Maturation, processing, and targeting of newly identified SRP-dependent proteins (de novo expression). (A-D) Immunoblot analysis of HeLa cell lysates expressing tetracycline-induced PRL-GFP, GUSB-GFP, PTX-GFP, and CRISP-GFP. Cells were treated with control or SRP small interfering RNAs (siRNAs) and processed for immunoblotting with the indicated antibodies. Experiment performed in triplicate. (E) Confocal microscopy images of HeLa cells depleted of endogenous SRP proteins by siRNA and stably expressing PTX3-GFP. Cells were immunostained with an anticalnexin antibody, and DNA was visualized with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 10 μm. Experiment performed in triplicate. (F) Confocal images of HeLa cells depleted of endogenous SRPs by siRNA and stably expressing CRISP3-GFP. Cells were immunostained with an anti-GM130 antibody and DNA was visualized with DAPI. Scale bar, 10 μm. Experiment performed in triplicate. (G) Quantification of the colocalization of CRISP3-GFP with GM130. Unpaired t test, 2-tailed (P < .0001 and P < .005). Quantitative analysis represents 3 independent experiments.