Gel detection system

RNA isolation from wt and PTX-res MCF-7 cells were performed using TRIPure (Roche, USA) according to the manufacturer’s indications. cDNA was synthesized from total by using iScript cDNA Synthesis Kit (Bio-Rad, USA). Twist, 18S rRNA genes were amplified using synthesized cDNA by RT-PCR (Mini Thermal Cycler, Bio-Rad, USA). Then, electrophoresis was performed at 1.5% agarose gel, visualized by Gel Detection System (Bio-Rad, USA).

To evaluate Hexon/E7 protein expression levels of Ad4-HPV16E7 virus, HEK293T cells were infected with recombinant virus Ad4-HPV16E7 and Ad4 (control group) respectively for 24 h, without viral infection were used as a blank control group, then the cell total protein was extracted with a cell lysis buffer (120 mM NaCl, 0.5% NP-40, 50 mM Tris–HCl pH 8.0, and 1 mM PMSF) and evaluated by BCA methods. Whole cell extracts (30–45 μg) were eparated by 12% SDS-PAGE and then transferred to NC membranes and incubated with 5% skim milk in TBST at room temperature for 1.5 h. After that, the membranes were probed with primary antibodies at a 1:200–1:500 dilution overnight at 4°C: Rabbit anti-HPV 16 E7 antibody (cat. no. 67017-1-Ig; ProteinTech Group, Inc., Chicago, IL, USA), mouse anti-Hexon antibody (generated in our laboratory), and mouse anti-GPDH antibody (cat. no. 4970; Cell Signaling Technology, Inc., Beverly, MA, USA), then washed with TBST and incubated with goat anti-rabbit (1:5,000; Sigma)/mouse (1:10,000; Sigma) horseradish-peroxidase conjugated secondary antibody for 1 h at room temperature for 2 h. Detection was performed by Gel Detection System (Bio-Rad, USA). Western blotting bands were quantified using ImageJ software.³

After the treatments, the cells were gently washed with PBS twice and total proteins were extracted and quantified as described above. Equal amounts of proteins were separated by sodium dodecyl sulfate gel electrophoresis and subsequently transferred to polyvinylidene difluoride membranes. After blocking with a PBST buffer containing 5% skim-milk at room temperature for 1 h, the blots were incubated with the indicated primary antibodies at 4 °C overnight. After three washes with tris-buffered saline, the blots were incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h. The antibodies used in this study are listed in Table S2. Immunoreactive bands were detected using an enhanced chemiluminescence kit (ECL-Plus; Thermo Fisher Scientific) and imaged using a Bio-Rad gel detection system.

The protocols of Western blot in this study were adapted from previous research [28 (link)]. Briefly, frozen LT muscles were ground to powder with liquid nitrogen to extract total protein using RIPA lysis buffer (Beyotime Biotech Inc, Shanghai, China). The concentrations of the protein samples were determined using the Pierce BCA protein Assay kit (Cat. No. 23227, Thermo-Fisher-Scientific, Waltham, MA, USA). Subsequently, the protein samples were loaded for SDS-PAGE. Proteins were then transferred to the PVDF membrane. β-actin protein was used as a control for equal protein loading. PVDF membrane was blocked by skimmed milk for one hour before incubation with primary antibody (anti-PGC1-α, Cat. No. 2178S, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. Subsequently, the PVDF membrane was incubated with secondary antibody at 4 °C for an hour. Finally, visualization was performed using the ECL kit and gel detection system (Bio-Rad, Hercules, CA, USA). Targeted bands were quantified using Image J software (NIH, Bethesda, MD, USA).