Antibody production, Western results and subcellular localization
Peptides from Acdp1 N- (TSFLLRVYFQPGPPATAAPVPSPT) and C- (TQQLTLSPAAVPTR) terminuses, conserved peptide from ACD domain of Acdp1 (HNIVDILFVKDLAFVDPDDCTPLLTVTRF) were commercially synthesized (Sigma Genosys). These antigenic sites were predicted by software from Sigma Genosys and polyclonal antibodies for each peptide were produced by immunizing rabbits. To test the specificity of the antibodies, we conducted Western-blot analysis of mouse brain tissue extracts. As shown in Fig. 6A, the antibody produced by C-terminal peptide specifically detected Acdp1 (lane 3). The antibody generated by N-terminal peptide recognized Acdp4 in addition to Acdp1, although the reactivity to latter was significantly higher (Fig. 6A, lane 2). As expected, the antibody produced by the conserved sequence peptide detected all Acdp proteins (Fig. 6A, lane 1). To further determine the specificity of the antibody against the Acdp1 C-terminus, we analyzed extracts of HEK293, 3T3 and PC12 cells. The results are shown in Fig. 6B. Apparently, this antibody detected a specific band of Acdp1 in all cell lysates. Of note, shown in Fig. 6B are signals of 10 μg extracts of HEK293 cell lysates, 100 μg extracts of 3T3 and PC12 cell lysates. Thus, the expression levels of Acdp1 in these cell types vary a lot, with the highest expression in HEK293 cells. Nevertheless, these immunoblot results support our analysis of brain tissue extracts that the antibody against Acdp1 C-terminus specifically recognizes Acdp-1.
The specificity of the Acdp1 C-terminus antibody suggests the possibility of using it to localize Acdp-1 within cells. Since Northern blot revealed almost exclusive expression of Acdp1 in the brain, we examined its subcellular localization in hippocampus neurons isolated from mouse embryos. The neurons were cultured on glass coverslips coated with a confluent monolayer of mouse cortical astrocytes in dishes. Immunostaining was using the Acdp1 C-terminus specific antibody. Confocal imaging revealed that Acdp1 is predominantly localized on the plasma membrane. A series of sections of a cell at the thickness of 0.5 micrometer clearly showed membrane location of Acdp1-immunoreactivity (Fig. 7), which is consistent with the observation of transmembrane domains within the Acdp proteins.
Figure 6  Fig. 6A: Immunoblot analysis of Acdp proteins in brain tissue extracts. Immunoblotting were carried out using a Western blotting detection system (ECL) (PIERCE). Lane 1, probed with antibody generated by conserved peptide. From top to bottom, each band corresponding to Acdp1 (115 kD), Acdp2 (100 kD), Acdp4 (90 kD) and Acdp3 (80 kD). Lane 2 and 3, probed with the Acdp1 antibodies generated by N-terminal and C-terminal peptides, respectively. Fig. 6B: Immunoblot analysis of Acdp1 in HEK293, 3T3 and PC12 cells. The blots were probed with the antibody against the C-terminus of Acdp-1. Lane 1, 10 μg of HEK293 cell lysates. Lane 2 and 3, 100 μg of 3T3 and PC12 cell lysates.
Figure 7  Subcellular localization of Acdp1 in hippocampus neurons. A series of confocal images from a cultured neuron stained with an anti-Acdp1 antibody. The step of each imaging section is 0.5 μm, from the surface of the neuron (0 μm) to the middle plan (4.5 μm).