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1.

Figure. From: The adaptor molecule Disabled-2 links the transforming growth factor ? receptors to the Smad pathway.

Fig. 6. Dab2 associates with Smad2 and Smad3 in vivo in a ligand-dependent manner. (A and B) Smad2 and Smad3 interact with Dab2 in vivo. COS7 cells were transiently transfected with Flag-tagged WT or F166V Dab2 and various Smads with or without the activated form of TβRI (TβRIAct). After 48 h, lysates were prepared and (A) immunoprecipitated with antibodies specific to each Smad [Smad2 and Smad3 (Zymed Laboratories), Smad4 and Smad7 (Santa Cruz)]. The presence of Dab2 in the complex was confirmed by western analysis with a monoclonal antibody to Dab2 (α-p96). Expression of Dab2 and the Smads (B) was determined by western analysis with anti-Flag antibody (M2; Sigma).

Barbara A. Hocevar, et al. EMBO J. 2001 June 1;20(11):2789-2801.
2.

Figure. From: The adaptor molecule Disabled-2 links the transforming growth factor ? receptors to the Smad pathway.

Fig. 7. Endogenous Dab2 associates with endogenous Smad2 and Smad3. (A) Dab2 interacts with Smad2 in a time- and ligand-dependent manner. BAHgpt cells were treated with TGFβ for 1 h followed by immunoprecipitation with anti-Smad2 (Transduction Laboratories) antibody. The presence of Dab2 in the immunoprecipitates was confirmed by western analysis for Dab2 (α-p96). Comparable Dab2 levels are shown by western analysis in the lower panel. (B) BAHgpt cells stably expressing WTDab2 were treated with TGFβ for various times and lysates subjected to immunoprecipitation with anti-Smad2 (Transduction Laboratories) or anti-Smad3 (Zymed Laboratories) antibodies or pull-down assay with the GST–MH2 Smad2 domain or control GST beads. The presence of Dab2 in the precipitate was confirmed by western analysis with a monoclonal antibody to Dab2 (α-p96). Equal expression of Dab2 at the various time points was verified by western analysis using the same antibody (lower panel).

Barbara A. Hocevar, et al. EMBO J. 2001 June 1;20(11):2789-2801.
3.

Figure. From: The adaptor molecule Disabled-2 links the transforming growth factor ? receptors to the Smad pathway.

Fig. 5. Dab2 associates with Smad2 and Smad3 in vitro. (A) Dab2 interacts with the Smads in vitro. Full-length WTDab2 or the various deletion constructs depicted in Figure 4A were synthesized in vitro using [35S]methionine. Equal amounts of 35S-labeled reaction products were incubated with bacterially expressed GST constructs correspond ing to GST protein alone (GST), full-length GST fusion proteins of Smad1, Smad2, Smad3 and Smad4, or GST fusion proteins of the MH1 and MH2 domains of Smad2. Following extensive washing, bound proteins were analyzed by SDS–PAGE, subjected to fluorography and visualized by autoradiography. An aliquot (10%) of 35S-labeled reaction product input is shown in the control lane (Cont.). (B) Equal loading of the bacterially expressed GST fusion proteins utilized in (A) is demonstrated by Coomassie Blue staining of the gel following SDS–PAGE.

Barbara A. Hocevar, et al. EMBO J. 2001 June 1;20(11):2789-2801.
4.

Figure. From: The adaptor molecule Disabled-2 links the transforming growth factor ? receptors to the Smad pathway.

