Fig. 3.
SMAD4 Leu97 variant reduces TGFβ signaling. a The effect of SMAD4 rare variants on the TGFβ was assessed in COS7 cells signaling. The SMAD4 activity of Leu97 alteration to induce 3TP luciferase activity was reduced compared to WT. COS7 cells were co-transfected with 3TP-lux plasmid and plasmids expressing WT or rare variants in SMAD4, followed by TGFβ1 treatment for 24 h. The 3TP luciferase activities were measured with renilla luciferase as internal control. The enhancement of luciferase activity in response to TGFβ1 treatment in SMAD4 Leu97 was significantly lower than that of SMAD4 WT, Val24, Thr246. b Immortalized SMCs (SMC11023) expressing control vector, SMAD4 WT or SMAD4 Leu97 were treated with TGFβ1 for 4 h and the expression of p15INK4b was measured by real-time qPCR and with GAPDH as internal control. c, d SMAD4 Leu97 variant reduced TGFβ1-induced smooth muscle specific gene expression and protein levels. SMC11023 cells infected with control vector, Flag-tagged SMAD4 WT or Leu97 variant were treated with TGFβ1 for 48 h followed by Western blot (c) and real-time qPCR (d). Asterisks indicate a p value <0.05 when compared to the SMAD4 WT