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Fig. 4. Inhibition of Bmp4 or Tgfß1 signaling by HtrA1. (A) Inhibition of Bmp4 signaling by HtrA1. C2C12 cells were transfected in 24-well plates with 250 ng/well pGL3-Id985WT and 50 ng/well expression vectors for Smad1, Smad4 and Bmp4. The expression vectors for HtrA1 (pcDNA3-HtrA1) or noggin (pEF-Bosnoggin) were co-transfected as indicated below the figure. (B) Inhibition of Tgfß1 signaling by HtrA1. C2C12 cells were transfected in 24-well plates with 250 ng/well pGL3ti-(SBE)4 and 50 ng/well Smad1 expression vector. Recombinant human Tgfß1 protein (10 ng/ml) was added 24 hours after transfection. The expression vectors for HtrA1 and noggin were co-transfected as indicated below the figure. (C) Inhibition of Bmp4 signaling by various HtrA1 mutants. The expression vectors for {Delta}FS, {Delta}FS/PDZ, {Delta}linker/FS/PDZ or S328A were co-transfected as in A. Insets show results of western blots, showing the expression levels of wild-type HtrA1 and its mutants, other than {Delta}FS/PDZ, at 50, 100 and 200 ng DNA/well. Data are the average of three experiments. Standard deviations are shown as a line on each bar. (D) HtrA1 did not inhibit the signal that originated from the constitutively active Bmp receptor. C2C12 cells were transfected as described in A, except that the expression vector of Bmp4 was replaced with pEF-Bos-caBMPR-IB (3 ng/well). The amount of pEF-Bos-caBMPR-IB was adjusted so that it gave similar transcriptional activation to that of the Bmp4 vector (50 ng/well; see the right half of the panel).