Ecl western blotting analysis system

The cells were treated as mentioned above and lysed in cell lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing 1 μM phenylmethylsulfonyl fluoride, 1.5 μM pepstatin A, and 0.2 μM leupeptin. The samples were separated by 10% SDS-polyacrylamide gel electrophoresis and were then transferred to polyvinylidene fluoride membranes, which were blocked with 5% dried milk protein using the techniques listed below as described previously [19 (link)]. After blocking, the blots were incubated with primary antibodies. After being washed with TBST, the membranes were incubated at 37°C for 1 h in blocking buffer that contained the secondary antibodies and were visualized via a chemiluminescence ECL Western blotting analysis system (BD, USA). The protein levels were quantified using ImageJ software (NIH, USA) and are expressed as percentages of the control after normalization to the housekeeping protein β-actin.

Lysing of the cells was conducted in a cell lysis buffer (Beyotime, Shanghai, China) that contained 0.2 μM leupeptin, 1.5 μM pepstatin A, and 1 μM phenylmethylsulfonyl fluoride. The 5% nonfat milk was utilized to block proteins loaded onto a nitrocellulose membrane after they had been separated on an SDS-denatured polyacrylamide gel, followed by the incubation of membranes at 4°C over the night with rabbit primary antibodies (anti-HS3ST1, anti-cleaved caspase-3, anti-caspase-3, anti-cleaved caspase-8, anti-caspase-8,anti-GAPDH, anti-Bax, anti-Bcl-xl, anti-SPOP, anti-FADD, anti-p-p65, and anti-p65, Sigma-Aldrich, St. Louis, MO, USA). The following day, the membranes were rinsed before being incubated with secondary antibody (Sigma-Aldrich, St. Louis, MO, USA). Visualization was achieved utilizing a chemiluminescence enhanced chemiluminescence (ECL) western blotting analysis system (B&D, San Jose, CA, USA). After being normalized to GAPDH, the protein levels were determined utilizing ImageJ software (NIH, USA).

The cells were lysed in cell lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing 1 μmol/L phenylmethyl-sulfonyl fluoride, 1.5 μM pepstatin A and 0.2 μM leupeptin. Protein concentrations were quantified by the Bradford assay. The samples were separated by 10% SDS-polyacrylamide gel electrophoresis and were then transferred to a polyvinylidene fluoride membrane, which was blocked with 5% dried milk protein; after blocking, the blots were incubated with monoclonal antibodies. After being washed with TBST, the membranes were incubated at 37°C for 1 h in blocking buffer that contained the secondary antibodies, which included goat anti-rabbit IgG or rabbit anti-mouse IgG, and were visualized by a chemiluminescence ECL western blotting analysis system (BD, USA). The protein levels were quantified using ImageJ software (NIH) and were expressed as percentages of the control after being normalized with the housekeeping protein β-actin.