Activation of Smurf E3 Ligase Promoted by Smoothened Regulates Hedgehog Signaling through Targeting Patched Turnover
Figure 1
Identification of Ptc as a Smurf-interacting protein.
(A) A yeast two-hybrid screen was performed that identified Ptc as a Smurf-interacting protein. The AH109 yeast strain was transformed with the indicated plasmids and plated at permissive (−Leu/−Trp) and restrictive (−Ade/−His/−Leu/−Trp) medium. Full-length smurf were cloned into the bait vector, pGBKT7, as the bait for library screen; and pACT2 is the prey vector. B4/5 (aa 1174–1286) and L1 (aa 1198–1286) were two independent Ptc clones from the screen. (B and C) S2 cells were transfected with combinations of DNA constructs as indicated. After 48 h transfection, lysates from transfected S2 cells were immunoprecipitated with anti-Flag M2 affinity gel (B) or anti-Myc affinity gel (C). Western blots were performed to analyze the presence of Flag- or Myc-tagged proteins. (D) S2 cell lysates were immunoprecipitated with mouse anti-Ptc antibody or control mouse IgG. Western blots were performed to analyze the presence of Ptc or Smurf proteins. (E and F) Schematic drawings of Ptc (E) and Smurf (F) and their deletion constructs. (G–I) S2 cells were transfected with combinations of DNA constructs as indicated. After 48 h transfection, lysates from transfected S2 cells were immunoprecipitated with anti-Flag M2 affinity gel. Western blots were performed to analyze the presence of Flag- or Myc-tagged proteins.