FIG. 2.

The nuclear export of p37 protein is mediated by both the CRM1-dependent and CRM1-independent pathways. Subconfluent cultures of Vero cells were transiently transfected with the plasmids encoding GFP, GFPp37, GFP(1-160), GFP(158-234), or GFP(231-372). Forty-eight hours posttransfection, the subcellular localization of the different fusion proteins was visualized by fluorescence confocal microscopy (magnification, ×600) under different experimental conditions: (a) untreated cells; (b) cells incubated in energy depletion medium for 3 hours (−ATP); (c) cells treated with leptomycin B (20 ng/ml) for 3 hours (+LMB); and (d) cells simultaneously transfected with siRNA for CRM1 depletion and plasmids encoding the different GFP fusion proteins (+CRM1 siRNA). Experiments were performed at least three times, and representative images are shown. (e) Western blot analysis of CRM1 expression in Vero cells following transfection with siRNA for depletion of CRM1 (CRM1 siRNA) or with a nonsilencing control siRNA. Forty-eight hours after transfection, the cells were harvested, and Western blot analysis was performed using anti-CRM1 and anti-α-tubulin antibodies as described in Materials and Methods.