Phosphor erk1 2

After treatment, cells were harvested and lysed, and protein concentrations were measured by using the Bio-Rad protein assay kit (Bio-Rad, Richmond, CA, USA). All samples were separated on SDS-PAGE gel and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). After blocking the membranes with 5% (w/v) non-fat dry milk in TBS containing 0.1% Tween 20 (TBS-T) for 1 hour at room temperature, they were immunoblotted with the following monoclonal primary antibodies: vimentin, E-cadherin, MMP-2, MMP-9, phosphor-ERK1/2, total-ERK1/2, NF-kB subunit p65, β-actin, and GAPDH (all from Cell Signaling Technology, Beverly, MA,USA). Appropriate horseradish peroxidase-conjugated secondary antibodies, including mouse IgG and rabbit IgG antibodies (Abcam, Cambridge, MA, USA), were applied and incubated for 1 h at room temperature. Specific signals were visualized using a chemiluminescence (ECL) detection kit (Millipore).

For western blot analysis, differentially treated cells were washed with cold PBS and then lysed in lysis buffer containing protease and phosphatase inhibitor. Insoluble cell fragments were removed by centrifugation. Cells were separated by SDS-PAGE electrophoresis and next transferred to PVDF membranes. Membranes were incubated overnight with the primary antibodies against phospho-AKT (Cell Signaling Technologies), ERK-1/2 (Cell Signaling Technologies), phosphor-ERK-1/2 (Cell Signaling Technologies), STAT3 (Cell Signaling Technologies), phospho-STAT3 (Cell Signaling Technologies), phospho-IGF-I (Cell Signaling Technologies, Danvers, MA, USA), JAK1, phospho-Jak1 (Tyr1022/1023) (Cell Signaling Technologies), JAK2 (Cell Signaling Technologies), phospho-Jak2 (Tyr1007/1008) (Cell Signaling Technologies), IGF-I Receptor β (Cell Signaling Technologies), AKT (Cell Signaling Technologies), epithelial-mesenchymal transition (EMT) antibody sampler kit (Cell Signaling Technologies). Fluorescent secondary anti-mouse (Thermo Scientific, Waltham, USA) and anti-rabbit (Thermo Scientific, Waltham, USA) antibodies were used and were detected using Odyssey Sa infrared imager (LI-COR Biosciences, Lincoln, USA).

The materials for cell culture were purchased from Gibco (Grand Island, NY, USA). MAPK inhibitor, including PD98059 for ERK1/2, SP600125 for JNK, SB203580 for p38, and LY294002 for Akt, and AP-1 inhibitor, i.e., Tanshinone IIA, were purchased from Sigma (St. Louis, MO). Rabbit antibodies against MSH2, phosphor-ERK1/2, ERK1/2, phosphor-Akt, Akt, and β-actin were purchased from Cell Signaling Technology (Beverly, MA). The control-, MSH2-, and c-jun-siRNA were purchased from Thermo (Waltham, MA). All others chemicals were obtained from Sigma (St. Louis, MO).

The selective antibiotics, zeocin was purchased from Invitrogen (CA, USA). 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and MitoTracker were purchased from Molecular Probes (Invitrogen, CA, USA). Antibodies against PGC-1α, Keap1, and Nrf-2, were purchased from Santa Cruz (Dallas, Texas, USA). Antibody against Hrd1 was purchased from Novus Biologicals (Littleton, CO, USA). Antibodies against caspase 3, cleaved caspase 3, Bax, Bcl2, phosphor-p53, total-p53, phosphor-GSK3β, totoal-GSK3β, phosphor-p38, phosphor-ERK1/2, phosphor-JNK, total-p38, total-ERK1/2, total-JNK, HO-1 and c-myc were all purchased from Cell Signaling Technology (Danvers, MA, USA). SB203580 and PD98059 were purchased from Calbiochem (Cat#559398 for SB203580 and Cat#513001 for PD98059, Darmstadt, Germany). N-acetyl-L-cysteine (NAC, Cat#A7250) and Thiazolyl Blue Tetrazolium (Cat#M2128) for MTT assay was purchased from sigma-aldrich. For over-expression of human PGC-1α in HK-2 cells, human PGC-1α/pCDNA4 plasmid was purchased from Addgene (Cat#10974, Cambridge, USA). Nrf-2 specific siRNA (Cat# sc-37030) and control siRNA (Cat# sc-37007) were purchased from purchased from Santa Cruz (Dallas, Texas, USA). DhamaFECT 1 Transfection reagent was purchase from GE Healthcare (Cat# T-2001-02, USA).

The cells were lysed in NP40 lysis buffer containing 1mM PMSF, 1‰ protease inhibitor and 1% phosphorylase inhibitor on ice for 30 min. After centrifuged, the supernatant was acquired and separated by 12% SDS-PAGE, and then transferred to nitrocellulose membrane. The nitrocellulose membrane was blocked with 4% skim milk powder in PBST for 1 h, and incubated with anti-tilapia IFN-γ mAb or 1:1000 diluted primary antibody against S6, 4EBP1, phospho-S6 (Ser240/244), phospho-4EBP1 (Thr37/46), phosphor-STAT5 (Tyr694), phosphor-Erk1/2 (Thr202/Tyr204), phosphor-Akt (Thr308), phosphor-Akt (Ser473), and β-actin from Cell Signaling Technology at 4°C overnight, and further incubated with goat anti-rabbit or mouse IgG H&L 800 or 680 (Abcam). To detect the overexpressed IFN-γ in 293T cells, the nitrocellulose membrane was incubated with anti-tilapia IFN-γ mAb, followed by Ap-conjugated goat anti-mouse IgG secondary antibody (Southern Biotech), and visualized with freshly prepared substrate solution (100 mM NaCl, 100 mM Tris, and 5 mM MgCl₂; pH 9.5) containing nitroblue tetrazolium (Sigma) and 5-bromo-4-chloro-3-indolylphosphate (Sigma). Images were acquired by Odyssey CLx Image Studio.

Pun (>90%) was purchased from Beijing Huanyu Kechuang Biological Company (Beijing, China). LPS (Escherichia coli O55:B5) was purchased from Sigma–Aldrich (Darmstadt, German). Mouse tumor necrosis factor-α (TNF-α, Batch No 978941030), interleukin (IL)-10 (IL-10, Batch No 132111031116), IL-6, (Batch No 1321213119) and transforming growth factor-β1 (TGF-β1, Batch No 9711891510) were purchased from Wuhan Boster Biology Technology Co. Ltd. (Wuhan, China). Total-antioxidant capacity (T-AOC; Batch No A024) and malondialdehyde (MDA; Batch No A003-1) were purchased from Nanjing Jiancheng Biotechnology Company (Nanjing, China). Mouse anti-human nuclear factor-κB p65 (NF-κB p65, #8242), phosphorylated nuclear factor-κB p65 (pNF-κB p65, #3033), IκBα (#9242), phosphor IκBα (#2859), ERK1/2 (#9102) and phosphor ERK1/2 (#9101) were purchased from Cell Signaling Technology; JNK (sc-7345), phosphor JNK (sc-6254), p38 (sc-7972) and phosphor p38 (sc-7973) antibody were purchased from Santa Cruz (CA, U.S.A.). β-actin antibody was purchased from Sungen (China). Anti-rabbit IgG, HRP-linked antibody (7074S) and anti-mouse IgG, HRP-linked antibody (7076S) were purchased from Cell Signaling Technology. Enhanced chemiluminescence detection kit was obtained from Merck Millipore (Darmstadt, Germany).