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. 2005 Aug;25(15):6578–6591. doi: 10.1128/MCB.25.15.6578-6591.2005

FIG. 7.

FIG. 7.

Silencing function of wild-type and mutant Psc proteins in vivo. (A) Wing imaginal disks immunostained for Abd-B (red) and GFP (green), which serves as a marker for wild-type cells. Abd-B is not expressed in wild-type cells (top left) but is strongly misexpressed in clones lacking Psc and Su(z)2 (marked by lack of GFP signal) in the absence of a Psc transgene (no TG); the mutant clones also display a tumor-like phenotype (5). Transgenes expressing wild-type Psc or PSC1-872 restore silencing of Abd-B and prevent the tumor-like phenotype in the mutant clones. In comparison, PSC1-572 and PSC456-1603 are ineffective. Psc− Su(z)2− mutant clones sort out from the surrounding wild-type tissue and form vesicles; in the “no TG,” “hs-Psc1-572,” and “hs-PSC456-1603” images, all cells in each of the clones express Abd-B, but not all cells are visible in this single confocal section. (B) Ectopic expression of Abd-B in some cells in the nervous system (arrows) (not all cells showing ectopic expression are visible in this focal plane) when PSC1-572, but not wild-type Psc, PSC1-872, or PSC456-1603, is overexpressed in embryos. Note that under the conditions used to express Psc proteins in imaginal disks in the experiment shown in Fig. 7A (see Materials and Methods), expression of PSC1-572 does not result in ectopic expression of Abd-B protein in wild-type (GFP-positive) imaginal disk cells. (C) Expression of mutant forms of Psc in transgenic embryos. Western blot analysis of embryonic extracts (EE) from transgenic embryos expressing PSC1-572, PSC1-872, and PSC456-1603, respectively. Embryos were not heat shocked (−hs) or were heat shocked (+hs) for 30 min and then were incubated for another 30 min prior to extract preparation. In each case, a Psc protein signal of the expected size is present only in extracts from heat-shocked embryos; the corresponding recombinant Psc proteins (RP), purified from baculovirus, were loaded as size references. The blot was stripped and reprobed with an antibody against alpha-tubulin to demonstrate comparable loading of extracts. We could not detect the endogenous wild-type Psc protein, suggesting that transgene-encoded Psc proteins are expressed at significantly higher levels.