α tubulin

For Western blot analysis, total protein was prepared from cell lines. Immunoblotting were performed according to standard procedures with p53 (sigma and Cell signaling), Bad, Bax, Bcl-2, VDAC and NDRG2 (Cell Signaling), α-tubulin and β-actin (Boster) at room temperature for 1 hour and then at 4°C overnight. All blots were detected using the enhanced chemiluminescence (ECL) detection method.

Western blotting was carried out as previously described [24 (link)]. The primary antibodies were purchased from Bethyl Laboratories (QKI), Cell Signaling Technology (E-cadherin, N-cadherin, Vimentin, Snail, Slug, p-β-catenin (Ser33/37/Thr41) and p-GSK3β (Ser9)), Santa Cruz Biotechnology (β-catenin, α-tubulin, β-actin, c-myc, cyclin D1, MMP2, DKK3, DVL1, DVL3, GSK3β), Boster (GAPDH) and Sigma (flag).

Whole-cell lysates were prepared with RIPA lysis buffer of appropriate volume. Proteins were separated by 10% SDS-PAGE gel and transferred to PVDF membrane, following membrane blocking with 5% Albumin from bovine serum (BSA). Then the membranes were incubated in primary antibodies against targeted protein at 4 °C overnight, and after further incubated with HRP-conjugated secondary antibody, the bands were scanned. Antibodies used in experiments included: anti-TGFBR2 (Cell Signaling Technology, USA), anti-EZH2 (Abcam, USA), anti–HIF1A (Cell Signaling Technology, USA), anti-HIF2A (Cell Signaling Technology, USA), and anti-H3K27me3 (Abcam, USA). All primary antibodies except for internal control were used at 1:1000 dilution. GAPDH and α-tubulin (Boster, China) were used as the internal control at 1:5000 dilution.

Cell lysates were separated on SDS–PAGE gels, and proteins were detected on nitrocellulose blots with enhanced chemiluminescence reagents (GE Healthcare). The following antibodies were used: TNF-α, diluted 1:500 (Beyotime, China); IL-6, diluted 1:500 (Beyotime, China); Mcl-1, p38 MAPK and phospho-p38 MAPK (1:1000, Cell Signaling, USA); Bax (1:500, Abcam, USA); complement C3 (1:500, Novus Biologicals, USA); α-tubulin (1:1000, Boster Biological Technology, China); and GAPDH (1:1000, bs-2188R; BIOSS, China). Membranes were blocked with 5% nonfat milk dissolved in Tris-buffered saline solution for 1 h, followed by overnight incubation with the primary antibody [26 (link)]. Detailed information about antibodies is shown in the attached list (Table 1).

Total tissue or cell lysates were prepared with a detergent lysis buffer. Western blot was performed using the indicated primary antibodies: NF-κB p65 (1:1000, Cell Signaling Technology, no. 8242, MA, USA), phospho-p65 (Ser536) (1:1000, Cell Signaling Technology, no. 3031, MA, USA), ERK (1:1000, Cell Signaling Technology, no. 9102, MA, USA), phospho-ERK (p44/p42) (Thr202/Tyr204) (1:1000, Cell Signaling Technology, no. 9102, MA, USA), GFAP (1:1000, Cell Signaling Technology, no. 3670, MA, USA), MMP9 (1:1000, Abcam, no. 38898, Cambridge, UK), NR2B (1:1000, Abcam, no. 65783, Cambridge, UK), phospho-NR2B (1:1000, Abcam, no. 3856, Cambridge, UK), IL-6 (1:1000, Abcam, no. 208113, Cambridge, UK), TNF-α (1:500, Santa Cruz Biotechnology, sc52746, Santa Cruz, CA), Iba1 (1:500, Santa Cruz Biotechnology, sc32725, Santa Cruz, CA), ZO-1 (1:1000, Thermo Fisher, no. 617300, US), Claudin-5 (1:1000, Thermo Fisher, no. 35-2500, US), PPARγ (1:1000, Proteintech, 16643-1-AP, US), anti-GAPDH (1:1000, Boster Biotechnology, BM1985, Wuhan, China), and α-tubulin (1:1000, Boster Biotechnology, BM3885, Wuhan, China). Each blot was repeated three times.

All the cells were gathered and total proteins were extracted using RIPA lysis buffer supplemented with protease inhibitor Cocktail (Roche, Switzerland). Protein concentrations were calculated by using BCA protein assay kit (Beyotime, China). Equivalent amounts of protein samples (50 μg) were separated by 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. Nonspecific binding was blocked by incubating the PVDF membranes with 5 % bovine serum albumin (BSA) (Sigma-Aldrich, USA) for 2 h at room temperature. The membrane was then incubated with primary antibodies included p53 (1/1000) (Cell Signaling Technology, USA), p21 (1/2000) (Cell Signaling Technology, USA), Cyclin D1 (1/2000) (Affinity, USA), CDK4 (1/1000) (Affinity, USA), CDK6 (1/2000) (Affinity, USA), E-cadherin (1/1000) (BD Biosciences), β-catenin (1/500) (Boster, China), Vimentin (1/500) (Boster, China), ZEB1 (1/1000) (Cell Signaling Technology, USA), GAPDH (1/500) (Boster, China) and α-tubulin (1/500) (Boster, China) at 4 °C overnight. After several washes, the membranes were incubated with corresponding secondary antibody and detected by enhanced chemiluminescence (ECL) assay kit (Millipore, USA).