Anti tgf beta

The following antibodies and materials were used: anti-GAPDH (Calbiochem), anti-EGFR (Millipore), anti-CREB, anti-phospho CREB (S133), anti-STAT3, anti-phospho STAT3 (Y705), anti-c-Jun (Cell signaling), and anti-TGF-beta (Abcam). Electrophoresis reagents were purchased from Biorad.

The chemical substances, antibodies, and reagents used herein were obtained from various sources. Dulbecco's modified Eagle medium (DMEM), phosphate buffer saline (PBS), Collagenase, fetal bovine serum (FBS), and Lipofectamine 3,000 were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany); Mitochondrial division inhibitor 1 and MitoSOX Red Mitochondrial Superoxide Indicator were purchased from MedChemExpress(Shanghai); TRIzol reagent and mRNA qRT-PCR Sybr Green Detection Kit were purchased from Invitrogen (USA). An Annexin V-FITC, Propidium Iodide (PI) Detection Kit,m and A terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) kit were purchased from BD Biosciences (New Jersey, USA); The following primary antibodies were used in this experiment: anti-GAPDH (1:100, Cell Signaling Technology, USA), anti-UCP2 (1:5,000, R&D Systems, Inc, USA), anti-MMP9 (1:1,000, R&D Systems, Inc. USA), anti-TGF-beta (1:1,000, Abcam, Cambridge, Britain), and anti-p-DRP1 (1:1,000, Cell Signaling). MTT assay (Beyotime); LDH assay (Beyotime).

Mesangial primary cells were fixed with 4% formaldehyde for 10 min, and immunohistochemistry was performed with goat polyclonal anti-alpha-8 integrin antibody (R&D Systems, no. AF4076), rabbit polyclonal anti-S100A4 antibody (Cell Signaling Technology, no. 13018), rabbit polyclonal anti-podocin antibody and mouse monoclonal anti-Na-K ATPase antibody (Santa Cruz Biotechnology, no. sc-21712). To confirm that primary culture cells do not contain proximal tubular cells, immunofluorescence staining was performed with FITC conjugated lotus tetragonolobus lectin. To confirm the specificity of the anti-alpha-8 integrin antibody and anti-S100A4 antibody, immunofluorescent staining of C57BL/6 J mice kidney fixed with 4% paraformaldehyde was performed. To evaluate the fibrosis of glomeruli in db/db mice, immunohistochemistry was performed with rabbit polyclonal anti-TGF-beta (Abcam, no. 92486), mouse monoclonal anti-smooth muscle actin (DAKO, no. M0851), and rabbit polyclonal anti-type IV collagen (R&D Systems, no. NB120-6586) antibodies