Phosphor p38

Western blot analysis was conducted as previously described [21 (link)]. Phospho-ERK1/2 (#9101), total ERK (#9102), phosphor-JNK (#9251), total JNK (#9252), phosphor-p38 (#4631), total p38 (#9212) and β-actin (#4967) were obtained from Cell Signalling. TXNIP (NBP1-54578) and AP-1 (ab21981) were purchased from Novus Biologicals and Abcam, respectively. Protein expression was measured by a ChemiDoc system (Bio-Rad, Hercules, CA, USA).

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).

After being pretreated with ISE, RAW264.7 macrophages were stimulated with LPS or L1CM for 30 min, and then harvested in RIPA lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP40, 1 × protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN), and 1 × phosphatase inhibitor cocktail (Sigma-Aldrich). Proteins were resolved in 7.5% acrylamide gels and then transferred to nitrocellulose membranes. The membrane was blocked with 5% non-fat milk in Tris-buffered saline before being incubated, respectively with specific primary antibodies for the following proteins: IκB-α (1:1000), phosphor-p44/p42 (Thr202/Tyr204) (p-ERK) (1:1000), phosphor-p38 (Thr202/Tyr204) (p-p38) (1:1000), and Phospho-SAPK/JNK (Thr183/Tyr185) (p-JNK) (1:1000), phosphor-STAT3 (p-STAT3) (1:1000), ERK (1:1000), JNK (1:1000), p38 (1:1000), STAT3 (1:1000) (all from Cell Signaling Technologies, Danvers, MA), and β-actin (1:5000, Sigma-Aldrich). The membranes were next incubated with horseradish peroxides (HRP)-conjugated secondary antibodies followed by exposure to enhanced chemiluminescent reagents (Millipore, Burlington, MA).

Cells were harvested in lysis buffer [50 mM Tris-HCl, pH 7.4, 1.5% NP-40, 0.1% SDS, 150 mM NaCl, 50 μg/ml PMSF, with fresh proteinase inhibitor cocktail (Roche)]. Proteins from total cell lysates were separated by SDS-PAGE, transferred to PVDF membranes, blocked in 5% bovine serum albumin (BSA) in TBST, and blotted with primary antibody according to the manufacturer's instructions [GAPDH, 1:5000, Sigma; Erk, 1:1000, Cell Signaling Technology; Phosphor-Erk, 1:1000, Cell Signaling Technology; P38, 1:1000, Cell Signaling Technology; Phosphor-P38, 1:1000, Cell Signaling Technology; NF-κB p65, 1:1000, Cell Signaling Technology; Phosphor-NF-κB p65(Ser536), 1:1000, Cell Signaling Technology]. Thereafter, the membranes were washed three times with TBST and incubated with appropriate secondary antibodies for 1 h at room temperature. The ECL chemiluminescence system was used to detect the signals.

Total tissue and cell lysates were assayed by Western blot according to our previous research 10. The antibodies Nrf2, Histione, extracellular signal‐regulated kinase (ERK), phosphor‐ERK, Jun N‐terminal kinase (JNK), phosphor‐JNK, p38, phosphor‐p38, anti‐rabbit‐HRP, anti‐goat‐HRP and anti‐mouse‐HRP were from Cell Signaling Technology (Danvers, MA, USA), and β‐actin, c‐fos, phosphor‐c‐fos, c‐jun, phosphor‐c‐jun, MMP‐1, procollagen type I, TGF‐β1, elastin and DLD‐E3 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Each experiment was repeated at least three times.

Western blot analysis was performed as previously described[7 (link)]. Total protein was extracted from the cultured cells using a cell scraper and RIPA buffer [1×PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate and 0.1% sodium dodecyl sulfate, pH 7.6 with 1×Protease inhibitors (Roche) and 100 μg/ml of PMSF]. The protein samples were separated by SDS-PAGE and then transferred to nitrocellulose blotting membranes. The membranes were blocked in 5% non-fat milk for 1 hour, probed with primary antibodies overnight at 4°C, and then incubated with secondary antibody for 1 hour at room temperature. The membrane was then infiltrated with chemiluminescence to detect the target protein signal.
Primary antibodies against DR5, DcR1, NF-κB p65, phosphor-NF-κB p65, IκBα, phosphor-IκBα, phosphor-IKKα/β, JNK, phosphor-JNK, phosphor-p38, Erk, or phosphor-Erk were purchased from Cell Signaling (USA). Antibodies against p38 and horseradish peroxidase conjugated anti-mouse-IgG or anti-rabbit-IgG were from Santa Cruz (USA). The primary antibodies were diluted to 1:1000 and the secondary antibodies were diluted to 1:2000.