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. Author manuscript; available in PMC: 2020 May 1.
Published in final edited form as: Exp Cell Res. 2019 Feb 25;378(1):11–20. doi: 10.1016/j.yexcr.2019.01.026

Figure 4. Noggin is required for TGFβ to inhibit BMP signaling.

Figure 4.

(A) C3T10H1/2 cells were treated with vehicle (CTRL) or TGFβ1 for 8 hours. RNA was collected and relative levels of mRNA for Scx and Noggin were determined by qPCR and analyzed with REST software. n= 4 biological replicates, * = p < 0.05. REST analysis shown in Table S4A. (B) C3T10H1/2 cells were transfected 30 pmole of siRNA control (si-CTRL) or siRNA directed to noggin (si-NOG) then incubated for 48 hours. Immunoblot of Noggin protein levels confirmed knockdown by si-NOG. α-tubulin was used as a loading control. (C) C3T10H1/2 cells were transfected siRNA control (si-CTRL) or siRNA directed to noggin (si-NOG). After 48hrs, cells were treated with vehicle control (−) or TGFβ1 (T) for 8hrs. Protein extracts were collected and pSmad1/5 and Smad1 levels were determined by Immunoblot. α-tubulin was used as a loading control. Representative of n=5 replicates shown (D) C3T10H1/2 were transfected with si-CTRL or si-NOG for 48hrs. RNA was harvested after treatment with TGFβ1 for 8hrs. Relative mRNA levels of SCX, ADAMTSL2, and FMOD was measured with qPCR and normalized with HPRT. Data was analyzed using REST software, * = p< 0.05, (n=5). REST analysis shown in Table S4B. (E) TGFβ signaling was measured by immunoblot of pSmad2 and Smad2/3 in Si-CTRL and Si-NOG treated cells. Representative from n=3 replicates shown.