DEV00928 Supplemental Data

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• Figure 1 -

  Fig. S1. Stbm forms oligomers in vivo. (A) GST pull-down assays showing that the C-terminal intracellular tail of Stbm [GST-StbmIntra(303-584)] interacts in vitro with Stbm. GST-StbmIntra(303-584) also interacted with StbmDPBM, indicating that this direct interaction did not depend on the PBM of Stbm. This interaction was further confirmed using yeast 2-hybrid. Interaction between LexA-StbmIntra(303-584) and B42-Stbm(303-584) led to the expression of lacZ and LEU2 reporters in S. cerevisiae (see Materials and methods below). (B) Endogenous Stbm from pupal epithelia co-immunoprecipitated with GFP::Stbm using anti-GFP antibodies. Western blot analysis using anti-Stbm antibodies detected Stbm and GFP::Stbm in the lysates. Degradation products of GFP::Stbm were also detected in the immunoprecipitates. Co-immunoprecipitation of Stbm was not detected when extracts were prepared from a mixture of two distinct populations of pupae, one expressing GFP::Stbm but not Stbm (stbm^6c, arm-GFP::Stbm pupae), and the other expressing Stbm but not GFP::Stbm (wild-type pupae). Because interaction was not detected in extracts prepared from a mixture of flies expressing either only GFP::Stbm or only Stbm, we conclude that the Stbm-Stbm::GFP complex formed in vivo.

• Figure 2 -

  Fig. S2. Localization of Stbm, Pins and Dlg in dividing pI cells. z-section stack of the xy sections corresponding to Fig. 5C-E¢¢¢ showing the apicobasal and anteroposterior localization of Stbm (green), Pins (blue) and Dlg (red) in dividing pI cells at prometaphase.

  Materials and methods

  Co-immunoprecipitation was performed on pupal epithelia (head, legs, wings, notum) dissected from 20-26 hours after puparium formation (APF) pupae. For each genotype, 100 pupae were dissected and immediately frozen in dry ice. Tissues were homogenized in 0.5 ml of PBS, NP40 0.5%, PFA block (Boehringer Mannheim) and Protease Inhibitor Cocktail (Boehringer Mannheim). Clear lysates were obtained by centrifugation at 4°C (20 minutes, 12,000 rpm). Lysates were then pre-cleared with 30 ml of Protein A-agarose beads (Roche) pre-coupled with mouse anti-Cut antibodies (2B10 ascite, DSHB) for 1 hour at 4°C and then incubated with 30 ml of Protein A-agarose beads (Roche) pre-coupled with 5 mg of mouse anti-GFP (Santa Cruz; 4 hours at 4°C). Beads were then washed three times in lysis buffer. Immune complexes were analyzed by western blot analysis (anti-Stbm; 1:1000).

  The LexA yeast two-hybrid system originally developed in the Brent laboratory was used. Detailed protocols can be found at http://proteome.wayne.edu/finlabindex.html. A fragment of the stbm cDNA (amino acid 303-584) was sub-cloned into pJG4-5 and pEG202. The pJG4-5-Stbm plasmid was used to transform the RFY231 yeast strain while pJEG202-Stbm plasmid was introduced into the RFY206 strain. The pJG4-5-Cdi2 and pEG202-Cdc2 were used as control.