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. 1998 Jun 15;141(6):1415–1422. doi: 10.1083/jcb.141.6.1415

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The BRUCE protein is associated with endomembranes. (A) Western blot of membrane fractions from mouse brain, mouse AtT20 (neuroendocrine), mouse N2a (neuroblastoma), and rat PC12 (neuroendocrine) cell lines probed with BRUCE-specific antibodies (antibody C). (B) Immunoprecipitation of in vivo–radiolabeled proteins with BRUCE-specific antibodies (antibodies C and N) from cytosolic (S, supernatant) and membrane (P, pellet) fractions (see Materials and Methods). BRUCE is enriched in membrane fractions (see below) and can be extracted by 0.5 M NaCl (compare lanes S and P) or buffer (PBS; compare lanes S and P). Antibodies specific for the ER-lumenal protein disulfide isomerase (PDI) and synaptophysin, an integral membrane protein of synaptic vesicles, were used for detection by Western blotting from the identical protein fractions. These proteins used as controls were found in the pellet fraction. A portion of lumenal PDI was also detectable in the cytosolic fraction (S, first lane), presumably because some microsomal vesicles were “inside-out” or disintegrated. (C) In contrast to lumenal PDI, BRUCE is sensitive to proteinase K treatment at membranes in vitro, indicating that BRUCE faces to the cytosol. (D) Confocal micrographs of rat PC12 cells and primary rat neurons stained with antibodies (indirect immunofluorescence) specific for BRUCE (green, antibodies C and N), and the marker proteins (red) TGN38 (marker for TGN; Humphrey et al., 1993), MAP2 (marker for dendrites), and TAU (marker for axons). Note that BRUCE appears in a punctated cytosolic distribution and is present in the TGN, axons, and dendrites. Colocalization of BRUCE with marker proteins is shown by superimposition (yellow). The BRUCE-specific antibodies used in these studies (A–D) are affinity-purified, and the signals are specific since they could be completely eliminated by competition with epitope-containing protein fragments (not shown).

The BRUCE protein is associated with endomembranes. (A) Western blot of membrane fractions from mouse brain, mouse AtT20 (neuroendocrine), mouse N2a (neuroblastoma), and rat PC12 (neuroendocrine) cell lines probed with BRUCE-specific antibodies (antibody C). (B) Immunoprecipitation of in vivo–radiolabeled proteins with BRUCE-specific antibodies (antibodies C and N) from cytosolic (S, supernatant) and membrane (P, pellet) fractions (see Materials and Methods). BRUCE is enriched in membrane fractions (see below) and can be extracted by 0.5 M NaCl (compare lanes S and P) or buffer (PBS; compare lanes S and P). Antibodies specific for the ER-lumenal protein disulfide isomerase (PDI) and synaptophysin, an integral membrane protein of synaptic vesicles, were used for detection by Western blotting from the identical protein fractions. These proteins used as controls were found in the pellet fraction. A portion of lumenal PDI was also detectable in the cytosolic fraction (S, first lane), presumably because some microsomal vesicles were “inside-out” or disintegrated. (C) In contrast to lumenal PDI, BRUCE is sensitive to proteinase K treatment at membranes in vitro, indicating that BRUCE faces to the cytosol. (D) Confocal micrographs of rat PC12 cells and primary rat neurons stained with antibodies (indirect immunofluorescence) specific for BRUCE (green, antibodies C and N), and the marker proteins (red) TGN38 (marker for TGN; Humphrey et al., 1993), MAP2 (marker for dendrites), and TAU (marker for axons). Note that BRUCE appears in a punctated cytosolic distribution and is present in the TGN, axons, and dendrites. Colocalization of BRUCE with marker proteins is shown by superimposition (yellow). The BRUCE-specific antibodies used in these studies (A–D) are affinity-purified, and the signals are specific since they could be completely eliminated by competition with epitope-containing protein fragments (not shown).