Pvdf membrane

Cells were harvested and lysed with RIPA buffer (Cell signaling, 9806) in ice for 30 min and centrifuged at 12000× g for 10 min. Supernatant was collected and protein concentration was measured using BCA method (Thermo Fisher Scientific). Protein samples were boiled in 1x NuPAGE LDS sample buffer (Invitrogen) at 70℃ for 10 min. Twenty microgram protein samples were subjected to 7.5% - 15% gradient SDS-PAGE gel and transferred to PVDF membrane (MACHEREY-NAGEL, Germany). The membranes were blocked in 1x Roti-Block (Carlroth, Germany) at room temperature for 1 h and were then probed with specific primary antibodies overnight at 4°C. Blots were incubated with HRP-conjugated secondary antibody (Invitrogen, 31430 and 31460) for 1 h at room temperature. Bands were visualized by SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, USA) and detected using the ChemoStar ECL Imager (Intas Science Imaging, Germany).

Protein preparation and western blotting analysis were performed using standard methods. HeLa cells were inoculated into 6-well plates at a density of 1 ×10⁵ cells per well and incubated at 37 °C with 5% CO₂ for 24 h. VO(hntdtsc)NPIP at final concentrations of 0, 0.5, 1.0, and 2.0 μM were added to each well. After incubation for 24 h, the cells were digested with 0.25% trypsin and centrifuged for 5 min at 2000 rpm, then rinsed twice with ice-cold PBS, and lysed in 100.0 μl RIPA lysis buffer (Solarbio, R0010, China) containing 10% protease inhibitor for 30 min at 4 °C. The protein concentrations were determined by BCA protein assay kit (Solarbio, PC0020, China). Then, protein extracts (30 μg) were resolved in 8%–10% SDS-PAGE (SDS-PAGE; Bio-Rad, Hercules, CA, United States) and transferred to a polyvinylidene difluoride (PVDF) membrane (Macherey-Nagel). Subsequently, the PVDF membranes were blocked in 5% nofat milk for 1 h and incubated with primary antibodies Bax, Bcl-2, cytochrome c, cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, p16, cyclin D1, CDK4, p-Rb, and β-actin with gentle rotation overnight at 4 °C and were next incubated with the secondary antibody consisting of horseradish peroxidase (HRP) conjugated for 2 h. The enhanced chemiluminescent (NEN Life Science Products, Boston, MA, USA) detection system was used for immunoblot protein detection.

Cells grown in confluent monolayers were rinsed twice with cold PBS, re-suspended in lysis buffer (1% Triton X-100; 50 mM Tris pH 7,5; 150 mM NaCl; 0.5% Nonidet P-40) containing the cocktail of protease inhibitors (Roche) and cleared by centrifugation. Proteins were quantified using the BCA protein assay (Pierce). Protein extracts (100 μg/lane) were resolved in 10% SDS-PAGE and transferred to a PVDF membrane (Macherey-Nagel). The proteins were detected with appropriate antibodies and visualized using an enhanced chemiluminescence kit (GE Healthcare Bio-Science). Far Western blotting was performed as described in the legend to the Supplemental Figure S1 according to [55 (link)].