Tris buffered saline tween 20 tbst

Total proteins were extracted from SK-N-SH and SK-N-BE cells using RIPA lysis buffer (Beyotime, Shanghai, China). Equal amount (20 µg) of total proteins were separated by gel electrophoresis with 12% SDS-polyacrylamide gel (SDS-PAGE), and then transfected onto the PVDF membranes (polyvinylidene fluoride; Millipore, Bedford, MA, USA). Subsequently, membranes were incubated with Tris-Buffered Saline Tween-20 (TBST; Solarbio, Beijing, China) supplemented with 5% bovine serum albumin for 1 h at room temperature. Then the membrane was cultivated overnight at 4 °C with primary antibodies purchased from Abcam (Abcam, Cambridge, MA, USA) which was diluted in TBST solution. The primary antibodies used in this research were shown as below: Cleaved-caspase 3 (Cleaved-casp-3; 1:500, ab13847), B-cell lymphoma-2 (Bcl-2; 1:1000, ab32124), Bcl-2-Associated X (Bax; 1:7000, ab32503), GPNMB (1:100, ab227695), PCNA (1: 1000, ab18197) and GAPDH (1:1000, ab8245). Subsequently, the membranes was washed with TBST for three times and then incubated with corresponding secondary antibody Goat Anti-Rabbit IgG H&L (1: 20,000, ab205718, Abcam) for 1 h at room temperature. Then the bands were visualized using High sensitivity ECL chemiluminescence detection kit (Vazyme, Nanjing, China) and quantified by Quantity One software version 4.6 (Bio-Rad Laboratories). GAPDH was used as the internal control.

OPCs (CAS#: 4852-22-6) extracted from grape seeds were provided by Shanghai Yuanye (China). LPS and dexamethasone (DEX) were provided by Macleans (China). MAC-T cells were purchased from BMCC (China). Nuclear protein extraction kits and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazole bromide (MTT) were purchased from Solarbio (China). Fluorescence quantification kits were provided by Takara (China). Primary antibodies against phosphorylated p65 (p-p65), p-ERK1/2 p-IκBα, p65, IκBα and ERK1/2 were obtained from Bioss (China), and antibodies against JNK1/2, p38, p-JNK1/2, and p-p38 were from Wanleibio (China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (China). NF-κB Activation Nuclear Transport Test Kit and ELISA Kit were supplied by Bevotime (China) and Sinobestbio (China), respectively. Tris-buffered saline + Tween 20 (TBST) was supplied by Solarbio.