Fig. 8. Dab2 associates with the TGFβ receptor complex in vivo. (A and B) Dab2 interacts with the TGFβ receptor complex. COS7 cells were transiently transfected with HA-tagged wild-type (WT), kinase-deficient (KR) or activated (Act.) type I TGFβ receptors or HA-tagged wild-type (WT) or kinase-deficient (KR) type II TGFβ receptors as indicated. Cells were untreated or treated with TGFβ for the various times, as indicated, and lysates were subjected to immunoprecipitation with control IgG or antibodies to endogenous Dab2 (p96). The presence of co-precipitating TβRI or TβRII was confirmed by western analysis (α-HA 12CA5). Expression levels of the type I and type II TGFβ receptors in (A) were confirmed by western analysis of cell lysates using anti-HA antibodies (B). The positions of TβRI and TβRII are indicated by arrows. (C) Dab2 interacts with the TGFβ receptor complex. Mv1Lu, BAHgpt and 903 cells were incubated with [125I]TGFβ1. Following crosslinking, cells were lysed and an aliquot analyzed for receptor expression and crosslinking efficiency, left panel. The remainder of the lysate was immunoprecipitated with control mouse IgG or a monoclonal antibody to Dab2 (α-p96). Receptor complexes were resolved by SDS–PAGE and visualized by autoradiography. The positions of TβRI and TβRII are indicated by arrows.

Barbara A. Hocevar, et al. EMBO J. 2001 June 1;20(11):2789-2801.
5.

Figure. From: The adaptor molecule Disabled-2 links the transforming growth factor ? receptors to the Smad pathway.

Fig. 2. Mutant Dab2 exhibits decreased protein stability. (A) Steady-state levels of Dab2 are decreased in mutant 903 cells. Fifty micrograms of total cellular protein from BAHgpt, 903 and 903WTDab2 cells were subjected to western analysis utilizing a monoclonal antibody to Dab2 (α-p96). (BDe novo Dab2 synthesis is decreased in mutant 903 cells. BAHgpt and 903 cells were labeled with [35S]methionine for 4 h prior to lysis and immunoprecipitation with either non-immune mouse IgG or a monoclonal antibody to Dab2 (α-p96). Dab2 levels were visualized by autoradiography. (C, D and E) Mutant Dab2 is less stable than WTDab2. Pulse–chase analysis (C) was performed on COS7 cells transiently transfected with Flag-tagged WT or Mut Dab2 as described in Materials and methods. Following immunoprecipitation with anti-Flag antibody, [35S]Dab2 was visualized by autoradiography. Analysis of steady-state levels of WT or Mut Dab2 (D) was performed by western analysis utilizing anti-Flag antibody. (E) Levels of Dab2 proteins in (C) were determined by densitometric scanning and analysis with NIHImage software. Levels are expressed as a percentage of protein remaining at the various time points, with levels at time 0 designated as 100%.

Barbara A. Hocevar, et al. EMBO J. 2001 June 1;20(11):2789-2801.
6.

Figure. From: The adaptor molecule Disabled-2 links the transforming growth factor ? receptors to the Smad pathway.

Fig. 3. Dab2 expression restores TGFβ-mediated responses to mutant 903 cells. (A and B) Dab2 expression alters TGFβ-mediated induction of the endogenous genes fibronectin (A) and PAI-1 (B). BAHgpt, BAHgpt cells stably transfected with Dab2 (BAHgptWTDab2), 903 and 903WTDab2 cells were examined for [35S]fibronectin (FN) secreted to the media (A) or [35S]PAI-1 deposited to the extracellular matrix (B) following TGFβ treatment, as described in Materials and methods. FN and PAI-1 are indicated by the arrows. (C) Dab2 expression restores TGFβ-stimulated Smad2 phosphorylation to the mutant 903 cell line. BAHgpt, BAHgptWTDab2, 903 and 903WTDab2 cells were untreated or treated with 5 ng/ml TGFβ for 1 h as indicated. Phosphorylated Smad2 was identified by western analysis using an antibody specific only for the TGFβ-stimulated phosphorylated form of Smad2 (anti-phospho Smad2 465/467; Upstate Biotechnology). (D) Equivalent expression of cellular Smad2 in BAHgpt, BAHgptWTDab2, 903 and 903WTDab2 cells was confirmed by western analysis using an anti-Smad2 antibody (Zymed Laboratories). (E and F) Dab2 restores TGFβ-stimulated Smad2 (E) and Smad3 (F) nuclear accumulation in 903 cells. Nuclear proteins were isolated from BAHgpt, 903 and 903WTDab2 cells untreated or treated with TGFβ for 1 h, as described in Materials and methods. The accumulation of Smad2 and Smad3 in the nucleus was confirmed by western analysis using anti-Smad2 (Transduction Laboratories) and anti-Smad3 antibodies (Zymed Laboratories). The positions of Smad2 and Smad3 are indicated by the arrows.

