PATJ enhances Myosin phosphorylation by inhibiting Myosin phosphatase. (A) Endogenous MBS coimmunoprecipitates with endogenous PATJ from embryonic lysates. The figure represents blots from different gels with 5% (PATJ blot) and 95% of the immunoprecipitation (IP) loaded. (B) MBS-myc coimmunoprecipitates with PATJ-GFP from lysates of transfected S2R+ cells. (C) PATJ binds directly to MBS. GST-PATJ and MBP-MBS were expressed in E. coli and purified. GST alone served as negative control. Inputs are shown on Coomassie-stained gel. (D and E) Localization of endogenous MBS during cellularization (D) and in mature epithelial cells of the epidermis (E; junctional MBS is marked by arrows). (F) Overexpression of PATJ-HA in stripes using an engrailed::GAL4 driver line stabilizes/recruits Sqh-GFP in the embryonic epidermis. Sqh-GFP was expressed under its endogenous promoter (Royou et al., 2002). Here, we used an insertion on the third chromosome, resulting in a rather low protein expression. Similar results were obtained using a ubiquitous promoter (polyubiquitin; not depicted). (G) Segmental overexpression of PATJ-HA results in an increased phosphorylation of Sqh in embryos heterozygous for mbs^T541. (H) Follicle cell clones for PATJ^Δ1 showing decreased phosphorylation of Sqh at the apical junction (arrows). PATJ mutant clones are marked by the absence of GFP. UAS, upstream activation sequence. Bars: (D–F) 5 µm; (G and H) 10 µm.