Fig. 6. Block of membrane fusion arrests NPC assembly at a defined stage. (A) Nuclear formation assays were performed as before with extract and sperm chromatin. Reactions were run after addition of buffer or 100 µM -SNAP_L294A with or without sperm chromatin, as indicated. After 60 minutes, reactions were diluted, chromatin recovered by centrifugation through a sucrose cushion and analyzed by western blotting with antibodies specific to proteins indicated on the right. (B) The experiment as shown in A was repeated, but this time including a reaction lacking membranes. Nup107 was detected as indicated. LAP2 served as a membrane marker and the DNA-binding protein MCM3 as a control for equal chromatin recovery. (C) Samples from an identical experiment to that in A were fixed. Nup107- and FxFG-containing nucleoporins were detected by immunofluorescence microscopy using Nup107-specific or mAb414 antibodies, respectively, as indicated.