Nuclear localization of Annexin A7. (A) Extraction of Annexin A7 from nuclei of neuronal (PC-12, N2A) and an astroglial (C6) cell line using a hypotonic buffer. Samples of total cells (c), and corresponding amounts of nuclear membranes (m) and nucleoplasm (p) were subjected to SDS-PAGE and Western blotting. Annexin A7 (AnxA7) isoforms are extractable from the nucleus of the indicated cells (47 kDa and 51 kDa isoform, arrowhead). For control, immunoblotting of LAP2α (75 kDa; non-membrane-bound isoform), Emerin (EM, 34 kDa), and tubulin (TB, 55 kDa) which are specific for nucleoplasm (LAP2α), nuclear membranes (Emerin), and the cytoplasm (tubulin) are shown. Note that the nucleoplasm is only partially extractable. (B) Immunofluorescence images of the PC-12 cells used. Cells were fixed with paraformaldehyde, permeabilized with Triton X-100, pretreated with trypsin as indicated, and stained with Annexin A7 specific mAb 203–217, DAPI in blue, bar 10 μm.