UBPY regulates Smo ubiquitination and cell surface expression.

<p>(A) Myc-Smo expressing cells were treated with or without Hh-conditioned medium in the presence of UBPY or Luc dsRNA. After treatment with MG132, cell extracts were prepared and immunoprecipitated with anti-Myc antibody, followed by Western blot analysis with anti-Ub or anti-Myc antibody. Of note, shorter exposure was used for Western blot analysis of samples derived from cells not treated with Hh (left). (B) S2 cells were transfected with Myc-Smo and HA-tagged Ub (HA-Ub) and with or without Flag-tagged UBPY (Fg-UBPY). After treatment with MG132, cell extracts were prepared and immunoprecipitated with anti-Myc antibody, followed by Western blot analysis with anti-HA or anti-Myc antibody. (C–D) S2 cells were transfected with Fg-UBPY and Myc-tagged wild type Smo or the indicated Smo variants and treated with or without Hh-conditioned medium. Western blot analyses were carried out on cell lysates or immunoprecipitates using the indicated antibodies. Asterisks indicate monomeric forms of Myc-Smo and Myc-Smo^ΔCT. (E–E”) Large magnification view of a wing disc carrying <i>UBPY</i> mutant clones and immunostained to show the expression of Smo (red channel) and GFP (green channel). <i>UBPY</i> mutant clones are marked by the lack of GFP expression. Posterior <i>UBPY</i> mutant clones had reduced cell surface accumulation of Smo (arrows). (F–J”) Wild type wing discs (F–F”, I–I”) or wing discs expressing <i>UAS-UBPY</i> alone (G–G”, J–J”) or together with <i>UAS-Smo-RNAi</i> (H–H”) under the control of <i>MS1096</i> were immunostained to show the expression of Smo (red), Ci (green), and <i>dpp-lacZ</i> or <i>ptc-lacZ</i> (blue). (K) Confocal images of S2 cells expressing Myc-Smo (red) alone or together with Fg-UBPY (green). Top panels show cell surface staining while bottom panels show regular staining.