To verify the presence of endogenous Annexin A7 in nuclei, we used a biochemical approach and purified nuclei from neuro-2a, PC-12, and C6 cells and treated them with a hypotonic extraction buffer to obtain the nucleoplasm (Fig. 7A). The nucleoplasm and the remaining nuclei, from which the nucleoplasm has been extracted partially, were subjected to SDS-PAGE and Western blotting. Western blots probed with mAb 203–217 showed Annexin A7 in the nucleoplasm of neuronal (neuro-2a, PC-12) and astroglial (C6) cell lines (Fig. 7A), however, the large isoform could only be clearly extracted with nucleoplasm from C6 cells. For control, antibodies against LAP2α, Emerin and tubulin were used to verify that the nucleoplasm (marker:LAP2α, which is anon-membrane-bound isoform of LAP2) was successfully extracted and also not contaminated by nuclear membranes (marker:Emerin) or by cytoplasm (marker:tubulin). Likewise, in these neuronal and astroglial cell lines Annexin A7 had been observed in the nucleus by immunofluorescence (Fig. 7B and data not shown). We noticed no difference between PC-12 and neuro-2a cells, however, as for the brain sections, we found an increase of the Annexin A7 staining after pre-treatment with trypsin.