billerica, MA, USA), anti-PPARα (1 : 1000; GeneTex, Irvine, CA, USA), or anti β-actin (1 : 4000; GeneTex) antibodies at 4°C overnight. The membranes were incubated with anti-mouse IgG or anti-rabbit IgG secondary antibodies and washed thrice for 5 min each time. Protein band images were detected and captured using the UVP Biospectrum image system (Level, Cambridge, UK). Finally, all relevant protein expressions were normalized with β-actin.

Cells were harvested in lysis buffer (Tris-EDTA 10 mM, 0.5% NP40, NaCl 150 mM), containing a protease inhibitor, and boiled for 5 min at 90 °C. The protein extracts were run on SDS-polyacrylamide gel (SDS-PAGE) and transferred to Polyvinylidene Difluoride (PVDF) sheets (Merck Millipore, Darmstadt, Germany). Membranes were blocked for 40 min in 5% non-fat milk powder (Sigma-Aldrich, St. Louis, MO, USA) in PBS containing 0.1% Tween-20 (PBS-Tween) and then incubated overnight at 4 °C with one of the following primary antibodies: anti-LC3B (1:1500, Sigma-Aldrich, St. Louis, MO, USA), anti-PI3 Kinase p85 (dilution 1:800, Cell Signaling, Danvers, MA, USA), anti-Phospho AKT^Thr308 (1:800, Cell Signaling, Danvers, MA, USA), anti-AKT pan (1:800, Cell Signaling, Danvers, MA, USA), anti-Phospho AMPKα1/2^Thr172 (dilution 1:800, Cell Signaling, Danvers, MA, USA), anti-AMPKα1 (dilution 1:1000, Immunological Science, Milan, Italy), anti-Phospho p70 S6 Kinase^Thr389 (dilution 1:600, Immunological Science, Milan, Italy), anti-p70S6 Kinase (dilution 1:1000, Immunological Science, Milan, Italy), anti-Caspase 9 (dilution 1:2000, Immunological Science, Milan, Italy), anti-Caspase 3 (dilution 1:2000, Immunological Science, Milan, Italy), anti-β-Actin (1:2000, Immunological Science, Milan, Italy). β-Actin was used as a reference protein for loading control.

Human IGF-1 (catalog 13769) was obtained from Sigma-Aldrich. Primary antibodies used were as follows: anti-Phospho-Akt (Ser473) (catalog 4060), anti-Akt (catalog 9272), anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (catalog 4377), anti-p44/42 MAPK (Erk1/2) (catalog 9102), anti-PI3 Kinase p85(catalog 4292) and anti-IGF-1 Receptor β-subunit (D23H3) (IGF-1R β, catalog 9750) from Cell Signaling Technology; anti-TRAF4 (catalog ab108991) and anti-IRS-1 (Phospho Y896, catalog ab 4873) from Abcam; anti-Ubiquitin (catalog sc-8017) and anti-GAPDH (catalog sc-32233) from Santa Cruz Biotechnology Inc; anti-IRS-1 (21HCLC) (catalog 71009) and anti-Phospho-IRS-1(Y612) (catalog 44-816G) from Thermo Fisher Scientific; anti-Phosphotyrosine (pTyr, clone 4G10, catalog 05-321) from Sigma-Aldrich. HRP-conjugated secondary anti-mouse (catalog 1706516) and anti-rabbit (catalog 1706515) antibodies were obtained from Bio-Rad. Monoclonal ANTI-FLAG M2-peroxidase (HRP) antibody (catalog 8592A) and EZview Red ANTI-FLAG M2 Affinity Gel (catalog F2426) were obtained from Sigma-Aldrich. Protein A-Agarose beads (catalog sc-2001) were obtained from Santa Cruz Biotechnology. Recombinant human IRS-1 protein (catalog ab70538) was obtained from Abcam. This truncated recombinant protein has amino acids from 600 to 1245, which includes TRAF4-targeted lysines for ubiquitination.