Localization of the recombinant SET protein after its internalization. Recombinant SET protein (4 μM) was internalized for 3 h and 5 h 30 min in mouse primary neurons (A, B) and for 5 h 30 min in mouse brain slices (E, F). Confocal microscopy shows immunocytochemical staining of recombinant SET carried-out with an anti-tag HA antibody. Nuclei were labeled with TO-PRO-3. Four examples at 3 h (A) and at 5 h 30 min (B) are shown. At 3 h, recombinant SET protein is mostly located in the cytoplasm around the nucleus, whereas at 5 h 30 min SET is distributed in both the cytoplasm and nucleus. One representative immunocytochemical staining out of three independent experiments is presented. Scale bar, 10 μm. (C) Electrophoresis of tag-HA recombinant SET protein (10 μg of protein was loaded). Coomassie blue shows a major band at 49 kDa, as expected. D-F) Tag-HA recombinant SET protein (0.4 μg) was detected by western blotting with a tag-HA antibody (D). Sub-cellular fractions were prepared from brain slices treated for 5 h 30 min with recombinant SET protein and 10 μg of proteins were loaded in each lane. Recombinant SET was detected with a tag-HA antibody (E). APP was used as a cytoplasmic (Cyt) control and histone was used as a nuclear (N) control. Recombinant full-length SET protein is observed at 49 kDa in nuclear and cytoplasmic fractions as in (B). Unspecific bands recognized by the secondary antibody are shown in (F). For (D-F), one representative immunoblot out of five independent experiments is shown and for (C), one protein staining of recombinant SET out of three independent experiments is presented (C).