XB-IMG-172764
Xenbase Image ID: 172764
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FIGURE 4 Zbtb14 regulates BMP and Wnt signaling. (a) Zbtb14 decreases endogenous levels of phosphorylated Smad1/5/8 in Xenopus ectodermal cells. zbtb14-GR mRNA (250 pg) was injected into the animal region of 4- to 8- cell- stage embryos. Embryos were treated with DEX, and ectodermal explants were isolated at the blastula stage and cultured until the early gastrula stage (stage 10.5). Whole- cell lysates from explants were immunoblotted with anti- phospho Smad1/5/8 (pSmad1/5/8), anti- Smad1 (MO5), anti- HA, and anti- Tubulin antibodies, respectively. Zbtb14- GR was tagged at the C- terminus with the HA epitope. Tubulin was used as a loading control. (b) Zbtb14 decreases levels of pSmad1/5/8 induced by the constitutive active BMP receptor HA- CA- Alk6 in cultured HeLa cells. The effect of Zbtb14 depends on the proteasomal degradation pathway, which is inhibited by the proteasome inhibitor MG132. HeLa cells were transfected with the indicated combination of expression constructs, and then treated with or without MG132 for 6 hr before cell harvest. Whole- cell extracts were immunoblotted with the indicated antibodies. Tubulin was used as a loading control. Graph (right) shows quantification of the pSmad1/5/8 immunoblot which was normalized to Tubulin (left). (c) Zbtb14 decreases protein levels of BMP- regulated Smads (Smad1 and Smad8), Co- Smad (Smad4), TGF- β- regulated Smad (Smad2), and I- Smads (Smad6 and Smad7). COS cells were transfected with the indicated combination of expression constructs. Whole- cell extracts were immunoblotted with the indicated antibodies. Tubulin was used as a loading control. (d) Zbtb14 interacts with the R- Smad Smad3 and I- Smads (Smad6 and Smad7). COS cells were transfected with the indicated combination of expression constructs, and then treated with MG132 for 6 hr before harvest. Flag- tagged Smads (F- Smads) were immunoprecipitated (IP) from cells lysates with anti- Flag antibodies, and the precipitates were immunoblotted (IB) with anti- Myc antibodies (top). Input cells lysates were immunoblotted with anti- Myc (middle) or anti- Flag (bottom) antibodies. Interactions of Zbtb14 with Smad3, Smad6, and Smad7 were repeatedly observed in independent experiments (e and g; data not shown). (e) Zbtb14 interacts with Smurf1 and Smurf2. COS cells were transfected with the indicated combination of expression constructs, and then treated with MG132 for 6 hr before harvest. Flag- tagged Smads were immunoprecipitated from transfected cells with anti- Flag antibodies, and the precipitates were immunoblotted with anti- Myc antibodies (top). Input cells lysates were immunoblotted with anti- Myc (middle) or anti- Flag (bottom) antibodies. (f) Zbtb14 stabilizes cytosolic β- Catenin, which transduces canonical Wnt signaling. COS cells were transfected with Myc- tagged Zbtb14 and cell lysates were incubated with concanavalin A (Con A) beads to enrich free cytosolic β- Catenin by removing cadherin- bound β- Catenin. Input extracts were immunoblotted with anti- Myc (middle) or anti- Tubulin as a loading control (bottom). Graph (bottom) shows quantification of the immunoblot (top) which was normalized to Tubulin. (g) Zbtb14 interacts with β- TrCP as well as with Smad7. COS cells were transfected with the indicated combination of expression constructs, and then treated with MG132 for 6 hr before harvest. Flag- tagged constructs were immunoprecipitated from cells lysates with anti- Flag antibodies, and the precipitates were immunoblotted with anti- Myc antibodies (top panel). Input extracts were immunoblotted with anti- Myc antibodies (second panel) or anti- Flag antibodies (bottom panel). Extracts were also immunoprecipitated with anti- Flag and then immunoblotted with anti- Flag antibody (third panel), in order to detect β- TrCP, which is expressed at low levels. Image published in: Takebayashi-Suzuki K et al. (2018) Copyright © 2018. Image reproduced with permission of the Publisher, John Wiley & Sons. Larger Image Printer Friendly View |