Rabbit anti gapdh

Animals were sacrificed after deeply anesthetized with isoflurane, total proteins from the lumbar enlargements of spinal cords were extracted by ice-cold Radio Immunoprecipitation Assay (RIPA) lysis. Proteins were separated on SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membrane (Merck Millipore, United States). After blocking with 5% nonfat milk, the samples were incubated with rabbit anti-β-actin (1:3000, Abcam, United States), rabbit anti-GAPDH (1:3000, Merck Millipore, United States), mouse anti-VGluT2 (1:2000, Abcam, United States), goat anti-Iba-1 (1:2000, Wako, Japan), rabbit anti-GFAP (1:2000, Abcam, United States), rabbit anti-BDNF(1:2000, Abcam, United States), or rabbit anti-TrkB (1:2000, Abcam, United States) at 4 °C overnight. The next day, the corresponding rabbit, goat, or mouse Horseradish peroxidase (HRP)-conjugated secondary IgG (1:20000, Jackson ImmunoResearch, United States) was incubated for 1 h at room temperature. The gray value of the protein on the membrane of the corresponding molecular size was detected using western blotting detection kit (WesternBright Sirius, Advansta, United States) and ChemiDoc XRS imaging system (Bio-Rad). Image Lab 3.0 system (Bio-Rad) was utilized to perform standardized analysis on the test results.

Gemcitabine (Hospira UK Limited) was dissolved in water to a concentration of 10 mg/ml. BAY 11-7082 (Calbiochem, La Jolla, CA) was dissolved in dimethyl sulfoxide, stored at −20 °C, and protected from light. The following antibodies were used in western blot analysis: Rabbit anti-Cleaved Caspase-3 (Asp175) (Ref.9661, Cell Signaling Technology, MA, USA), Rabbit anti-PARP (Cat. No.11835238001, Roche), Rabbit anti-GAPDH (Ref.ABS16, Merck Millipore, Germany), HRP-conjugated Goat anti-Rabbit (DakoCytomation, Denmark).

Proteins from hippocampal tissue were isolated from the phenol–ethanol supernatant saved from the RNA isolation using the TRIzol™ Reagent (Thermo Fisher) protocol, according to the manufacturer's directions. From neuronal cultures, protein extracts were obtained by lysing cells in lysis buffer (50 mM Tris‐pH: 7.5, 150 mM NaCl, 1% Triton‐X100, 1× Complete Protease Inhibitor Cocktail (Roche)). Typically, 10 μg of protein sample mixed with 4× Laemmli Sample Buffer (Bio‐Rad) were separated on a 10% SDS‐PAGE. After electrophoresis, proteins were transferred to a nitrocellulose membrane using a Trans‐Blot Turbo system (Bio‐Rad), according to the manufacturer's protocol. Membranes were blocked in 5% milk prepared in Tris‐buffered saline containing 0.1% Tween20 and incubated in primary antibody solution overnight. Antibody dilutions (rabbit anti‐CACNB2 (1:1,000, Abcam), rabbit anti‐GAPDH (1:2,000, Millipore), and mouse anti‐GFP (1:1,000, Novus Biological)) were prepared in blocking solution. Membranes were washed five times in 5% milk before incubation in HRP (horseradish peroxidase)‐conjugated secondary antibody prepared in blocking solution for 1 h. Following incubation, membranes were washed five times in TBS‐T, developed with the Clarity™ Western ECL Substrate (Bio‐Rad) according to manufacturer's guidelines, and visualized with the ChemiDocTM MP, Imaging System (BioRad).