Hrp conjugated secondary antibody

A nuclear extract kit (Active Motif, Tokyo, Japan) was used to extract cytoplasmic and nuclear proteins of kidney tissues, according to the manufacturer's protocol. Protein extracts separated upon 7.5% SDS-PAGE were transferred to 0.45 m PVDF membrane (Bio-Rad, California, Hercules, USA). The membranes were incubated with TBST (20 mM Tris-HCL, pH 7.5, 150 mM NaCl, 0.1% Tween 20) with 5% nonfat milk powder for 2 hours before Western blotting overnight at 4°C with rabbit polyclonal antibodies against mouse Nrf2, HO-1, NQO-1, GSR (Cell Signaling Technology, USA), and GCLc (av54576, Sigma, USA). Histone H3 and β-actin (Cell Signaling Technology, USA) were used as a protein control to normalize the volume of protein expression. After being washed for 3 × 10 minutes with TBST, the membranes were incubated with HRP-conjugated secondary antibody (1 : 2000, Cell Signaling Technology, USA) for 1.5 hours. Then, after the membranes were washed 3 times with TBST, immunoreactive bands were visualized with electrochemiluminescence reagent (Amersham, Uppsala, Sweden). Densitometric and ImageQuant analyses were subsequently quantified using Image Lab Software (Bio-Rad, USA).

Whole cell lysates were separated in SDS/PAGE gels and transferred to polyvinylidenediflouride (PVDF) membranes (Invitrogen, Carlsbad, CA, USA). After blotting membranes were blocked in 5% BSA (Sigma–Aldrich, St. Louis, Missouri, USA) in Tris-Buffered Saline (TBS) containing 0,1% Tween 20. Membranes were incubated with one or more of the following antibodies as specified in the figures and figure legends: antibodies to detect phosphorylated and total STAT5, were obtained from (Cell Signaling, Danvers, MA, USA). Antibody dilution is 1:1000 and membranes incubated with primary antibody at 4°C overnight. When indicated, incubations with the appropriate HRP-conjugated secondary antibody (Cell Signaling) were conducted with dilution of 1:5000 for 1 hour at room temperature. Membranes were visualized with the ECL Western blotting detection system (Millipore) according to the manufacturer's instruction and were analysed by using a DSS camera (Bio-Rad).

Tissues or cells were harvested and lysed with ice-cold RIPA buffer (Beyotime, Haimen, China) containing protease and phosphatase inhibitors (Roche). Lysates were clarified by centrifugation at 15,000 × g for 30 min. The protein concentration of the supernatant fraction was determined by the Bradford assay (Thermo Fisher Scientific, Waltham, MA, USA). Protein samples were diluted in 4× SDS loading buffer (TaKaRa), heated to 95 °C for 5 min and separated in a 10% SDS-polyacrylamide gel. The proteins were electroblotted onto polyvinylidene fluoride membranes and incubated for 1 h in 5% bovine serum albumin in phosphate-buffered saline (PBS) or non-fat dry milk dissolved in PBS containing 0.1% Tween-20 (PBST) at room temperature. The blotting membranes were incubated with primary antibodies to BEX1 (kindly provided by Professor Frank L. Margolis), CDKN1a, B-cell lymphoma-2 (Bcl-2), poly ADP-ribose polymerase (PARP), cleaved PARP and PPARG (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C, extensively washed in PBST, incubated with HRP-conjugated secondary antibody (Cell Signaling Technology) for 1 h at room temperature and washed again with PBST. The blotting membranes were developed with chemiluminescent reagents (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. The densitometry of the bands was quantified using ImageJ software.

RAW 264.7 macrophages were cultured in DMEM containing 10% (v/v) FBS at 70–80% confluency. The cells were then either primed or left unprimed with 10 ng/mL IFN-γ for 1 h, before each sample and LPS (1 μg/mL) were added for 24 h. The cells were then lysed with 1× cell lysis buffer (Cell Signaling Technology), and protein concentration was measured using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Equal quantities of total protein were loaded onto a 10% SDS polyacrylamide gel (Bio-Rad Laboratories) for separation. The separated proteins were transferred to Immobilon P membranes (Millipore, Billerica, MA, USA). Nonspecific proteins were blocked with 5% fat-free milk for 1 h, before the primary antibody was treated at 4°C overnight. Protein bands were detected with a chemiluminescence detection kit (GE Healthcare, NJ, USA) after hybridization with an HRP-conjugated secondary antibody (Cell Signaling Technology). The COX-2, iNOS, and p-IκB-α protein levels were expressed as a relative value to that of β-actin. P-JNK, p-ERK, p-p38, and p-MKK4 levels were expressed as a relative value to that of JNK, ERK, p38, and MKK4, respectively. Relative protein levels were quantified by using ImageJ (National Institutes of Health, Bethesda, MD, USA).