Polyvinylidene fluoride pvdf membrane

YQFM was purchased from Tasly Pharmaceutical Co., Ltd. (Tianjin, China, batch number 20121210). Dantrolene (Dan) was purchased from Sigma (St. Louis, MO, USA). Hoechst 33342 (bisbenzimide) and enhanced chemiluminescence (ECL) reagents were obtained from Beyotime Biotechnology (Shanghai, China). Polyvinylidene fluoride (PVDF) membranes were purchased from Millipore (Bedford, MA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Ameresco (Ameresco, OH, USA). The primary antibodies, horseradish peroxidase- (HRP-) conjugated goat anti-rabbit and anti-mouse IgG, were purchased from Bioworld Technology, Inc. (St. Louis Park, MN, USA).

Western blot analysis was performed to determine the expression levels of various proteins in cells. Cells were treated with RY-2f or DMSO at different concentrations for 24 h. Cells were harvested, washed with cold 1 × PBS, and lysed with RIPA lysis buffer (Beyotime) for 30 min on ice, then centrifuged at 12,000 g for 15 min at 4°C. The total protein concentration was determined by BCA protein assay kit (Beyotime). Equal amounts (30 μg per load) of protein samples were subjected to SDS-PAGE electrophoresis and transferred on to polyvinylidene fluoride (PVDF) membranes (Millipore). The blots were blocked in 10% non-fat milk, and incubated with primary antibodies, followed by incubation with secondary antibodies conjugated with horseradish peroxidase (HRP). The protein bands were developed with the chemiluminescent reagents (Millipore). Antibodies to p21, Bcl-2, Bad, Bax, cyclin A, cyclin B1, CDK2, pAKT(Ser437), AKT, PI3K (p110 α), mTOR, PTEN were from Santa Cruz Biotechnology. The Antibody to cleaved-PARP-1 was purchased from Cell Signaling Technology. The antibody to β-Actin was purchased from Sigma-Aldrich.

Cell lysates were prepared in RIPA buffer (Solarbio, Beijing, China) containing phenylmethylsulfonyl fluoride (PMSF). Lysates were incubated on ice for 30 min and centrifuged at 12,500 g for 10 min at 4 °C. Protein concentrations of the supernatants were determined with BCA protein assay kit (ThermoFisher Scientific, Waltham, MA, USA). Protein samples were denatured using Loading buffer (with DTT). Proteins were then separated on precast 10% PAGE gels (Yeason, Shanghai, China) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Boston, MA, USA). After blocking in blocking buffer (5% skim milk in TBS with 0.5% Tween-20), membranes were incubated with the primary antibody (listed in the Supplementary materials and methods) overnight at 4 °C. Then the membranes were washed three times with TBST buffer, incubated with the HRP-conjugated appropriate secondary antibodies for an hour at room temperature. Chemiluminescent detection was performed using a ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA) after adding ECL solution (ThermoFisher Scientific) on the membrane.

Striatal tissues were collected in cold lysis buffer containing 1% Triton X-100, 1 mM EDTA in phosphate-buffered saline (PBS), protease inhibitor cocktail, homogenized, and centrifuged at 10,000 × g for 10 min at 4°C. Protein concentration was determined using a BCATM protein assay kit (Thermo Fisher Scientific, Waltham, MA, United States) and assessed by Western blotting. Equal aliquots of the samples were denatured at 100°C, separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and blotted onto polyvinylidene fluoride (PVDF) membranes (Millipore Corporation, Billerica, MA, United States). Membranes were incubated in a blocking buffer containing 5% BSA in TTBS for 1-h at room temperature. Immunodetection was performed by incubating membrane blots overnight at 4°C separately with the following primary antibodies (1:1000): anti-CRMP-2 (IBL, Gunma, TS, Japan), anti-Cdk5-p35/25, anti-calpastatin, and anti-synapsin-II (Cell Signaling, Dallas, TX, United States). For chemiluminescent detection, membrane blots were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000) for 2-h at room temperature. Data collection and processing of the integrated optical density of the bands were performed with a luminescent image analyzer (LAS-3000) and IMAGE GAUSE software (Fuji Photo Film, Japan).

Materials were purchased respectively as follows: EGM-2 medium kit from Lonza Cambrex (Nottingham, UK), enhanced chemiluminescence (ECL) reagent from AbClon (Seoul, South Korea), Griess reagent from Promega Co. (WI, USA), LeGene Premium Express 1st Strand cDNA Synthesis System from LeGene Biosciences (CA, USA), polyvinylidene fluoride (PVDF) membranes from Millipore (MA, USA), pyridine-d5 from Cambridge Isotope Laboratories Inc. (MA, USA), RNAiso PLUS from TAKARA Korea Biomedical Co. (Seoul, South Korea), thin-layer chromatography (TLC) silica gel 60 F₂₅₄ from Merck (Darmstadt, Germany), and TOPreal™ qPCR 2× PreMIX SYBR green from Enzynomics (Seoul, South Korea). N(G)-nitro-L-arginine methyl ester (L-NAME), fetal bovine serum (FBS), and silica gel resin were purchased from Sigma-Aldrich (MO, USA). All other chemicals were of ultra-pure grade. The primary antibodies (eNOS, phospho-eNOS Ser¹¹⁷⁷, Akt, phospho-Akt Thr³⁰⁸, and GAPDH) and horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit and anti-mouse) were obtained from Merckmillipore (CA, USA). All other chemicals were of ultra-pure grade.

For FUNDC1 and DRP1 immunoprecipitation, cell extracts were prepared using IP lysis buffer (50 mM Tris-HCl at pH 7.4, 150 mM NaCl, 0.5% Nonidet P-40, 10% glycerol, and 1 mM EDTA) and fresh protease inhibitors. The lysates were precleared by centrifugation for 10 min. Ten percent of the supernatants were reserved for measuring protein input. The remaining lysate was incubated with IgG-coupled magnetic beads (Pierce™ Protein G Magnetic Beads) overnight at 4 °C on a rotator wheel. Following five washes with lysis buffer, the immune complex beads were heated at 100 °C in 1× loading buffer (75 mM Tris-HCl at pH 6.8, 10% glycerol, 2% SDS, 0.05% bromophenol blue, and 2.5% β-mercaptoethanol) prior to being loaded on 12% SDS-PAGE gels. After electrophoresis, the separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA) in transfer buffer (25 mM Tris, 0.192 M glycine, and 20% methanol), and the transferred membranes were immunoblotted with antibodies.