Coomassie blue protein assay

Protein samples were isolated using radioimmunoprecipitation assay (RIPA) lysis buffer, followed by centrifugation at 12,000 ×g for 15 min at 4°C. After supernatant collection, the protein concentrations were evaluated by the Bio‐Rad Coomassie Blue protein assay (Bio‐Rad, Hemel Hempstead). Equal amounts of protein (40 μg) were separated on SDS‐PAGE, followed by Western blotting as previously described (Hao et al., 2019).

The expression of XPC in DPCs with XPC overexpression at P2, P3, and P7 was examined using real-time PCR and western blot 48 h after transfection as mentioned above. The nontransfected cells and cells transfected with vector served as control groups. Briefly, quantification of XPC and 18s mRNA was performed using ABI Prism 7000 sequence detection system (Applied Biosystems, NY, US). Primers used for detection were listed in Table 1. Delta-delta Ct method was performed to analyze the result. The total protein was measured by a Bio-Rad Coomassie Blue protein assay (Bio-Rad Laboratories, Richmond, CA, US). Proteins were detected with mouse monoclonal antibodies against human XPC (1 : 500, Abcam, MA, US) and GAPDH (1 : 1000 dilution, Santa Cruz Biotechnology, CA, US).

Western blot was performed as described previously [9 (link)]. Briefly, total protein of DPCs at P1 to P7 was measured by Bio-Rad Coomassie Blue protein assay (Bio-Rad Laboratories, Richmond, CA, USA), whereas only the representative results of P3 and P7 were presented in Results. Twenty micrograms of protein was diluted by 10% bromophenol blue and boiled before being separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The membranes were blocked in 5% low-fat milk at room temperature for 1 h, rinsed, and incubated with monoclonal antibodies mouse against human XPC (1 : 500 dilution, Abcam, MA, USA), Oct-4 (1 : 100, Chemicon, MA, USA), Sox2 (1 : 50, R&D System, MN, USA), c-Myc (1 : 50, Santa Cruz, CA, USA), or human GAPDH (1 : 1000 dilution, Santa Cruz, CA, USA) overnight at 4°C. After washing, the membrane was incubated with the HRP-conjugated secondary antibody (1 : 5000 dilution, Jackson, USA) at room temperature for 1 h. Immunoreactive proteins were then visualized by incubating membranes with electrogenerated chemiluminescence plus detection agents (GE Healthcare, USA).