Nestin

The cells were harvested in radioimmunoprecipitation assay buffer and lysed on ice. From each sample, equal amounts of protein were employed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to PVDF membranes. After being blocked with 5% nonfat milk/TBST, the membrane was successively incubated with the appropriate primary antibody and a secondary antibody. Every incubation was followed by 3 washes with TBST. Protein signals were determined using a chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA, USA). Primary antibodies: MSH6, N-cadherin, Vimentin, E-cadherin, p-AKT, p-STAT3, p-Smad2/3, p-ERK, p-p38, p-JNK, p-p65, STAT3, Smad2/3, ERK, p38, JNK and p65 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA); Ki67, Nestin, CD133, SOX2, Cyclin D1, Bax, Bcl-2, MMP2, MMP9, HIF1A, VEGFA, Snail, Slug, Twist, ZEB1, ZEB2, AKT, TGFB1 and β-actin antibodies were supplied by Proteintech (Wuhan, Hubei, China); CXCR4 antibody was obtained from Abcam (Shanghai, China). Secondary antibodies: horseradish peroxidase (HRP)-conjugated affinipure goat anti-mouse IgG and HRP- conjugated affinipure goat anti-rabbit IgG were purchased from Proteintech (Wuhan, Hubei, China).

Antibodies for glial fibrillary acidic protein (GFAP), Nestin, and Caspase-1 were from Proteintech (Chicago, Illinois, USA). Antibodies against interleukin-1β (IL-1β), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and horseradish peroxidase conjugated antirabbit and mouse IgG were acquired from Cell Signaling Technology (Boston, Massachusetts, USA). Anti-Claudin-1 and anti-Synapsin-1 were purchased from Bioworld (Dublin, Ohio, USA), whereas anti-Occludin was from Thermofisher (USA). Anti-NLRP3 (nucleotide-binding domain-like receptor protein3) and anti-P2Y12 receptors were purchased from NOVUS (Cambridge, UK) and Abcam (Cambridge, UK), respectively.

Total protein was extracted from cells using ice-cold radioimmunoprecipitation assay (RIPA) buffer (Sevenbio, Beijing, China) supplemented with protease inhibitors (10 mg/mL aprotinin, 10 mg/mL phenyl-methylsulfonyl fluoride [PMSF], and 50 mM sodium orthovanadate). The BCA protein assay kit (Beyotime Institute of Biotechnology) was used to determine the protein concentration of the supernatant. Equal amounts of protein samples (30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrically transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore, Shanghai, China). Non-specific binding was blocked by incubation with 5% fat-free milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) at room temperature for 2 h.
The membranes were subsequently incubated with primary antibodies as follows: Nestin (1:500; Proteintech, Chicago, IL, USA), P21 (1:500; Proteintech), Cyclin D1 (1:5000; Proteintech), and GAPDH (1:3000; Affinity, Cincinnati, OH, USA) at 4 °C overnight. The membranes were washed and incubated with HRP-conjugated secondary antibodies (Sevenbio, Beijing, China), diluted at 1:3000 at room temperature for 2 h. Immunoblots were visualized using an enhanced chemiluminescence kit (ECL; Sevenbio, Beijing, China) and detected by Bio-Rad Chemi-Doc (Bio-Rad, CA, USA) and then normalized to that of GAPDH or β-actin.