Barbara A. Hocevar, et al. EMBO J. 2001 June 1;20(11):2789-2801.
7.

Figure. From: The adaptor molecule Disabled-2 links the transforming growth factor ? receptors to the Smad pathway.

Fig. 1. Dab2 expression mediates functional complementation of the 903 TGFβ-signaling mutant cell line. (A) Diagrammatic representation of the genetic complementation approach. The parental cell line BAHgpt expresses the E.coli gpt gene driven by the 3TP promoter. In media containing 6TG, TGFβ treatment leads to cell death, indicative of TGFβ responsiveness. Mutant cell lines that do not respond to TGFβ maintain growth in media containing 6TG plus TGFβ. (B) Transfection of Dab2 into 903 cells restores TGFβ responsiveness. Parental BAHgpt, and mutant 903, 903L2 and 903 cells transfected with human wild-type Dab2 (903WTDab2) were plated into media containing 6TG (30 µM) and 6TG (30 µM) plus 5 ng/ml TGFβ. After 5 days, cells were fixed with methanol and stained with hematoxylin to visualize growth. (C and D) Dab2 expression restores TGFβ-mediated induction of the 3TPLux (C) and SBE-Luc (D) reporter constructs. BAHgpt, 903, 903L2 and 903WTDab2 cells were transiently transfected with 3TPLux or SBE-Luc and SV40-RL as a control for transfection efficiency. Cells were treated with TGFβ for 18 h, followed by lysis and determination of luciferase activity. Luciferase activity is expressed as a ratio of 3TPLux luciferase activity divided by the SV40-RL activity. Shown is the mean ± SD of duplicates from a representative experiment. Open bars represent untreated cells while black bars denote TGFβ treatment.

Barbara A. Hocevar, et al. EMBO J. 2001 June 1;20(11):2789-2801.
8.

Figure. From: The adaptor molecule Disabled-2 links the transforming growth factor ? receptors to the Smad pathway.

Fig. 4. Restoration of TGFβ signaling requires both the PRD and PTB domain of Dab2. (A) Diagrammatic representation of various constructs of Dab2. Depicted are full-length and deletion constructs of Dab2 containing the N-terminal PTB domain and C-terminal PRD (gray shading). The asterisk designates the S634N mutation present in mutant 903 Dab2 (Mut-Dab2) and the introduced F166V mutation in full-length WTDab2 (F166V-Dab2). (B) Expression levels of various Flag-tagged Dab2 constructs from stable cell lines were determined by western analysis with α-Flag antibody (M2; Sigma). Lysate from the original mutant 903 cell line was used as a control (Cont.). (C and D) TGFβ-stimulated transcriptional activation requires both the PRD and PTB domain of Dab2. BAHgpt, 903 and 903 cells stably expressing the various forms of Dab2 described above were transiently transfected with the reporter constructs 3TPLux (C) and SBE-Luc (D), and SV40-RL as a control for transfection efficiency. After 24 h, cells were untreated (open bars) or treated (black bars) with TGFβ for 18 h. Following lysis, luciferase activity was determined and expressed as the ratio of specific luciferase activity divided by the SV40-RL activity. Shown is the mean ± SD of duplicates from a representative experiment.

Barbara A. Hocevar, et al. EMBO J. 2001 June 1;20(11):2789-2801.

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