rabbit polyclonal tgfβr1  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Endothelial SIRT3 regulates myofibroblast metabolic shifts in diabetic kidneys

doi: 10.1101/2020.12.15.422824

Figure Lengend Snippet: (a) Schematic diagram showing the isolation of endothelial cells from the non-diabetic and diabetic mice. (b) Western blot analysis of TGFβR1, smad3 phosphorylation, total smad3, α-SMA, HK2, PKM2 and PDK4 in the lysates of isolated endothelial cells from non-diabetic and diabetic kidneys of control littermates and eEx mice. Representative blots are shown. Densitometry calculations were normalized to β-actin. N=6 were analyzed in each group. (c) Western blot analysis of TGFβR1, smad3 phosphorylation, total smad3, α-SMA, HK2, PKM2 and PDK4 in the lysates of isolated endothelial cells from non-diabetic and diabetic kidneys of control and eKO mice. Representative blots are shown. Densitometry calculations were normalized to β-actin. N=5 for non-diabetic group, N=6 for diabetic group. (d) Glutaryldehyde chemical cross-linking experiment for PKM2, was performed in the isolated endothelial cells from the non-diabetic and diabetic kidneys of control and eEx mice. The representative blot from five blots is shown. N=5/each group. (e) Glutaryldehyde chemical cross-linking experiment for PKM2, was performed in the isolated endothelial cells from the non-diabetic and diabetic kidneys of control and eKO mice. The representative blot from five blots is shown. N=5/each group. (f) Western blot analysis of CPT1a and PGC1α in the lysates of isolated endothelial cells from the non-diabetic and diabetic kidneys of control and eEx mice. Representative blots are shown. Densitometry calculations were normalized to β-actin. N=5/group. Data in the graph are shown as mean ± SEM. (g) Western blot analysis of CPT1a and PGC1α in the lysates of isolated endothelial cells from the non-diabetic and diabetic kidneys of control and eKO mice. Representative blots are shown here. Densitometry calculations were normalized to β-actin. N=5 non-diabetic group, N=6 diabetic group. Data in the graph are shown as mean ± SEM.

Article Snippet: Rabbit polyclonal anti-TGFβR1 antibody (SAB4502958) was obtained from Sigma (St Louis, MO).

Techniques: Isolation, Western Blot

Journal: bioRxiv

Article Title: Endothelial SIRT3 regulates myofibroblast metabolic shifts in diabetic kidneys

doi: 10.1101/2020.12.15.422824

Figure Lengend Snippet: (a) Western blot analysis of SIRT3, α-SMA and TGFβR1 in the scramble siRNA, sirt3 siRNA-transfected and TGFβ2-stimulated HUVECs. (b) Smad3 phosphorylation and total smad3 in the scramble siRNA, sirt3 siRNA-transfected and TGFβ2-stimulated HUVECs. Representative blots are shown. Densitometry calculations are normalized by β-actin. Three independent experiments were performed. (c) Design of conditioned media experiment. Renal tubular epithelial cells (HK2 cells) were cultured in the conditioned media either from scramble siRNA- or sirt3 siRNA-transfected endothelial cells (HUVECs). (d) Representative western blotting images of the indicated molecules from three independent experiments are shown. Densitometric analysis of the levels relative to β-actin is shown. Data in the graph are shown as mean ± SEM. (e) Immunofluorescent analysis for vimentin/E-cadherin in the kidneys of nondiabetic and diabetic, control littermates and eEx mice. Representative pictures are shown. N=7/each group. Scale bar: 50 μm. (f) Immunofluorescent analysis for vimentin/E-cadherin in the kidneys of nondiabetic and diabetic, control littermates and eKO mice. Representative pictures are shown. N=7/each group. Scale bar: 50 μm.

Article Snippet: Rabbit polyclonal anti-TGFβR1 antibody (SAB4502958) was obtained from Sigma (St Louis, MO).

Techniques: Western Blot, Transfection, Cell Culture