Rabbit monoclonal antibody against human sod2

Cells were seeded in a 6-cm culture plate for 12 h and treated with CAM and DDP separately or combined for 48 h. Then cells were collected and lysed with RIPA lysis buffer. Cellular proteins were quantified using a DC Protein Assay Kit (Bio-Rad, Richmond, CA, USA). Next, 50 μg of the extracted proteins were loaded onto a 10% SDS-PAGE gel and transferred onto a PVDF membrane (Millipore Corp., Bedford, MA, USA)). First, the membrane was blocked with 5% BSA for 1 h and then incubated overnight at 4 °C with primary antibodies including rabbit monoclonal antibody against human activated caspase-3 (1:1000 dilution; Cell Signaling Technology, Inc., Beverly, MA, USA), rabbit polyclonal antibody against human cleaved-PARP (1:1000 dilution; Cell Signaling Technology, Inc., Beverly, MA, USA), and rabbit monoclonal antibody against human SOD2 (1:800 dilution; Proteintech Group, Inc., Wuhan, China). GAPDH was used as an internal control (rabbit monoclonal antibody against human GAPDH, 1:2000; Cell Signaling Technology, Inc., Beverly, MA, USA). Chemiluminescence was detected using an imaging system (Bio-Rad, Hercules, CA, USA).

Clarithromycin (CAM) was purchased from Selleck Chemicals (Houston, TX, USA); it was dissolved in dimethyl sulfoxide (DMSO). DDP was obtained from Sigma-Aldrich (St. Louis, MO, USA); it was dissolved in sterile filtered RNA-free water. N-acetyl cysteine (NAC) was purchased from Beyotime Biotechnology (Shanghai, China). Antibodies used for analysis included the following: primary rabbit polyclonal antibody against human cleaved-PARP (Epitomics, Burlingame, CA, USA), rabbit monoclonal antibody against human activated caspase-3 (Cell Signaling Technology, Inc., Beverly, MA, USA), rabbit monoclonal antibody against human SOD2 (Proteintech, Wuhan, China), goat monoclonal antibody against human 8-OHdG (Millipore, Bedford, MA, USA), and mouse monoclonal antibody against human γ-H2AX (Millipore, Bedford, MA, USA). The secondary antibody for GAPDH was obtained from Santa Cruze Biotech (Santa Cruz, CA, USA).