Phosphor 4e bp1

Metformin was purchased from Aladdin chemistry Co.Ltd and was diluted across a range of concentrations in culture media. Antibodies against total Akt, phosphor-Akt(Ser473), total Erk1/2, phosphor-Erk1/2, phosphor-Acetyl-CoA Carboxylase (Ser79), total Acetyl-CoA Carboxylase (Ser79), total p70 S6 kinase (Thr389), phosphor-p70 S6 kinase (Thr389), total AMPKα (Thr172), phosphor-AMPKα (Thr172), total 4E-BP1, phosphor-4EBP1, FASN and β actin were purchased from Cell Signaling Technology. AZD6244 and MK-2206 were from Selleckchem (Houston, Texas, USA).

Antibodies against phosphor-AMPKα, AMPKα, phosphor-ACC, ACC, phosphor-mTOR, mTOR, phosphor-p70S6K, p70S6K, phosphor-4E-BP1, 4E-BP1, phorphor-NFκB p65, NFκB p65, IκBα, COX2, phosphor-STAT3, STAT3, Mcl-1, Bcl-xL, Bim, Bak, Bax, Bid, Puma and Bad were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against Bcl-2 and Protein A/G Agarose beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Metformin and aspirin as well as interleukin-6 (IL-6) were purchased from Sigma-Aldrich (St Louis, MO, USA). AZD-8055 was purchase from Invitrogen (Carlsbad, CA, USA).

Lanatoside C (lana.C), rottlerin, Ro318220, Gö6983 were purchased from Sigma Chemical Co. (St Louis, MO, USA). The above drugs were all dissolved in dimethylsulfoxide (DMSO). The non-conjugated primary antibodies were against C23 (nucleolin, Sigma Chemical Co., St Louis, USA), BID, caspase-8, caspase-9, PKCδ (Thr505), PKCδ, caveolin, phoshpo-p44/42 MAP kinas (Thr202/Tyr204), p44/42 MAP kinas (Thr202/Tyr204), phosho-SAPK/JNK (Thr183/Tyr185), p38 MAP kinase, phosho-mTOR, phosphor-p70 S6 kinase (Thr421/Ser424), phosphor-4E-BP1 (Thr37/46), phosho-eIF4E (Ser209), AKT (Cell signaling Technology, Beverly, MA, USA), AIF, Bcl-xl, Mcl-1, α-tubulin, PARP-1/2 (Santa Cruz Biotechnology Corp., Santa Cruz, CA, USA), caspase-2, caspase-6, caspase-7 (BD Biosciences, San Jose, CA, USA), phosho-AKT (Ser473, Epitomics Inc., Burlingame, CA, USA), caspase-3 (Imgenex, San Diego, CA, USA), β-actin (Millipore, Temecula, CA, USA). The secondary horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit IgGs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). z-VAD-fmk was purchased from R&D system (Minneapolis, MN, USA).

Cells were lysed in a RIPA buffer (150 mM NaCl, 1.0% IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0.) with Halt Protease and Phosphatase Inhibitor Cocktail (Pierce) and then cleared by centrifugation. Protein concentration was estimated with a BCA assay (Pierce). Proteins present in cell lysates (30 μg) were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membrane. Membranes were stained with Ponceau S to confirm equal loading and probed, stripped with NewBlot™ PVDF Stripping Buffer (LI-COR), and reprobed with antibodies specific for phospho-AKT (Cell Signaling), AKT(Cell Signaling), phosphor-ERK (Cell Signaling), ERK (Cell Signaling), phospho-HER2 (Cell Signaling), HER2 (Sigma-Aldrich), phospho-HER3 (Cell Signaling), HER3 (Cell Signaling), AKT1(Cell Signaling), phosphor-4EBP-1(Cell Signaling) or actin (Santa Cruz). Immunoreactive proteins were detected and quantified using infrared fluorescence, IRDye® secondary antibodies, and Odyssey® imagers (LI-COR).

Cells were harvested and lysed with RIPA buffer (Beyotime, Shanghai, China). Cell lysates were subjected to standard Western blotting using antibodies against GRP78, XBP1s, ATF-4, CHOP, p62, phosphor-mTOR (Ser 2448), mTOR, phospho-p70 S6 Kinase (p70S6K, Thr389), p70S6K, phosphor-4EBP1(Thr 37/36), 4EBP1 (Cell Signaling Technology, Danvers, MA, USA), XDH (Abcam, Cambridge, UK), and GAPDH (Proteintech, Rosemount, IL, USA). The β-actin and LC3 antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA).