FIG. 2.

Immunolocalization of Sec13 to both sides of the NPC in addition to cytoplasmic and intranuclear sites. (A) Immunoblot analysis of HeLa cell lysates performed with anti-Sec13 antibodies and probed with either preimmune serum (control, lane 1) or antibodies against Sec13 (lane 2). (B) Immunofluorescence and confocal microscopy of HeLa cells, which were fixed, permeabilized with saponin, and labeled with anti-Sec13 antibodies and mAb414 (labels some FG repeat-containing Nups). (C) HeLa cells transfected with GFP-Sec13 and analyzed by confocal microscopy show staining similar to the observed labeling of endogenous Sec13 shown in B and D. (D) HeLa cells were processed as in B except that anticalnexin antibodies were used. Endoplasmic reticulum labeling is observed. The merged image shows partial colocalization of Sec13 with calnexin at the endoplasmic reticulum. (E) Isolated rat liver nuclear envelopes were probed with rabbit anti-Sec13 antibodies and mouse anti-RanGAP1 monoclonal antibody 19C7. Rabbit and mouse antibodies were detected with 10-nm and 5-nm gold-coupled secondary antibodies, respectively. Envelopes were processed for thin sectioning and observed by electron microscopy. C, cytoplasmic side; N, nucleoplasmic side.