Fig. 7. Role of Drosophila Cni (dCni) in Grk processing and secretion. (A) S2 cells were transfected to express Grk, Star, B-Rho and/or a secreted form of Grk (secGrk). Grk expression was visualized by immunoprecipitation and/or blotting with an antibody against the Grk ectodomain. (B) dCni enhanced the level of non-processed Grk. S2 cells were transfected to express Myc-Grk, Star, B-Rho and/or dCni-Flag. Myc-Grk in cell lysates and Grk in the media were visualized using anti-Grk ectodomain antibody. (C) Silencing of dCni in S2 cells causes accumulation of processed Grk (pGrk) in the presence of Star and B-Rho, and reduces Grk secretion. Top panel: downregulation of endogenous dCni mRNA expression by dsRNA-dCni, as assessed by RT-PCR. Lower panel: S2 cells were transfected to express Grk, B-Rho and/or Star, and treated with dsRNA-dCni. Grk expression was visualized, as in B. Accumulation of pGrk with dsRNA-Cni treatment was 2.7 fold and secretion of secGrk was 0.5 fold. Treatment with a lysosome inhibitor (chloroquine; 0.5 mM) does not affect pGrk relative accumulation and secretion. (D) Effect of dsRNA-Star on Grk processing and secretion. Top panel: downregulation of endogenous Star mRNA by dsRNA-Star, assessed by RT-PCR. Lower panels: S2 cells were transfected to express Grk, B-Rho, Star and/or dCni-Myc, and treated with dsRNA targeting Star. Grk was visualized using anti-Grk ectodomain antibody. (E) Silencing of dCni does not affect the general secretory pathway in cells transfected with secretable GFP (Grk-signal peptide-GFP). Dividing vertical white lines indicate parts from the same gel (A,E).