Skip to main content
. Author manuscript; available in PMC: 2010 Nov 13.
Published in final edited form as: Cell. 2009 Nov 13;139(4):757–769. doi: 10.1016/j.cell.2009.09.035

Figure 2. Requirements for Smad ALP.

Figure 2

(A) Immunoblot analysis of wild-type, Smad1C and Smad1L mutant knock-in MEFs treated with BMP or UV, with antibodies against the indicated proteins. (B, C) SW480 Smad4-null cells, or cells stably expressing Smad4, were treated with BMP (B) or TGFβ (C). Nuclear and cytoplasmic fractions were analyzed using the indicated antibodies. (D) Chromatin immunoprecipitation of BMP and TGFβ stimulated C2C12 cells with the indicated antibodies. Precipitates were subjected to qRT-PCR of the BMP and TGFβ responsive regions of the indicated genes with unresponsive regions of the same genes serving as negative controls. Data show the mean ± S.D. of triplicates and are representaive of at least two independent expreriments. (E) Immunoblot analysis of BMP treated HaCaT cells in the absence or after addition of α-amanitin for the indicated times. Total cell extracts were analyzed using the indicated antibodies. (F) HaCaT cells were stimulated with BMP for 1 h in the absence or presence of MG132 or of a siRNA against Smurf1. Cells were harvested at the indicated times after BMP removal and total cell extracts were analyzed by immunoblot. (G) As in (F) but cells were stimulated with TGFβ and siRNA against Nedd4L was used. (H) Schematic model of the sequential steps leading to Smad-ALP and binding of ubiquitin ligases.