polyclonal rabbit anti tgfβ ri  (Santa Cruz Biotechnology)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: ALG3 contributes to stemness and radioresistance through regulating glycosylation of TGF-β receptor II in breast cancer

doi: 10.1186/s13046-021-01932-8

Figure Lengend Snippet: ALG3 enhances radioresistance via regulation of TGFBR2 glycosylation. ( a ) Downshift of TGFBR2 bands in ALG3-sg cells was detected by Western blot. But not TGFBR1 bands ( b ) Representative immunofluorescence images of TGFBR2 expression level in cytoplasmic and membrane fractions. ( c ) A schematic model of different subtypes of N-glycans. The round spots are mannose, the square ones are acetylglucosamine, and the red spot is the initial of the N-glycosylation site, which is initiated by ALG3. ( d ) TGFBR2 band shift could be seen in ALG3-sg cells or cells treated by tunicamycin. And downregulation of ALG3 reduced the expression level of p-SMAD2. ( e ) Representative immunofluorescence images of p-SMAD2 expression level in cytoplasmic and nuclear fractions. Nuclear translocation of p-SMAD2 was significantly decreased in ALG3-sg and tunicamycin treatment groups. ( f ) The co-immunoprecipitation between TGFBR1 and TGFBR2, TGFBR1 and p-SMAD2 could be detected in ALG3-control group, but not tunicamycin treatment, and ALG3-sg groups. ( g ) TGFBR2 inhibitor (LY2109761) in ALG3-transduced cells decreased the surviving fraction of breast cancer cells after radiation treatment, which were detected by CCK-8 assays. Data were analyzed by two-way ANOVA. Each bar represents the mean ± SD of three independent experiments. ( h ) Inhibition of TGFBR2 in ALG3-transduced cells decreased the number of colonies after radiation treatment. ( i ) Inhibition of TGFBR2 in ALG3-transduced cells decreased the proportion of CD44 + CD24 − cells, which were detected by flow cytometry. “ns” no significance, * P < 0.05

Article Snippet: Then, incubated cell extracts with the polyclone rabbit antibody to TGFBR1 (NO. sc518045, Santa Cruz Biotechnology, Inc., California, USA) or TGFBR2 (NO. ab225902, 1:100, Abcam, Inc., Cambridge science park, UK) for 16 h at 4 °C.

Techniques: Glycoproteomics, Western Blot, Immunofluorescence, Expressing, Membrane, Electrophoretic Mobility Shift Assay, Translocation Assay, Immunoprecipitation, Control, CCK-8 Assay, Inhibition, Flow Cytometry

Journal: The Journal of Neuroscience

Article Title: Peripheral Nerve Injury Alters Blood–Spinal Cord Barrier Functional and Molecular Integrity through a Selective Inflammatory Pathway

doi: 10.1523/JNEUROSCI.1642-11.2011

Figure Lengend Snippet: Experimental protocols for immune modulation of BSCB integrity

Article Snippet: Microvessels were fixed with 4% PFA for 30 min and incubated overnight at 4°C with rabbit anti-TGF-β-RI polyclonal antibody (1:250; Santa Cruz Biotechnology) and rat anti-CD31 (for endothelial cells; 1:250; BD Biosciences), followed by a 60 min incubation at room temperature in fluorochrome-conjugated goat secondary antibody.

Techniques: Recombinant, Derivative Assay

Journal: The Journal of Neuroscience

Article Title: Peripheral Nerve Injury Alters Blood–Spinal Cord Barrier Functional and Molecular Integrity through a Selective Inflammatory Pathway

doi: 10.1523/JNEUROSCI.1642-11.2011

Figure Lengend Snippet: Inflammatory mediators modulate BSCB integrity. A, Neutralizing endogenous MCP-1 with a MCP-1 antibody (3 μg for 3 d) significantly reduced the leakage of EB in the spinal cord observed at day 3 after injury. B, Intrathecal infusion of recombinant murine MCP-1 (2.5 μg for 3 d) produced an increase of EB content in the spinal cord in naive animals, similar to that seen in nerve-injured animals. Intrathecal catheterization provoked a slight, but nonsignificant increase of EB content in the spinal cord. C, IL-1β injected intravenously caused a dose-dependent impairment of BSCB permeability to EB. D, Intrathecal administration of antiinflammatory cytokines TGF-β1 (2 μg) and IL-10 (3 μg) for 3 d significantly reduced the EB content in the spinal cord increased by peripheral nerve injury. TGF-β1 did not affect the BSCB permeability in naive rats. N = 3–6/group. Values are presented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 versus naive rats without any treatment; #p < 0.05 versus injured saline-treated.

