Figure 2: TRAF6 promotes Lys63-dependent polyubiquitination of wt TβRI but not of the E161A mutant TβRI.

(a) PC-3U cells transiently transfected with C-terminally HA-tagged wt TβRI, or E161A mutant TβRI deficient in association with TRAF6, were treated or not with TGFβ, whereafter ubiquitination of TβRI was examined by an in vivo ubiquitination assay. Complexes immunoprecipitated (IP) with anti-HA antibodies were immunoblotted (IB) with Lys63 (K63)-linked polyubiquitin-specific antibody. A light-chain specific secondary antiserum was used to avoid cross-reaction with IgG heavy chain. Molecular weight markers are indicated. Another part of the corresponding total cell lysates (TCL) was subjected to immunoblotting for HA to detect the TβRI-ICD fragment, and activation of p38 and Smad2, by phospho-specific antisera. The filters were then reprobed with total p38 or Smad2, to verify specificity of phospho-specific antisera and actin served as internal control for equal loading of proteins. (b) In vivo ubiquitination assays performed in PC-3U cells transiently transfected with HA-tagged wt, K63- and K48-only ubiquitin. Cell lysates were immunoprecipitated with V22 antibody against TβRI, and K63-dependent polyubiquitination was visualized by immunoblotting with P4D1-antiserum. A light-chain specific secondary antiserum was used to avoid cross-reaction with IgG heavy chain. The TCL-filter was subjected to immunoblotting with an HA-antibody to verify equal expression levels of ubiquitin. (c) PC-3U cells ectopically expressing C-terminally HA-tagged wt TβRI or the corresponding E161A mutant were stimulated with TGFβ for 0.5 h and thereafter stained with an HA antibody. Staining with DAPI was used to visualize cell nuclei. Scale bar 20 μm.