acvrl1 fc  (R&D Systems)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: The TGFβ type I receptor TGFβRI functions as an inhibitor of BMP signaling in cartilage

doi: 10.1073/pnas.1902927116

Figure Lengend Snippet: TGFβRI associates with ACVRL1 and ACTRIIB to block ACVRL1/ACTRIIB complex formation. ( A ) TGFβRI associates most with ACVRL1. Lysates prepared from ATDC5 cells were subjected to IP with an anti-TGFβRI antibody cross-linked to magnetic beads. Immunoprecipitated proteins were then probed with anti-ACVRL1, anti-ACVR1, anti-BMPR1A, and anti-BMPR1B antibodies. ( B ) Quantification of protein levels shown in A . Three biological replicates were analyzed. The average input protein level is normalized to 1. Immunoprecipitated protein level (IP-TGFβRI) was normalized using input and plotted relative to input + SD ( n = 3). Significance was established using 2-way ANOVA and t test. * P < 0.013. ( C ) Of the tested type II BMP receptors, TGFβRI associates almost exclusively with ACTRIIB. Immunoprecipitated proteins were probed with anti-TGFβRII, anti-ACTRIIA, and anti-ACTRIIB antibodies. Note that ACTRIIB, but not TGFβRII or ACTRIIA, could be coimmunoprecipitated with TGFβRI. ( D ) Quantification of protein levels shown in C . Three biological replicates were analyzed as described in B . * P < 0.017. ( E ) Western blot images confirm that TGFβRI protein is deleted by Crispr-cas9 in Tgfbr1 mutant ( Tgfbr1 △/△ ) ATDC5 cells, and that these cells exhibit elevated basal pSMAD1/5/8 levels. ( F and G ) The Western blot images of TGFβRI ( F ) and pSMAD1/5/8 levels ( G ) were quantified using ImageJ. Values of protein level were normalized using GAPDH and are plotted relative to control + SD ( n = 3 biological replicates). Significance was established using Student's 2-tailed t test. * P < 0.05. ( H ) Lysates from control or Tgfbr1 △/△ cells were subjected to IP with an anti-ACVRL1 antibody. Immunoprecipitated proteins were then probed with anti-TGFβRII, anti-ACTRIIA, and anti-ACTRIIB antibodies. In control cells, ACVRL1 associates with ACTRIIA. In Tgfbr1 △/△ cells, ACVRL1 associates with both ACTRIIA and ACTRIIB. ( I ) Quantification of protein levels shown in E . Three biological replicates were analyzed. Immunoprecipitated protein level was normalized using input and plotted relative to input + SD ( n = 3). Significance was established using Student's 2-tailed t test. * P < 0.01. Note that there is a significant increase of ACTRIIB/ACVRL1 association in Tgfbr1 △/△ cells compared with control cells. ( J ) Lysates from control or Tgfbr1 △/△ cells were subjected to IP with an anti-TGFβRII, anti-ACTRIIA, or anti-ACTRIIB antibody. Immunoprecipitated proteins were then probed with an anti-ACVRL1 antibody. In control cells, ACVRL1 associates with ACTRIIA. In Tgfbr1 △/△ cells, ACVRL1 associates with both ACTRIIA and ACTRIIB. ( K ) Quantification of protein levels shown in G . Three biological replicates were analyzed as described in I . * P < 0.05. Note that there is a significant increase in ACTRIIB/ACVRL1 association in Tgfbr1 △/△ cells compared with control cells.

Article Snippet: After 3 h of starvation, cells were treated with various ligands, Fc-proteins, and antibodies: TGFβ1, 5 ng/mL (R&D 240-B); BMP2, 200 ng/mL (R&D; 355-BM); BMP9, 100 ng/mL (Biolegend; 553102); NOGGIN, 400 ng/mL (R&D; 1967-NG); anti-BMP9, 400 ng/mL (R&D; MAB3209); ACVRL1-Fc, 400 ng/mL (R&D; 370-AL); ACTRIIB-Fc, 400 ng/mL (R&D; 3725-RB); anti-ACVRL1, 400 ng/mL (Santa Cruz; 19546); and anti-ACTRIIB, 400 ng/mL (Santa Cruz; 25453).

Techniques: Blocking Assay, Magnetic Beads, Immunoprecipitation, Western Blot, CRISPR, Mutagenesis