Anti tgf β

The harvested PBMCs were washed with phosphate buffered saline (PBS) and lysed with a protease inhibitor cocktail in a radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China). Total proteins in the lysate were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bradford, MA, USA). Subsequently, the membranes were blocked with 5% non-fat milk in Tris-Buffered Saline Tween (TBST) (50 nM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.5) for 1 h at room temperature, followed by incubation with primary antibodies at 4°C for overnight. The primary antibodies were anti-VDR (1:1,000, Cell Signaling Technology, MA, USA), anti-TGF-β (1:1,000, Cell Signaling Technology, MA, USA), and anti-β-actin (1:1,000, Cell Signaling Technology, MA, USA), and anti-β-actin antibody was used as a control. The blots were washed with TBST three times and then incubated with secondary horseradish peroxidase-conjugated antibody (1:5,000 dilution, Biotechnology, Santa Cruz, CA, USA) for 4 h at room temperature. An enhanced chemo-luminescence detection kit (ECL Advance, UK) was used to expose the film and visualize chemiluminescence. Densitometry of all proteins was normalized against the loading control. Protein bands were calculated using the ImageJ software (NIH, Bethesda, MD, USA).

The SH-SY5Y cells were treated as mentioned above, and proteins were extracted with RIPA lysis buffer (Beyotime, Shanghai, China). The following antibodies were used: anti-GAPDH, anti-Bax, anti-ACY-1, anti-phospho-ERK1/2, anti-ERK1/2, anti-TGF-β, anti-Bcl-2, and anti-Bad (Cell Signaling Technology, Danvers, MA, USA). The samples were visualized using an enhanced chemiluminescence system, and the bands were analyzed with the Image J software.

Western blot was performed as previously described (20 (link),21 (link)). Cell proteins were extracted with RIPA lysis buffer (KeyGene Biotech, Nanjing, China), and the protein concentration was determined by BCA (KeyGene Biotech). After SDS-PAGE electrophoresis and membrane transfer, the membranes were blocked with 5% non-fat milk for 1 h and then incubated with primary antibody overnight at 4 °C. After incubation with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h, the membranes were visualized using enhanced chemiluminescence reagents (Beyotime). The primary antibodies used in the study included Bax, caspase-3, Bcl-2, E-cadherin, matrix metalloproteinase (MMP)-2, MMP-9, and β-actin (purchased from ProteinTech, IL, USA), anti-Snail, anti-vimentin, anti-N-cadherin, anti-β-catenin, anti-TGF-β, α-SMA, anti-p-Smad2, anti-Smad2, anti-p-Smad3, anti-Smad3, and anti-cleaved caspase-3 were purchased from Cell Signaling Technology (MA, USA). Anti-ATP1A2 was supplied by Abcam (Cambridge, UK).