Polyvinylidene difluoride membrane

Total protein from cells or tissue was extracted using a protein extraction kit (Vazyme, Nanjing, China). Nuclear and cytoplasmic proteins were extracted according to the instructions of the Nuclear and Cytoplasmic protein extraction kit (Sangon Biotech, Shanghai, China). The protein was separated by SDS–PAGE (5–12%), transferred onto polyvinylidene difluoride membranes (Solarbio, Beijing, China) and blocked in 10% nonfat milk in TBST for 2 h at RT. The membranes were successively incubated with primary antibodies overnight at 4 °C. Following three washes with TBST, the membranes were then incubated with secondary antibodies at RT for 2 h. After three washes, the membranes were subjected to chemiluminescence using Clarity Western ECL Substrate (Affinity, Changzhou, China). β-actin and Lamin B1 were used as standard proteins for cytoplasmic and nuclear proteins, respectively. Protein expression was detected using an enhanced chemiluminescence detection system (ImageQuant LAS 4000 mini, USA). Antibody information is provided in Table S1.

Cells of logarithmic phase growth were plated in 50ml culture bottle. After incubated with DMEM for 24h, the cells were treated with Hcy or/ and NaHS at different concentrations for 48 h. Then the treated cells were resuspended in 100 uM cell lysis buffers and PMSF incubation on ice for 30 min. The supernatant was collected after centrifuged at 12,000 rpm for 10 min at 4˚C. The protein concentrations were detected by BCA method (Solarbio, Beijing, China). Equal amounts of total protein extracts were electrophoresed through 10% or 12% SDS-PAGE gel, then transferred to polyvinylidene difluoride membranes (Solarbio, China). After blocked with TBST (50 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.1% Tween-20) containing 5% BSA (Sigma, USA) for 2 h, the membranes were incubated with primary antibodies including monoclonal antibody for P16^INK4a, P21^CIPL, and SIRT1(dilution, 1:1,000) at 4˚C overnight. After washed with TBST for 5 min 5 times, the membranes were incubated with secondary antibodies including Goat anti-rabbit (Proteintech, USA) (dilution, 1: 5,000) at room temperature for 2 h. After washed for three times with TBST for 3 times, the membranes were visualized with Western Blotting Chemiluminescence Reagent (Solarbio, Beijing, China), followed by apposition of the membranes with autoradiographic films (Kodak, China). The expression of β-actin for each sample was used as a control.

As for cells and tumor tissue, protein was extracted using RIPA lysis buffer (P0013E, Beyotime, Shanghai, China) on ice. The protein concentrations were determined by the BCA Protein Assay Kit (P0012, Beyotime, Shanghai, China) according to the manufacturer’s instructions. After boiling for 10 min at 98 °C, protein samples with loading buffer were loaded for electrophoresis with an appropriate voltage for 3 h.
The gels were transferred onto polyvinylidene difluoride membranes (Solarbio, Beijing, China) at 300 mA for complete protein transfer. The membranes were blocked with 5% skim milk and then incubated with primary antibodies at 4 °C overnight. After being rinsed 3 times with PBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit/mouse IgG (Zhongshanjinqiao, Beijing, China) for 1 h at room temperature. The immunoreactive bands were visualized by the ECL chemiluminescence system (Bio-Rad, CA, USA) after spreading SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, MA, USA) for a few seconds.