Sc 8007

The proteins were separated in a sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and transferred onto a polyvinylidene fluoride membrane (Millipore, MA, USA) by wet transfer at 100 V. For immunodetection, the membrane was blocked for 1 h at room temperature with 5% bovine serum albumin, incubated with the primary antibody overnight at 277.15 K, and then incubated with the secondary antibody for 2 h at room temperature. The primary antibodies included anti-Tau (1:100; sc-32274, Santa Cruz Biotechnology Inc.), anti-HK1 (1:1,000; 19662-1-AP, Proteintech, Wuhan, China), anti-VDAC I (1:1,000; AF1027, Beyotime Biotechnology), anti-Bax (1:200; sc-7480, Santa Cruz Biotechnology Inc.), anti-PARP-1 (1:200; sc-8007, Santa Cruz Biotechnology Inc.), anti-caspase-3 (1:1,000; 9662S, Cell Signaling Technology, MA, USA), and anti-β-actin (E-AB-48018, Elabscience, Wuhan, China). The secondary antibodies included anti-mouse secondary antibody (HS201-01, TransGen Biotech, Beijing, China) at a 1:1,000 dilution or anti-rabbit secondary antibody (HS101-01, TransGen Biotech) at a 1:1,000 dilution. The signals were detected via chemiluminescence (Tanon, Shanghai, China).

Protein lysates (20-25μg) were denatured using 1X Laemmli Buffer and run on 7.5, 10, or 16% SDS PAGE gels at 100V for about 2 hours. Proteins were then transferred onto a PDVF membrane (BioRad or Thermo Fisher Scientific) at 100V for 1 hour. Ponceau-S staining was done to verify transfer. Non-specific binding was blocked by incubating the membrane for at least one hour at room temperature or overnight at 4°C in 5% milk in TBS for non-phosphorylated proteins, or 5% Bovine serum albumin (BSA Fraction V, Fisher Scientific) in TBS for phosphorylated proteins. The membrane was then blotted with primary antibody diluted in 5% BSA or Milk in TBS supplemented with 0.1% Tween 20 (TBST). After an overnight incubation at 4°C, the membrane was washed with TBST and incubated in the secondary antibody for at least 2 hours at room temperature in 5% BSA or Milk in TBST. After washing out the secondary antibody in TBST, the membrane was exposed to BioRad's Clarity Western ECL substrate according to manufacturer's instructions. Signal was detected using UltraCruz Autoradiography Film (Santa Cruz Biotech). Antibodies for GAPDH and p-IκBα were obtained from Cell Signaling (Catalog # 14C10 and 14D4, respectively). BCL2α, Pro-caspase 3, PARP-1, and cytochrome c antibodies were obtained from Santa Cruz Biotech Catalog # SC-7382, SC-7148, SC-8007, and SC-271627, respectively.

Cells were lysed in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, and 1% sodium deoxycholate) in the presence of 1× protease and phosphatase inhibitor cocktails (Sigma-Aldrich, MO, USA). Proteins were resolved by SDS-PAGE and transferred to Hybond ECL membrane (GE Healthcare, CO, USA). The membrane was immunoblotted with antibodies against Poly(ADP-ribose) 10H (ALX-804-220-R100, 1:400, Enzo Life Sciences, NY, USA), PARP1 (sc-8007, 1:1000, Santa Cruz Biotechnology, TX, USA), MYCN (sc-53993,1:250, Santa Cruz Biotechnology) and TUBB (T8328, β-tubulin; 1:5000, Sigma-Aldrich), each diluted in 5% milk and incubated at 4°C overnight. After the application of the appropriate HRP-conjugated secondary antibody and further washes, the immunoreactive protein was visualised using ECL reagents (GE Healthcare, IL, USA) according to the manufacturer’s instructions.