44 The Journal of Cell Biology | Volume 156, Number 1, 2002 KH domain not found in KSRP, and significant sequence differences in the 5 end of the mRNA. ZBP2 is associated with the zipcode in chicken brain and fibroblasts To facilitate our studies on the role of ZBP2 for mRNA lo- calization, we generated antibodies by injecting gel-purified ZBP2 into rats. The specificity of ZBP2 antibodies was de- termined by Western blotting analysis (Fig. 4 A), in which the rat antibodies against ZBP2 specifically recognized ZBP2 in both fibroblast and brain extracts, as well as in af- finity-purified protein fractions (Fig. 4 A, lanes 1–3). To ensure that ZBP2 in brain extracts was the protein that formed complexes M and S with the zipcode, we immu- nodepleted ZBP2 from brain extracts and performed RNA mobility shift assays to detect RNA–protein complexes (Fig. 4 B). When the supernatants from an immunoprecipitation with ZBP2 antibodies were incubated with the zipcode, the specific RNA–protein complexes M and S were greatly re- duced (Fig. 4 B, lane 3). The immunoprecipitation with anti-ZBP2 antibodies appeared to be specific, as complex F was only slightly reduced (Fig. 4 B, lane 3). In contrast, when the RNA mobility shift assay was performed with the supernatants after immunoprecipitation by a control rat pre- immune serum, no reduction in the intensity of the ex- pected RNA–protein complexes was found (Fig. 4 B, lane 2). This confirmed that the formation of complexes M and S was dependent on the presence of ZBP2. The formation of complex F was likely dependent on ZBP1, which is not rec- ognized by the ZBP2 antibodies. To verify that ZBP2 was immunodepleted from extracts with anti-ZBP2 antibodies, the same supernatants used in the above mobility shift assay were analyzed by Western blotting (Fig. 4 C). The result showed that in contrast to control and rat preimmune se- rum–treated supernatants (Fig. 4 C, lanes 1 and 2), ZBP2 had been removed by the anti-ZBP2 antibodies (Fig. 4 C, lane 3). Developmental regulation of ZBP2 To determine the expression pattern of ZBP2 in developing neurons, protein extracts were prepared from different devel- opmental stages of chicken brain and analyzed for ZBP2 lev- els by Western blotting (Fig. 5). The highest expression of ZBP2 was seen in 6-d embryos (Fig. 5, 6 d), the level of ex- pression was reduced to 30% before hatching and remained stable thereafter (Fig. 5). In contrast, the amount of -actin protein is nearly constant in the same time points. This is consistent with the events during neural embryogenesis, when axons and dendrites are at maximal growth, and also coincides with the expression of ZBP1 (unpublished data). Cellular localization of ZBP2 To investigate the subcellular localization of ZBP2 and whether ZBP2 was associated with -actin mRNA in vivo in fibroblasts and neurons, we performed a combined assay, us- ing in situ hybridization with fluorescent-labeled oligo nu- cleotide probes for -actin mRNA (red; Fig. 6, A, left panel, and B), and with antibodies for ZBP2 (green; Fig. 6, A, middle panel, and B). ZBP2 is predominantly present in cell Figure 3. Complete sequence of ZBP2 compared with human KSRP. Proteins in the 2.0 KCl elution fraction of the RNA affinity column as shown in Fig. 2 were concentrated with a contricon-30 filter (Amicon) and electrophoresed in 10% SDS-PAGE. The proteins visualized by Coomassie blue staining were excised for microsequencing. The underlined sequences show the five microsequenced peptides of 16, 13, 13, 7, and 24 aa of purified ZBP2 (92 kD). The red regions indicate the four highly conserved K homology domains of the hnRNP. Note the proline, glycine-rich NH2-terminal, and glutamine-rich COOH-terminal domains of ZBP2. The identity of ZBP2 and KSRP is 86%. ZBP2 is available at GenBank/EMBL/DDBJ/accession no. AF461020. nuclei. However, occasionally a significant amount of the ZBP2 was detected by immunofluorescence in the leading edge of CEFs and the growth cone of chicken developing neurons. We superimposed their images (Fig. 6, A, right, and B) where ZBP2 and -actin mRNA were overlapped (yellow) in the leading edge of CEFs (Fig. 6 A, right) and the growth cone of neurons (Fig. 6 B, arrows). This provided evidence that ZBP2 and -actin mRNA were spatially asso- ciated in vivo in the cells examined. Although the protein and RNA are generally distributed in the same region of the neurites, occasionally they are overlapping. Both the -actin mRNA and the ZBP2 show a punctate distribution, particu-