Bax 2d2

Following quercetin treatment, HeLa and SiHa cells were lysed with RIPA buffer (Tris-HCl 50 mM, NaCl 150 mM, NP40 1%, sodium deoxycholate 0.5%, SDS 0.1%) containing protease inhibitor cocktail (Sigma, MO, USA). Protein concentration was determined by bicinchoninic acid method using Pierce^™ BCA Protein Assay kit (Pierce biotechnology, IL, USA). Equal protein amounts from all samples were loaded and resolved in SDS-PAGE 12% and transferred to PVDF membrane. The membranes were blocked with 5% milk in TBS-Tween-20 for 1 h, and then incubated overnight at 4°C with the following primary antibodies: p53 (DO-1, 1:1,000 dilution), HPV18 E6 (G-7, 1:200 dilution), HPV16/18 E6 (C1P5, 1:100 dilution), Bax (2D2, 1:500 dilution), Bcl-2 (C-2, 1:500), from Santa Cruz Biotechnology. The proteins were visualized using the ChemiDoc-XRS Imaging System (Bio-Rad Laboratories, Inc. CA, USA), using supersignal west femto maximum sensitivity substrate (Thermo Fischer Scientific, IL, USA) as a developer. GAPDH antibody primary (GA1R) from Santa Cruz Biotechnology was used as the loading control. The experiment was executed in triplicate and quantification was performed via densitometric analysis using ImageJ TM software. The densitometric data were normalized with the loading control. The level of the expression of the proteins was determined in function to the respective control.