Pvdf membrane

Treated samples were denatured and separated by SDS-polyacrylamide gel electrophoresis (Zhonghuihecai, Xian, China, PE008) and then transferred to PVDF membrane (Roche, Basel, Switzerland, 3010040001). The membrane was incubated with TBST containing 5% skimmed milk for 1 h at room temperature and reacted with the His monoclonal antibody (Abbkine, Wuhan, China, A02050) for 1 h. The membrane was incubated HRP-labeled secondary antibody. The signal was detected using superlumia ECL plus HRP substrate kit (Abbkine, Wuhan, China, K22030) according to manufacturer’s instruction.

Liver samples were snap-frozen and stored in liquid nitrogen until analysis. Liver proteins were prepared in Ripa buffer (supplemented with cOmplete™, EDTA-free Protease Inhibitor Cocktail tablets (Roche, Basel, Switzerland)) using an ice-jacketed Potter-Elvehjem homogenizer (1300 rpm; Thomas Scientific, Swedesboro, NJ, USA) according to our standard protocol [13 (link)]. Proteins (15 μg/μL) were resolved by SDS-PAGE and transferred onto a PVDF membrane (Roche, Basel, Switzerland). Membranes were blocked and incubated with primary antibodies (Supplementary Table S2) overnight at 4°C. For chemiluminescence detection, an appropriate horseradish peroxidase (HRP)-conjugated secondary antibody was used. AmidoBlack (Sigma Aldrich, St. Louis, MO, USA) was used for total protein normalization. The Alliance 4.7 Imaging System (UVITEC, Cambridge, UK) was used for the detection of immunoblots using an enhanced chemiluminescence kit (Thermo Fischer Scientific, Waltham, MA, USA).

Radio-immunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) was used to extract proteins from the cells and the PSG. The lysate was divided evenly and then centrifuged for 5 min at 12,000× g. Protease inhibitors were then added to the supernatants. Protein concentrations were measured using a bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China). The proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Roche, Basel, Switzerland). The PVDF membrane was blocked using 5% skimmed milk overnight at 4 °C and incubated with a primary antibody against BmJHBPd2 (1:10,000) for 2 h at 37 °C. After washing the PVDF membrane six times at 5 min intervals, the membranes were incubated with the secondary antibody goat anti-rabbit IgG (1: 20,000), labeled with horseradish peroxidase (HRP) (Sigma-Aldrich, St. Louis, MO, USA), and visualized with SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, USA) using the automatic exposing pattern of Genome XRQ (Gene Company, Hong Kong, China).

Total protein (tissue samples and cells) was extracted with the RIPA buffer containing a protease inhibitor cocktail (Roche, Basel, Switzerland). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (10%, SDS-PAGE, Sangon Biotech, Shanghai, China) was conducted to isolate total protein. Next, the isolated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Roche) and then blocked with tris buffered saline tween (TBST) buffer containing 5% skim milk. After washing with TBST, the membranes were incubated with primary antibodies at 4 °C overnight, including anti-Cleaved PARP (#9541, 1:1000), anti-B-cell lymphoma/leukaemia-2 (Bcl-2) (#4223, 1:1000), anti-Bcl-2-associated x (Bax) (#2772, 1:1000), anti-KIF2A (ab197988, 1:200, Abcam, Cambridge, MA, USA), and anti-β-actin (#4967, 1:1000). Subsequently, the membranes were incubated with goat anti-rabbit IgG (#7077, 1:2000). Protein bands were visualized with an ImmunoStar LD (Wako Pure Chemical, Osaka, Japan). β-actin was used as a loading control. All antibodies were purchased from Cell Signaling Technology (Santa Cruz, California, USA), except for KIF2A.

A549 cells were treated with various concentrations of CANE (25, 50, and 100 µg/ml) for 24 h, followed by protein isolation using RIPA lysis buffer. An equal amount of protein lysate (50 μg in each lane) was separated in reducing polyacrylamide gel and further transferred into a polyvinyldenefluoride (PVDF) membrane (Roche Diagnostics, Indianapolis, IN, USA) by electroblotting. The membrane was subsequently probed with appropriate primary antibodies, followed by horseradish peroxidase (HRP)-conjugated secondary antibody, and visualized by enhanced chemiluminescence (ECL) according to the recommended procedure (Amersham Pharmacia, Piscataway, New Jersey).