Ecl western blotting detection regents

Transmembrane TNF-expressing Jurkat cells were stimulated with 0.01 μM IFX for 2 h in the presence (Rhoi-IFX) or absence (IFX) of 0.01 μM of Y-27632. Stimulated or unstimulated cells were lysed in SDS sample buffer. Separated proteins by electrophoresis were then transferred onto nitrocellulose membrane. Then the membrane was incubated with rabbit anti-phospho SAPK/JNK (1:1000) or anti-JNK (1:1000) in Tris-Buffered Saline-Tween (TBS-T) with 5% bovine serum albumin at 4°C overnight. The membrane was washed three times with TBS-T and incubated with HRP-conjugated anti-rabbit IgG at room temperature for 1 h. The membrane was visualized with ECL western blotting detection regents (GE Healthcare Japan).

After the indicated treatments, total cell extracts were obtained and lysed by RIPA buffer (KeyGENBioTECH, Nanjing, China). Protein concentrations were determined according to BCA Protein Assay Kit (Beyotime, Shanghai, China). The extracted proteins in the cell lysates were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The primary antibodies were as follows: rabbit anti-EGFR monoclonal antibody (Cell Signaling technology, Danvers, MA, USA), anti-PTEN monoclonal antibody (Cell Signaling technology, Danvers, MA, USA), anti-p-AKT(Ser473) monoclonal antibody (Cell Signaling technology, Danvers, MA, USA), anti-AKT monoclonal antibody (Cell Signaling technology, Danvers, MA, USA), anti-cyclin D1 monoclonal antibody (Santa Cruz Biotechnology, Dallas, USA), anti-Bcl2 monoclonal antibody (Santa Cruz Biotechnology, Dallas, USA), anti-Bax monoclonal antibody (Santa Cruz Biotechnology, Dallas, USA), anti-CYP2E1 monoclonal antibody (Epitomics, CA, USA) and mouse anti-β-actin monoclonal antibody (BOSTER, Wuhan, China). Secondary antibodies include HRP-Conjugated AffiniPure Goat Anti-rabbit IgG (ZSGB-BIO, Beijing, China). Immunoreactive proteins were visualized using ECL western blotting detection regents (GE Health-care, Buckinghamshire, UK).

Whole cell lysates (5 μg) were electrophoresed through 10% SDS-PAGE gels and proteins were transferred to a polyvinylidene difluoride membrane using a semi-dry electroblot (Bio-Rad, Hercules, CA). The blot was probed with primary antibodies (anti-human SP1 antibody [#07-645; Millipore, Billerica, MA], anti-human GAPDH antibody [#ABS16; Millipore]) and secondary antibody (anti-rabbit IgG-Peroxidase [A9169; Sigma-Aldrich]), and then the binding to antibody was detected using ECL Western Blotting Detection Regents (General Electric Company, Fairfield, CT) and a Fuji image analyzer (LAS mini-3000; Fujifilm, Tokyo, Japan) according to the manufacturer's protocol.