Bca protein quantification assay

The cellular thermal shift assay (CETSA), as described previously (Guo et al., 2020 (link)), was employed to evaluate the binding of genistein and viral RPO30 protein in living cells. In brief, HEK293 cells were, respectively, transfected with pCMV-N-HA-ORFV RPO30 or pCMV-N-HA-ORFV RPO35 plasmid using LipoFiter 3.0 (Hanbio, China) for 40 h and exposed to 100 μM genistein or DMSO at 37°C with 5% CO₂ for 2 h. Subsequently, the cells were washed with PBS to remove excess drugs, harvested, and then lysed on ice for 30 min in RIPA lysis buffer. Protein concentrations were determined by the BCA protein quantification assay (Beyotime, China). Protein solutions were adjusted to 1 mg/mL, and 30 μL aliquots of supernatants were added to a Thermal Cycler (Bio-Rad, USA), heated at different temperatures for 5 min, and then chilled at room temperature for 3 min. After centrifugation at 13,000×g for 30 min at 4°C, supernatants were transferred to new tubes, separated by SDS-PAGE on 10% gels, transferred to PVDF membrane, and incubated with primary antibodies HA tag rabbit polyclonal antibody (Proteintech, China) or GAPDH/β-actin mouse monoclonal antibody (Proteintech, China) and appropriate secondary antibodies.

Mouse cochleae and HEI-OC1 cells were lysed in cold RIPA buffer (Beyotime) with a protease and phosphatase inhibitor cocktail (MCE) on ice for 30 minutes. Samples were centrifuged at 12,000 g for 10 minutes, and the supernatants were stored at -80°C for later use. Protein concentrations were qualified by BCA protein quantification assay (Beyotime). A total of 50 micrograms of protein for each sample were loaded into 10% or 12% SDS–PAGE gels, which were prerun at 70 V and then at 100 V. The proteins were next transferred to PVDF membranes using the Trans-Blot Turbo transfer system (Bio-Rad). The membranes were blocked with 5% BSA or milk in tris-buffered saline containing 0.05% Tween-20 (TBST) (pH 7.4), incubated with the diluted primary antibody in 5% BSA at 4°C overnight, and then probed with the appropriate secondary antibody at room temperature for 1 hour. Detected proteins were visualized via the chemiluminescence method or performed with an Odyssey CLX Imaging System (LI-COR).