Polyclonal anti rabbit horseradish peroxidase

PK-15 cells were collected with the cell lysis buffer containing protease inhibitor (Beyotime) on ice. Then the cell lysates were sonicated using a Snoics VCX105 sonicator and centrifuged at 12 000 rpm for 20 min at 4 °C. Protein concentration was determined by the BCA protein assay kit (Beyotime). Equal amounts of protein samples were diluted in 5 × SDS-PAGE loading buffer and heated at 95 °C for 5 min. The samples were separated on 12% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking for 1 h at RT in Tris-buffered saline (TBS) containing 5% nonfat milk powder and 0.1% Tween 20, the membranes were reacted with primary antibodies overnight at 4 °C. The membranes were washed and incubated in secondary antibody (polyclonal anti-rabbit–horse radish peroxidase from Sigma) at room temperature for 1 h. Blots were visualized according to the standard enhanced chemiluminescence system (Bio-Rad, Berkeley, USA). Quantification of protein blots was performed using the Image-Pro Plus 6.0 software (Media Cybernetics, Sarasota, USA), and images were acquired from an EU-88 image scanner (Seiko Epson Corporation, Suwa, Japan).

Cells were collected in 80 μl lysis buffer containing protease inhibitors (Beyotime, China) and were sonicated (SonicsVCX105, USA). The lysate was centrifuged at 12,000 rpm for 20 min at 4°C and the supernatant was immediately collected for use. Protein concentration was determined using the BCA kit (Beyotime, China). Fifty μg of protein were diluted in sample loading buffer and heated at 95°C for 5 min. The denatured proteins were resolved by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE), and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated for 2 h at RT in Tris-buffered saline (TBS) containing 5% milk (for SelS) or BSA (for β-actin, p38, p-p38, ERK1/2, and p-ERK1/2), and 0.1% Tween 20 (TBST), followed by overnight incubation at 4°C in specific primary antibodies (anti-SelS from Santa Cruz Biotechnology, diluted 1/500; anti-β-actin, anti-p38, anti-p-p38 anti-ERK1/2, and anti-p-ERK1/2 from Cell Signaling, diluted 1/1000). The membranes were washed and incubated in HRP-conjugated secondary antibody (polyclonal anti-rabbit–horseradish peroxidase from Sigma) at RT for 1h. The blots were visualized and analyzed by a Luminescent Image Analyzer (FUJIFILM LAS-4000) and expressed as a percentage respect to the control group.