Figure 1. A 50 kDa fragment of the cytoplasmic domain of ErbB3 is expressed in the nucleus of Schwann cells. A, Immunostaining of purified rat primary Schwann cells with ErbB3 antibodies specific for the intracellular domain (C-ErbB3) or the extracellular domain (N-ErbB3) of ErbB3. Both pictures represent median images at the level of the nucleus from z-stacks (12 μm total with 2 μm increments) at 20× magnification. C-ErbB3 displayed staining in Schwann cell cytoplasm, membrane, perinuclear region, and nucleus (inset shows distinct pattern of speckled nuclear staining at 60×), whereas N-ErbB3 staining was restricted to the membrane, cytoplasm, and perinuclear region (inset shows lack of nuclear staining at 60×). B, Total protein lysates of rat primary Schwann cells blotted with two antibodies against separate epitopes of the cytoplasmic domain of ErbB3. This reveals the full-length ErbB3 band at 180 kDa and additional bands at 120, 80, and 50 kDa. Incubation with an ErbB3 blocking peptide abrogates all bands, suggesting that these are ErbB3 specific. Densitometric analysis from three independent experiments shows that the 50 kDa band corresponds to ∼1/50th of the density of the full-length ErbB3 band. β-Actin was used a loading control antibody. C, Protein lysates of Schwann cells were separated into cytoplasmic and nuclear fractions and blotted with an antibody that recognizes the cytoplasmic portion of ErbB3. This revealed the existence of the full-length ErbB3 receptor (∼180 kDa band) in the cytoplasmic fraction (left panel) and the ∼50 kDa band in the nuclear fraction (right panel). The purity of the cytoplasmic and nuclear fractions was confirmed by the expression of ATPase and Lamin A/C in the corresponding lysates. Specificity of the ErbB3 antibody is verified with preincubation with an ErbB3 blocking peptide, which eliminates antibody binding in both the cytoplasmic and the nuclear lysates.