Phototope hrp chemiluminescent substrate system

OCI-ly8 cells were treated with different concentrations of BM-1197 (0, 0.25, 0.5, 1, 2 μM) for 24 h or with 2 μM BM-1197 for 0, 1, 3, 6, 12 and 24 h. After that, cells were collected and washed twice with cold PBS and lysed in 1 × cell lysis buffer, which was diluted from 10 × cell lysis buffer (Cell Signaling Technology). Then, 1x proteinase inhibitor cocktail was added in the lysis buffer. Lysates were centrifuged at 12000 g at 4 °C for 20 min. Supernatants were collected and stored at − 80 °C until used. The protein concentration of the supernatants was determined using BCA protein assay reagents. The relevant primary antibodies used included: Bcl-2, Bax (6A7) and PARP-1 polyclonal antibodies purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and anti-Mcl-1, PUMA, Bcl-xl, caspase 3, Caspase 9, cytochrome c (cyt c), and GAPDH monoclonal antibody purchased from Cell Signaling Technology (Danvers, MA). The COXIV polyclonal antibody was purchased from Biyun Tian Biotechnology Research Institute. Mouse and rabbit secondary antibodies were from Cell Signaling Technology (Danvers, MA). Antigen-antibody complexes were detected using the PhototopeTM-HRP Chemiluminescent Substrate System (Cell Signaling Technology) per the manufacturer’s instructions.

OCI-ly8 cells were treated with different concentrations of BM-1197 (0, 0.25, 0.5, 1, 2 μM) for 24 h or with 2 μM BM-1197 for 0, 1, 3, 6, 12 and 24 h. After that, cells were collected and washed twice with cold PBS and lysed in 1 × cell lysis buffer, which was diluted from 10 × cell lysis buffer (Cell Signaling Technology). Then, 1x proteinase inhibitor cocktail was added in the lysis buffer. Lysates were centrifuged at 12000 g at 4 °C for 20 min. Supernatants were collected and stored at − 80 °C until used. The protein concentration of the supernatants was determined using BCA protein assay reagents. The relevant primary antibodies used included: Bcl-2, Bax (6A7) and PARP-1 polyclonal antibodies purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and anti-Mcl-1, PUMA, Bcl-xl, caspase 3, Caspase 9, cytochrome c (cyt c), and GAPDH monoclonal antibody purchased from Cell Signaling Technology (Danvers, MA). The COXIV polyclonal antibody was purchased from Biyun Tian Biotechnology Research Institute. Mouse and rabbit secondary antibodies were from Cell Signaling Technology (Danvers, MA). Antigen-antibody complexes were detected using the PhototopeTM-HRP Chemiluminescent Substrate System (Cell Signaling Technology) per the manufacturer’s instructions.