Human TGF‑ beta  RII Antibody

TGF‑ beta 1 Inhibition of IL‑4-dependent Cell Proliferation and Neutralization by Human TGF‑ beta RII Antibody.

Recombinant Human TGF-beta 1 (Catalog # 240-B) inhibits Recombinant Human IL-4 (Catalog # 204-IL) induced proliferation in the TF-1 human erythroleukemic cell line in a dose-dependent manner (orange line). Inhibition of Recombinant Human IL-4 (5 ng/mL) activity elicited by Recombinant Human TGF-beta 1 (0.04 ng/mL) is neutral-ized (green line) by increasing concentrations of Goat Anti- Human TGF-beta RII Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-241-NA). The ND₅₀ is typically 10-20 µg/mL.

Detection of TGF-beta RII by Western Blot

SMAD-activation by recombinant GDF15 in myeloma cell lines.A. Phosphorylation of SMAD1/5 or SMAD2 was determined using immunoblotting in IH-1 cells treated with BMP-9 (0.5 ng/mL), activin A (25 ng/mL) or indicated concentrations of GDF15 (100–400 ng/mL) for 1 hour. B. INA-6 cells were treated with GDF15 (200 ng/mL) and the inhibitor SB431542 (0–2.5 μM) for 1 hour before immunoblotting with anti-phospho-SMAD2. C. INA-6 cells were transiently transfected with siRNAs targeting ACVR1B/ALK4, ACVR1C/ALK7, TGFBR1/ALK5 and a non-targeting control siRNA. Two days after transfection the cells were treated with GDF15 (200 ng/mL) for 1 hour before immunoblotting with anti-phospho-SMAD2. D. Knock-down of receptors by siRNA in cells used in (C) as shown by QRT-PCR. Gene expression was calculated with the comparative delta Ct-method with GAPDH as housekeeping gene. The error bars indicate SEM of three independent experiments. Asterisks above bars indicate the degree of significance for downregulation of each gene compared to control (*, P≤0.05; **, P≤0.01; and ***, P≤0.001). E. INA-6 cells were treated with GDF15 (100 ng/mL) and a neutralizing TGFBR2 antibody (10–15 μM) for 1 hour before immunoblotting with anti-phospho-SMAD2. F. INA-6 cells were treated with GDF15 (100 ng/mL) and the indicated soluble receptors (5 μg/mL for all except endoglin, which was 1 μg/mL) for 1 hour before immunoblotting with anti-phospho-SMAD2. Antibody staining towards GAPDH was used as loading control for all Western blots. The experiments were performed 2–3 times each. GDF15 used in this figure was from R&D Systems, Lot# EHF1713081. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/29161287), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of TGF-beta RII by Flow Cytometry

Blockade of transforming growth factor (TGF)-beta I-III did in part prevent regulatory T cells (Treg) induction. (A) CFSE-labeled CD4+CD25- T cells were cocultured with platelets in the ratio of 1:50 and were stimulated with anti-CD3 mAb (0.5 µg/mL) and anti-CD28 mAb (1.0 µg/mL) in the presence of either anti-TGF-beta I-III (10 µg/mL) or anti-TGF-beta receptor II (10 µg/mL) antibodies. Antibodies were added at day 0. The expression of Foxp3 and GARP and cell proliferation were determined on day 3 via flow cytometry. (B) Production of IL-2 and IFN-gamma was assessed by intracellular flow cytometry on day 6. The graphs show cells cultured in the presence of platelets normalized to CD4+CD25− T cells without platelets. Dot plots show one representative result of 10 independent experiments (n = 10, box and whiskers, medians ± min/max, * p < 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and n.s. determined by one-way ANOVA). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/33291452), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of TGF-beta RII by Flow Cytometry

Combining blockade of TGF-beta signaling and GARP led to a complete inhibition of platelet effects. (A) CFSE-labeled CD4+CD25− T cells were cocultured with platelets in the ratio of 1:50 and were stimulated with anti-CD3 mAb (0.5 µg/mL) and anti-CD28 mAb (1.0 µg/mL). CD4+CD25− T cells were incubated for 15 min with TGF-beta receptor II (10 µg/mL) antibody prior to coculture, as indicated. Excess antibody was removed. Pre-treated CD4+CD25− T cells were cultured in the presence of either anti-TGF-beta I–III (10 µg/mL) and/or anti-GARP Ab (10 µg/mL) antibodies. Antibodies were added at day 0. The expression of Foxp3, GARP and cell proliferation were determined on day 3 via flow cytometry. (B) Production of IL-2 and IFN-gamma was assessed by intracellular flow cytometry on day 6. The graphs show cells cultured in the presence of platelets normalized to CD4+CD25− T cells without platelets. Dot plots show 1 representative result of 10 independent experiments (n = 3, means ± SD, * p < 0.05, ** p ≤ 0.01, and n.s. determined by one-way ANOVA). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/33291452), licensed under a CC-BY license. Not internally tested by R&D Systems.