P jak2

Cells after treatment were lysed and centrifuged at 12,000 × g for 15 min. The supernatant was collected and the protein concentrations were detected using the BCA assay kit (Beyotime, Jiangsu, China). Equal amounts of total proteins (20–60 μg) were separated using SDS-polyacrylamide gel and electrotransferred to PVDF membranes. The membranes were initially blocked with 5% nonfat dry milk in PBS-Tween-20 (0.1%, v/v) at 4°C for 12 h and then probed with primary antibodies against cleaved caspase-3 (mouse IgG1), Bcl-2 (mouse IgG1 κ), Bax (mouse IgG1 κ), STAT3 (mouse IgG1 κ), p-STAT3 (mouse IgG1), p-JAK2 (Rabbit mAb), JAK2 (mouse IgG2b κ), p-Src (mouse IgG2a κ), Src (mouse IgG2a κ) or GAPDH (Rabbit mAb) (Santa Cruz, CA, United States), which were diluted following the manufacturer’s instructions at 4°C overnight. Then the membranes were blotted with the horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Beyotime, Jiangsu, China, 1:5,000) for 2 h at 37°C. Finally, the bands were visualized through the enhanced chemiluminescence protocol. Signals were densitometrically quantified and normalized to GAPDH. The results were analyzed using Image J software (NIH, United States).

Total lysates were prepared in radioimmune precipitation assay buffer and protein content was determined using the BCA method (Pierce, Rockford, IL, USA). Protein samples containing 15 μg of protein were separated in 7.5% SDS-polyacrylamide gels and transferred to PVDF membranes (Millipore, Billerica, MA). Membranes were blocked for 2 h in 5% milk powder in PBS and then incubated in primary antibodies against ER, FSHR, LHR, Bcl-2, Bax, p-JAK2, JAK2, p-STAT3, STAT3, β-actin (Santa Cruz Biotechnology), active caspase-3, FOXO1, and p-FOXO1 (Cell Signaling Technology, Beverly, MA) overnight at 4°C followed by HRP-conjugated secondary antibodies for 2 h at room temperature. After washing, proteins were detected using SuperSignal West Pico Chemiluminescent Substrate (Pierce). β-actin was used as the loading control.

Cell lysate extractions were prepared with radioimmunoprecipitation assay buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 150 mM NaCl, 50 mM Tris–HCl, pH 7.5, and 2 mM EDTA, pH 8.0) as described previously^{21 (link)–23 (link)}. Antibodies against C/EBPα, C/EBPβ, PPARγ, A-FABP, FASN, AKT, p-AKT, AMPKα1/2, p-AMPKα1/2, JAK2, p-JAK2, HSL, and PGC1α were purchased from Santa Cruz Biotechnology (Dallas, TX). Antibodies against ERK, p-ERK, STAT3, p-STAT3, p-HSL, PKA, p-PKA, and UCP1 were obtained from Cell Signaling Technology (Danvers, MA). The normalization control was anti-β-actin (Santa Cruz Biotechnology).

A total of 20–30μg of protein was loaded onto 4–12% Bis-Tris gels (NuPAGE, Life Technologies Corporation, Sweden). After transfer, the PVDF membranes were blocked in 5% Blotting Grade Blocker (Bio-Rad Laboratories AB, Sweden) in TBS supplemented with 0.1% Tween-20 (TBST, both from Merck Millipore, Germany) and incubated with the primary antibodies at 4°C overnight. After 1 h incubation with secondary antibodies (HRP-conjugated anti-mouse from Rockland Immunochemicals Inc., #20789 and HRP-conjugated anti-rabbit from Cell Signaling Technology Inc., USA, #7074), the proteins were detected by using an ECL solution (PerkinElmer Inc., Sweden). The following antibodies were from Cell Signaling Technology: STAT1(#9172, 1/1000), p-Y701-STAT1 (#58D6, 1/1000), STAT3 (#79D7, 1/1000), p-Y705-STAT3 (#D3A7, 1/1000), JAK1 (#3332, 1/1000), JAK2 (#3230, 1/1000). Anti-GAPDH antibody (ab9485, 1/5000) was purchased from Abcam; pJAK1 (#sc-16773-R, 1/500), pJAK2 (#sc-16566-R, 1/500)–from Santa Cruz Biotechnology.

Growth factors (HGF and EGF) and tyrosine kinase inhibitors (gefitinib and erlotinib) were purchased from PeproTech Inc. (Rocky Hill, CT, USA) and Wako Pure Chemical Industries Ltd. (Osaka, Japan), respectively. A6730, 5-FU, SU11274, U0126, and Stattic were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Antibodies were obtained from the following sources and used at the indicated dilutions: Sigma-Aldrich Co. (St. Louis, MO, USA), UPP1 (1:600), p-STAT3, and α-tubulin (1:10,000); Cell Signaling Technology, Inc. (Danvers, MA, USA), p-c-Met Tyr1003, Tyr1234/1235, Tyr1349, p-EGFR Tyr845, Tyr1068, Tyr1173, p-Akt1/2, and p-Jak2 (all 1:10,000); Santa Cruz Biotechnology (Dallas, TX, USA), p-Erk1/2 (1:2000). Other reagents and biochemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).