Pthr308 akt

The primary antibodies MAPK (Erk) (#9102, 1:1000), Akt (1:1000, #9272), Phospho-EGF Receptor (Tyr1068) (1:1000, #2234), p(Thr308)-Akt (1:1000, #9275), Phospho-Akt (Ser473) (1:1000, #9271), Phospho-MAPK (pERK) (1:1000, #9101), β-Actin (1:10000, #4967) were purchased from Cell Signaling (Leiden, The Netherlands) and anti-EGFR (1:1000, sc-03-G) was purchased from Santa Cruz Biotechnology(Texas, USA). Cetuximab (ERBITUX) was ordered from Merck (Dietikon, Switzerland). Gefitinib (Iressa) was bought from Sigma (Zwijndrecht, The Netherlands); sunitinib was purchased from LC Laboratories (Woburn, USA). Entinostat and SAHA were purchased from Selleckchem (Munich, Germany). Staurosporine and cisplatin were purchased from Sigma-Aldrich (Zwijndrecht, Nederland). Doxorubicin was purchased from Teva Pharmaceuticals. All drugs were aliquoted in DMSO and stored at -20°C. The human epidermal growth factor (hEGF) and platelet-derived growth factor (PDGF) were purchased from Sigma-Aldrich (Zwijndrecht, Nederland).

Antibodies against p-Tyr1022/1023-JAK1 (#3331), p-Tyr1007/1008-JAK2 (#3776), p-Tyr980/981-JAK3 (#5031), p-Tyr1054/1055-TYK2 (#9321), p-Tyr701-STAT1 (#9167), p-Tyr705-STAT3 (#9145), p-Tyr694-STAT5 (#9359), JAK1 (#3332S), JAK2 (#3230), JAK3 (#8863), TYK2 (#9312), STAT1 (#14994), STAT3 (#12640), STAT5 (#25656), c-Myc (#13987), cyclin D1 (#AF0931), Bcl-xL (#2764), p-Ser536-NF-κB (#3033), p-Thr308-AKT (#9275), p-Ser9-GSK-3β (#5558), p-Thr183/Tyr185-SAPK/JNK (#4668), NF-κB (#8242), AKT (#4691), GSK-3β (#9832), and SAPK/JNK (#9252) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against α-tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Protease inhibitor (B14001) and phosphatase inhibitor (B15001) were purchased from Bimake (Houston, TX, USA). Recombinant human STAT3 protein (ab268982) was obtained from Abcam (Cambridge, Britain). Recombinant human interleukin-6 (IL-6) protein (200-06) was purchased from PeproTech (Rocky Hill, CT, USA). Nitrocellulose membranes and chemiluminescent horseradish peroxidase (HRP) substrate were obtained from Millipore (Billerica, MA, USA). Gefitinib was purchased from Selleckchem (Houston, TX, USA). DAB color solution, hematoxylin solution, and eosin staining solution were purchased from Servicebio (Wuhan, China).

Cells were washed three times with ice-cold PBS and homogenized in 2% SDS by sonication. Insoluble material was removed by centrifugation at 21,000 × g. Protein concentration was determined by bicinchoninic acid method (Thermo Scientific). 10 μg of protein was resolved by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked in 5% skim milk powder in Tris-buffered saline for 1 h, followed by an overnight incubation at 4 °C with specific primary antibody solutions. Antibodies specific for pSer-473 Akt, pThr-308 AKT, pThr-642 TBC1D4, and Akt were from Cell Signaling Technology; GLUT4 antibody was made in-house; α-tubulin antibody was from Sigma; and 14-3-3β antibody was from Santa Cruz Biotechnology, Inc. Antibodies specific for PRDX2 were from Abcam, and PRDX3 antibody was from Abfrontier. Membranes were incubated with an appropriate secondary antibody for 1 h before signals were detected using ECL (Thermo Scientific or Millipore) on the Chemidoc MP (Bio-Rad). In some cases, IRDye700- or IRDye800-conjugated secondary antibodies were used and then scanned at the 700- and 800-nm channel using the Odyssey IR imager. Densitometry analysis of immunoblots was performed using ImageJ version 1.47.