Article Snippet: Microvessels were fixed with 4% PFA for 30 min and incubated overnight at 4°C with rabbit anti-TGF-β-RI polyclonal antibody (1:250; Santa Cruz Biotechnology) and rat anti-CD31 (for endothelial cells; 1:250; BD Biosciences), followed by a 60 min incubation at room temperature in fluorochrome-conjugated goat secondary antibody.

Techniques: Recombinant, Produced, Injection, Permeability, Saline

Journal: The Journal of Neuroscience

Article Title: Peripheral Nerve Injury Alters Blood–Spinal Cord Barrier Functional and Molecular Integrity through a Selective Inflammatory Pathway

doi: 10.1523/JNEUROSCI.1642-11.2011

Figure Lengend Snippet: TGF-β1 alters tight junction protein levels in spinal cord microvessels. Intrathecal administration of TGF-β1 (2.5 μg for day 0 to day 3) successfully prevents the decrease in tight junction protein levels ZO-1 (A, D) and occludin (B, D) observed after peripheral nerve injury. Levels of caveolae structural component caveolin-1, however, remained unchanged (C, D). The receptor for TGF-β1 (TGF-β-RI) is found in isolated spinal cord endothelial cells, confirmed by the colocalization with vascular marker CD31. DAPI staining (blue) was used for cellular identification (E). The protein levels of the TGF-β-RI remain unchanged after peripheral nerve injury, with or without TGF-β1 infusion (F). TGF-β1 treatment induced the phosphorylation of signaling pathway proteins Smad2/3 (pSmad2/3) in spinal cord microvessels (G). Three separate experiments in which each treatment group consists of pooled microvessels from two animals were included. Values are presented as means ± SEM. *p < 0.05 versus d3 + saline. Scale bar, 10 μm.

Article Snippet: Microvessels were fixed with 4% PFA for 30 min and incubated overnight at 4°C with rabbit anti-TGF-β-RI polyclonal antibody (1:250; Santa Cruz Biotechnology) and rat anti-CD31 (for endothelial cells; 1:250; BD Biosciences), followed by a 60 min incubation at room temperature in fluorochrome-conjugated goat secondary antibody.

Techniques: Isolation, Marker, Staining, Phospho-proteomics, Saline

Journal: The Journal of Neuroscience

Article Title: Peripheral Nerve Injury Alters Blood–Spinal Cord Barrier Functional and Molecular Integrity through a Selective Inflammatory Pathway

doi: 10.1523/JNEUROSCI.1642-11.2011

Figure Lengend Snippet: Peripheral nerve injury-induced disruption of BSCB provided access to circulating immune cells into the spinal cord parenchyma. A, GFP+ bone marrow-derived cells infiltrated into the spinal cords after PNI, which differentiated into Iba-1+ microglia. Intrathecal injection of TGF-β1 prevented the entrance of circulating immune cells at 14 d after nerve injury. Evidence on localization of infiltrated GFP+ bone marrow-derived cells in the parenchyma is shown by coimmunostaining with vessel marker Glut-1. Scale bars: left, 250 μm; middle, 10 μm; right, 20 μm. B, Quantitative analysis of GFP+ cells in the spinal cords of GFP chimeric mice after PNI and TGF-β1 treatment. C, To compare with naive animals, there was a marked infiltration of CD2+ and CD3+ lymphocytes into the ipsilateral side of spinal cords after peripheral nerve injury (white arrows). Intrathecal injection of TGF-β1 prevented the entrance of circulating lymphocytes at 7 d after nerve injury. Scale bar, 50 μm. D, Quantitative analysis of CD2+ and CD3+ cells in the dorsal horns of spinal cords of rats after nerve injury and TGF-β1 treatment. TGF-β1 successfully reduced the number of cells invading the spinal parenchyma. N = 6/group. Values are expressed as means ± SEM. *p < 0.05, **p < 0.01 versus controls (naive and sham); #p < 0.05, ##p < 0.01, ###p < 0.001 versus saline.

Article Snippet: Microvessels were fixed with 4% PFA for 30 min and incubated overnight at 4°C with rabbit anti-TGF-β-RI polyclonal antibody (1:250; Santa Cruz Biotechnology) and rat anti-CD31 (for endothelial cells; 1:250; BD Biosciences), followed by a 60 min incubation at room temperature in fluorochrome-conjugated goat secondary antibody.

Techniques: Disruption, Derivative Assay, Injection, Marker, Saline