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. Author manuscript; available in PMC: 2014 Oct 1.
Published in final edited form as: Cancer Discov. 2013 Jul 9;3(10):1172–1189. doi: 10.1158/2159-8290.CD-12-0499

Figure 4. DEAR1 binds to SMAD3 and induces SMAD3 ubiquitination.

Figure 4

(A) Western analysis of endogenous phospho-SMAD3, SMAD3, SMAD2 and DEAR1 expression in DEAR1-knockdown (DshR) and control (CshR) 76N-E6 HMECs. Cells were treated with TGFβ (2ng/ml) for different time points. Note dramatic increase in SMAD3 expression in DEAR1 knockdown cells. (B) Western analysis of endogenous SMAD3, SMAD2 and DEAR1 expression in DEAR1-knockdown (DshR) and control (CshR) 76N-E6 HMECs. Cells were treated with 100 ug/ml cycloheximide (CHX) or 25 uM MG132 for indicated time points. (C) Relative density analysis of Westerns (B) showing SMAD3 expression in DEAR1 knockdown versus controls at various time points following either CHX or MG132 treatment. (D) Co-immunoprecipitation (Co-IP) of HA/DEAR1 and Myc/SMAD proteins. HA/DEAR1 (HADR1) was transiently cotransfected with Myc/vector (Myc), Myc/SMAD2 (SM2), Myc/SMAD3 (SM3) or Myc/SMAD4 (SM4) into HEK293T cells shown in the left panel and Myc/SMAD3 was transiently cotransfected with HA/vector or HA/DEAR1 shown in the right panel. After 24 hours, cells were lysed in M-Per buffer (Pierce) and the lysates were immunoprecipitated with rabbit anti-HA antibody. SMAD proteins were blotted with mouse anti-Myc antibody. (E) High magnification deconvolution confocal images of immunofluorescence staining of HA/DEAR1 and Myc/SMAD2 and Myc/SMAD3. HA/DEAR1 was cotransfected with Myc/SMAD2 (panel i) or Myc/SMAD3 (panel ii) into HEK293T cells. Yellow arrows showed colocalization of DEAR1 and SMAD3. (F) Co-IP of endogenous SMAD3 and DEAR1 in HeLa cells which express both DEAR1 and SMAD3 proteins. Cell lysate was precipitated with either control anti-IgG or anti-SMAD3 (SM3) antibody. * indicates a band corresponding to the IgG heavy chain and (NS) indicates a non-specific band. (G) GST pull down assay indicates that DEAR1 directly binds SMAD3. GST-DEAR1 (GST/DR1) or control GST was added to HEK293T cell lysate expressing Flag/SMAD3 and GST was pulled down with anti-GST beads. SMAD3 was probed with a Flag antibody. (H) DEAR1 induces SMAD3 ubiquitination. DEAR1 (DR1) or empty vector (Vec) was transiently transfected with HA/Ubiquitin (HA-Ub) and Flag/SMAD3 (Fl/SM3) into HEK293T cells. Lysates were immunoprecipitated with anti-Flag antibody and probed for ubiquitination with an HA-antibody.