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Fig. 4. Expression of S182N stabilizes Ci in its unprocessed, 175 kDa form in wing imaginal discs. (A-D) Stabilization of Ci shown by immunostaining of discs using a monoclonal antibody recognizing the unprocessed form of Ci only (Ci^FL). (A) A wild-type disc showing normal levels of stabilization of Ci at the anteroposterior boundary. Ci^FL signal is strongest along the AP boundary (bracket). (B) Over-expression of Cos2 using the 71B Gal4 driver results in reduced levels of Ci^FL at the AP boundary when compared with wild type (bracket). (C) Expression of S182N using the 71B Gal4 driver results in a wider stripe of cells at the AP boundary positive for Ci^FL expression (bracket). Cos2-GFP expression driven by 71B Gal4 is shown in D. The same expression pattern is achieved for the expression of S182N driven by 71B Gal4 (not shown). (E) A protein blot of wing imaginal disc extracts probed with anti-Ci antibody 1C2 (a gift from Robert Holmgren), which recognizes both processed (75 kDa) and unprocessed (175 kDa) forms of Ci. Lane 1, wild-type imaginal disc extract; lane 2, extract from discs overexpressing Hh; lane 3, extract from discs overexpressing Cos2; lane 4, extract from discs simultaneously overexpressing Cos2 and Hh; lane 5, extract from discs overexpressing S182N; lane 6, extract from discs overexpressing S182T Cos2. 71B Gal4 was used to drive expression of all transgenes indicated. (F) Quantitation of three independent western blots is plotted as relative amounts of Ci^R to Ci^TOTAL (y-axis) in wing disc lysates corresponding to genotypes expressing the UAS transgenes indicated driven by 71B Gal4 (x-axis).

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