Goat anti rabbit secondary antibodies

The day before assay, RAW264.7 macrophages (5.0 × 10⁵ cells/well) were seeded in 6-well plates. Cells were left untreated or exposed to 1.25, 2.5 nM CdSe/ZnS QDs for 48 h. The medium was removed and cells were washed three times with PBS. Protein was extracted by the cold mammalian protein extraction reagent (Thermo, USA) according to the manufacturer’s instructions. A total of 15 μg proteins were resolved by 12 % SDS-PAGE and transferred to 0.22 μm polyvinylidene difluoride membranes (Millipore, USA). Membranes were soaked in blocking buffer (Skim Milk, China) for 1 h at room temperature and incubated with rabbit antibodies of GAPDH (1:1000, ABclonal, USA), Caspase-9 (1:1000, Proteintech, China), Caspase-3 (1:1000, Proteintech, China) at 4 °C overnight. Membranes were washed with TBST (10 mM Tris–HCL, 150 mM NaCl, and 0.05 % Tween 20) three times at 10 min interval. Membranes were incubated with goat anti-rabbit secondary antibodies (1:1000, ABclonal, USA) for 1 h. Then the membranes were washed three times with TBST. Protein bands were visualized with clarity western ECL substrate (BioRad, USA) and the pictures were obtained by chemiscope western blot imaging system (Clinx science Instruments, China).

Total protein was extracted from the tissues and cell lines using tissue extraction reagent and cell extraction buffer (Beyotime) purified and assessed qualitatively by Western blot (WB) analysis. Total protein extracts (30 µg) were shifted to 12% gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels to separate the proteins by different molecular weights and transferred onto nitrocellulose membranes for antigen–antibody reaction. The membranes were then blocked with 5% skimmed milk in TBST for 1 h and mixed with primary antibodies against RELA (1:1,000; ABclonal), Bax (1:1,500; ABclonal), Bcl2 (1:1,000; ABclonal), caspase 3 (1:1,000; ABclonal), cleaved caspase 3 (1:1,000; ABclonal), EGFR (1:1,000; ABclonal), CCND1(1:1,000; ABclonal), and GAPDH (1:1,000; ABclonal) and then incubated with goat anti-rabbit secondary antibodies (ABclonal) for 2 h at 37°C (Jiang et al., 2018 (link)).