Figure 4

(A) HEK293T cells were cotransfected with vectors encoding Myc-tagged ubiquitin, HA-tagged Nedd4L, and Flag-tagged Smad3 (WT or mutant). Whole cell extracts were subjected to anti-Flag immunoprecipitation, followed by immunoblotting with anti-HA and anti-Myc antibodies. (B) As in (A), except that wild-type (WT) or catalytically inactive mutant (DD) form of HA-Nedd4L was cotransfected with WT Flag-Smad3 and Myc-ubiquitin. (C) As in (A), except that full-length (FL), MH1+linker (NL), or linker+MH2 (LC) fragments of Smad3 was cotransfected with WT HA-Nedd4L and Myc-ubiquitin. (D) Wild-type (WT) or catalytically inactive (mut) HA-SCP2 was cotransfected with HA-Nedd4L, Flag-Smad3, and Myc-ubiquitin in HEK293T cells. Whole cell extract and Flag immunoprecipitates were immunoblotted with indicated antibodies. (E) Control HaCaT cells or cells expressing shRNA targeting Nedd4L were transfected with Myc-tagged ubiquitin. 24 h post-transfection cells were stimulated with TGFβ for 1 h. The Smad2/3 immunoprecipitates or whole cell lysates were subjected to western immunoblotting with the indicated antibodies. (F) As in (E), except that HaCaT cells stably expressing Flag-tagged Smad3 in wild-type, AY, or mut forms were used.