Figure 3

TGF-β1 enhanced the binding of IL-37 to the ALK1 receptor complex. (A) HUVECs were transfected with ALK1 siRNA or control (Ctrl) siRNA. Cell lysates were incubated with TGF-β and IL-37 and then subjected to co-immunoprecipitation with ALK1-specific antibodies. (B) Binding curves of IL-37 to immobilized TGF-β1. TGF-β1 (2 μg/mL), anti-IL-37 polyclonal antibodies (2 μg/mL) or BSA (2 μg/mL) were immobilized on 96-well plate and bound IL-37 was detected with antibodies. n = 4. (C) Interaction of IL-37 and TGF-β1 determined by in vitro pull-down assay. (D) TGF-β enhanced the binding between IL-37 and the ALK1 receptor complex (ALK1 Rec) in pull-down assay. The ALK1 receptor complex (pre-incubated ALK1-Fc and TβRII-Fc) or control Fc were conjugated to protein A/G beads, which were then incubated with IL-37 in the presence or absence of TGF-β1. (E) 96-well plates were coated with the ALK1 receptor complex, incubated with IL-37 and TGF-β, and bound IL-37 was detected by antibodies. (F) HUVECs were treated with IL-37 (1 ng/mL) in the presence or absence of TGF-β1 (5 ng/mL), and Smad1/5/8 phosphorylation was determined by Western blot. (G and H) IL-37 upregulated (G) mRNA levels and (H) protein levels of Id1 and Id3 in HUVECs. n = 4. Blots are representative of three experimental replicates. *P < 0.05, **P < 0.01, ***P < 0.001.