Figure 8

The activation of BMP type I receptor induces SNIP1 degradation which is co-regulated by Smad1, Smad4 and Az. A. SNIP1 degradation is lactacystin-sensitive and is dependent upon the co-expression of Smad1, Smad4 and Az in COS cell overexpression system. COS cells were transfected with the indicated plasmids. Lactacystin (10 μM) was added during the last 10 hrs. The expression of each protein was detected by Western blot. Cell lysates were also blotted with a monoclonal anti-α tubulin, which served as a control for loading (bottom panel). "*" in lanes 10 and 11 represents the mutant Smad1 (G419S). This construct is T7 tagged, thus did not show on the anti-Flag blot (top panel). Its expression was confirmed by anti-T7 on a separate blot (not shown). The right panel shows the Integrated Optical Density of SNIP1 obtained from quantification of SNIP1 signals using Densitometry. B. Increased expression of Smad1 enhances SNIP1 degradation induced by BMP type I receptor activation. COS1 cells were transfected with the indicated amount of DNA constructs of Smad1, Smad4, SNIP1, Az and ALK3Q233D. The protein levels were detected by Western blot. The IOD of SNIP1 is plotted in the right panel. For both panels, equal transfection efficiency was monitored by GFP and equal total protein loading was assured after protein concentration measurement. C. The degradation of endogenous SNIP1 is induced in a time-dependent fashion by BMP7 in HaCaT and L6 cells. HaCaT and L6 cells were treated with BMP7 (250 ng/ml) for the indicated time periods and cell lysates were separated on SDS-PAGE then blotted by anti-SNIP1 polyclonal antibody [25]. Two different forms of endogenous SNIP1 are indicated as SNIP1.A and SNIP1.B. The IOD of the combined IOD of these two forms are plotted in the right panel.