Ecl chemiluminescence substrate

Cells were lysed by RIPA buffer with protease inhibitors and phosphatase inhibitors according to the manufacturer’s protocol (Sigma Aldrich, St. Louis, MO, USA). For the isolation of nucleus and cytosol proteins, the nuclear protein isolation-translocation assay kit was employed according to the manufacturer’s protocol. The protein concentration was determined using the BCA protein assay reagent according to the manufacturer’s instructions (Thermo Scientific, Waltham, MA, USA). Cellular protein extracts were separated by electrophoresis using 8% SDS polyacrylamide gel and were electro-blotted onto PVDF membranes. The membranes were incubated with blocking solution for 1 h at room temperature, followed by incubation overnight with primary antibodies at 4 °C (1:1000). Blots were washed three times with Tris-buffered saline/Tween 20 (TBST) and incubated with a 1:5000 dilution of horseradish peroxidase conjugated secondary antibody for 1 h at room temperature. Blots were again washed three times with TBST and developed using an ECL chemiluminescence substrate (Thermo Scientific, Waltham, MA, USA). Band intensities were quantified using Alphaview SA software (Alpha Innotech Corporation, San Leandro, CA, USA).

Proteins were extracted from mouse liver tissue using RIPA lysis buffer that was suitably combined with a mixture of protease inhibitors and phosphatase inhibitors. Protein concentrations in the supernatant were quantified by using a BCA Protein Assay Kit (Beyotime, Shanghai, China). Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then wet electrotransferred to polyvinylidene fluoride (PVDF) transfer membranes for 90 min. Then, 5% skim milk in TBST (20 mM Tris pH 7.5, 150 mM NaCl, and 0.1% Tween 20) was used to block the PVDF membranes at room temperature for 2 h. Subsequently, the membranes were incubated with primary antibodies against ERK, JNK, p38, I-κB and NF-κB and phosphorylated ERK, JNK, p38, I-κB and NF-κB overnight at 4°C. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000) for 2 h at room temperature. Finally, an ECL chemiluminescence substrate (Thermo Scientific, USA) was used to develop the immunoreactivity of the membranes. Protein-antibody complexes were detected using an Azure c500 imaging system (Azure Biosystems, USA) and normalized using GAPDH as an internal control. Two duplicates were performed for each condition.

Cells were lysed by RIPA buffer containing protease inhibitors and phosphatase inhibitors, and the concentration of protein was measured by BCA protein assay kit according to the manufacturer’s instructions (Thermo Scientific). Equal amounts of cellular protein extracts were separated by SDS-PAGE. Afterwards, proteins were electrophoretically transferred to PVDF membranes. The membranes were incubated with blocking solution for 1 h at room temperature, followed by incubation overnight with primary antibodies at 4 °C. After washing three times with Tris-buffered saline/Tween 20 (TBST), the blots were hybridized with secondary antibody conjugated with horseradish peroxidase for 1 h at room temperature. Thereafter, blots were washed three times with TBST and developed using an ECL chemiluminescence substrate (Thermo Scientific). The signals were captured using Bio-Rad ChemiDOC XRS⁺ system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Cells were lysed by RIPA buffer with protease inhibitors and phosphatase inhibitors according to the manufacturer’s protocol (Sigma Aldrich, St. Louis, MO, USA). The protein concentration was determined using the BCA protein assay reagent according to the manufacturer’s instructions (Thermo Scientific, Waltham). Cellular protein extracts were separated by electrophoresis using 8% SDS polyacrylamide gel and were electroblotted onto PVDF membranes. The membranes were incubated with blocking solution for 1 hr at room temperature, followed by incubation overnight with primary antibodies at 4 °C (1:1000). Blots were washed three times with Tris-buffered saline/Tween 20 (TBST) and incubated with a 1:5000 dilution of horseradish peroxidase conjugated secondary antibody for 1 h at room temperature. Blots were again washed three times with TBST and developed using an ECL chemiluminescence substrate (Thermo Scientific). The signals were captured and the band intensities were quantified using Bio-Rad ChemiDoc XRS⁺ system (Bio-Rad Laboratories, Inc.).