1. Itch E3 Ligase-Mediated Regulation of TGF-β Signaling by Modulating Smad2 Phosphorylation 
Yongli Bai, Chun Yang, Kathrin Hu, Chris Elly, Yun-Cai Liu 
Molecular Cell 
Volume 15, Issue 5, Pages (September 2004)
DOI: /j.molcel

2. Figure 1
Reduced Cell Growth Inhibition of Itch−/− MEFs to TGF-β Treatment
(A) Equal numbers of MEF cells prepared from Itch+/− or Itch−/− embryo were plated on 96-well plate in triplicate. TGF-β (4 ng/ml) was added and cell proliferation was analyzed by measuring 3H-thymidine incorporation. Data are expressed as the percentage of cell growth with the value from untreated cells as 100%. Representatives of five repeated experiments are shown.
(B) Dose-dependent responses of MEFs. Cell proliferation was conducted as in (A). Representatives of three repeated experiments are shown.
(C) TGF-β, IL-1, or TNF-α was added to Itch+/− or Itch−/− MEFs. The effects of each cytokine on MEF proliferation were measured. The data are the mean of three experiments from three different pairs of MEF isolates plus standard errors.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2 Analysis of Signaling Molecules in Itch+/− and Itch−/− MEFs
(A) The Smad proteins are stable in Itch−/− MEFs. The protein levels of Smad2, Smad4, or Smad7 were examined in MEFs upon TGF-β stimulation. The equal loading of whole-cell lysates was confirmed with anti-actin antibody. Anti-Itch was also used to probe the blot to show Itch-positive MEFs.
(B) Reduced Smad2 phosphorylation in Itch−/− MEFs. Phosphorylation levels of Smad2 were detected by using anti-phospho-Smad2/3 antibody (top panel). The higher band comigrates with Smad2. The lower band may represent phospho-Smad3. The upper band was quantitated and the intensity at 0 hr for each sample was normalized as 1. The same blot was reprobed with anti-Smad2 (bottom panel).
(C) Accumulation of SnoN in Itch−/− MEFs. The protein level of SnoN was examined using anti-SnoN. The protein band corresponding to the molecular weight of SnoN is indicated (arrow). Actin was also probed to show equal input of total cell extract. The same membrane was reprobed with anti-Itch antibody.
(D) Half-lives of Smad2 are similar in Itch+/− and Itch−/− MEFs. Metabolically labeled MEF cells were subjected to immunoprecipitation using antibodies as indicated. The relative intensity of the radiolabeled proteins was quantitated and indicated below each lane. The intensity at the start of chase (0 hr) for each sample was normalized as 1.
(E and F) The induction of PAI-1 mRNA (E) and its protein expression (F) in MEFs upon TGF-β treatment (11 hr). The total RNA was serially diluted in (E).
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3 Ubiquitination of Smad2 by Itch E3 Ubiquitin Ligase
(A) HEK 293T cells were transfected with plasmids encoding HA-Ub (1 μg), Flag (F)-Smad2 (1 μg) in the absence or the presence of Itch or Itch CA plasmid (200 ng). Transfected cells were left unstimulated or were stimulated with TGF-β for 30 min. The cell lysates were subjected to immunoprecipitation with anti-Flag antibody, and the immunoprecipitates were blotted with anti-HA. The slow-migrating protein smears indicated polyubiquitinated Smad2 (Ub-Smad2). The same membrane was reprobed with anti-Smad2. Aliquots of cell lysates were immunoblotted with anti-Itch antibody.
(B) Cells were transfected with the indicated plasmids. The samples of anti-Smad2 immunoprecipitates were immunoblotted with anti-Smad antibody. The Ub-conjugated Smad2 ladders are indicated. Aliquots of cell lysates were blotted with anti-Smad2 or anti-Itch as indicated.
(C) In vitro ubiquitination of Smad2 by Itch. Flag-Smad2 was purified with anti-Flag agarose from Smad2-overexpressing 293T cells. Ubiquitination reactions were conducted in the presence of purified GST or GST-Itch as well as E1, E2, and ATP. Beads were washed three times and resolved with SDS-PAGE, followed by immunoblotting with anti-Smad2 or with anti-Ub antibody.
(D) Endogenous ubiquitination of Smad2 in MEFs. Itch+/− or Itch−/− MEFs were transfected with HA-Ub plasmid. Lysates prepared from control cells or cells stimulated with TGF-β for 1 hr were immunoprecipated with anti-Smad2. The immunoprecipitates were blotted with anti-HA antibody, and the same membrane was probed with anti-Smad2. Aliquots of cell lysates were immunoblotted with anti-Itch antibody.
(E) Itch associates with Smad2 in MEFs. MEF cells with or without TGF-β stimulation were immunoprecipitated with anti-Smad2 or with anti-CD28 as a negative control, and the samples were immunoblotted with anti-Itch antibody. Aliquots of cell lysates were immunoblotted with anti-Itch antibody.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4 Positive Regulation of Smad2 Function by Itch in 293 Cells
(A) Itch promotes TGF-β-mediated gene transcription. HEK 293H cells were transfected with plasmid containing a 3TP-lux reporter (800 ng) plus empty vector, Itch or Itch CA plasmid (200 ng), and Smad2 plasmid (50 ng), together with a reporter plasmid for β-galactosidase (200 ng) as a transfection control. The cells were left untreated or were stimulated with TGF-β for 24 hr. TGF-β responsiveness was evaluated by measurement of the relative luciferase activity normalized to the β-galactosidase absorbance at OD405 nm and presented as arbitrary units. Data represent three repeated experiments.
(B) Receptor, Smad2, and Itch complex formation in 293T cells. 293T were transfected with indicated plasmids, and the cells were stimulated with TGF-β for 30 min before lysis. Anti-Flag immunoprecipitates were immunoblotted sequentially with antibodies as indicated. The cell lysates were also immunoblotted with the indicated antibodies.
(C) Itch associates with the TGF-β receptor in 293T cells. Cells were transfected as in (A), and the cell lysates were immunoprecipitated with anti-TGF-β receptor I and immunoblotted with anti-Itch antibody.
(D) Itch HECT domain is required for Itch-augmented TGF-β receptor-Smad2 complex formation. Plasmids containing Flag-Smad2 (1 μg), a HA-tagged constitutively active form of TGF-β receptor (TGF-β R*, 100 ng), Itch (200 ng), or Itch mutant lacking the HECT domain (ΔHECT, 200 ng) were cotransfected into 293 cells. The immunoprecipitates of anti-Flag antibody were immunoblotted with antibodies as indicated. Aliquots of cell lysates (CL) were also probed with corresponding antibodies.
(E) Itch enhances Smad2 phosphorylation in 293 cells. The cells were transfected with plasmids containing Flag (F)-Smad2 (1 μg), Itch, or Itch CA cDNA (200 ng), and the transfected cells were stimulated with TGF-β. Phosphorylation of Smad2 was examined by immunoblotting the cell lysates with anti-phospho-Smad2/3 antibody. The same membrane was reprobed with anti-Smad2 or anti-Itch.
(F) Ubiquitination of Smad2 lacking the phosphorylation sites SSXS. Smad2 or Smad2 deficient in the C-terminal SSXS (Smad2Δ) was transfected into 293T cells plus 5 ng of plasmids containing TGF-β receptors I and II. Anti-Flag immunoprecipitates were immunoblotted with anti-HA to detect Smad2 ubiquitination. The cell lysates were also immunoblotted with anti-phospho-Smad2 or anti-Itch as indicated.
Molecular Cell  , DOI: ( /j.molcel )