A nuclear protein affecting -actin mRNA localization | Gu et al. 45 Figure 4. Characterization of antibodies to ZBP2. Antibodies against ZBP2 were made by injecting SDS-PAGE gel purified ZBP2 into rats. (A) Western blotting to ZBP2. (Lane 1) Crude brain extract. (Lane 2) Crude fibroblast extract. (Lane 3) 1.0 M KCl affinity- purified protein fraction of brain extract. The arrow indicates ZBP2. (B) Antiserum against ZBP2 or preimmune serum was incubated with brain extract for 2 h followed by incubation with agarose-protein G beads for 2 h at 4 C. The supernatants, after centrifugation at 1,500 rpm for 10 min, were analyzed by motility shift assay for their ability to form an RNA–protein complex. (Lane 1) Control brain extract. (Lane 2) Brain extract incubated with rat preimmune serum. (Lane 3) Brain extract incubated with anti-ZBP2 serum. Arrows denote the RNA–protein complexes identified in Fig. 1 A. (C) The same samples as in B were used for Western blotting. (Lane 1) Control brain extract. (Lane 2) Brain extract incubated with rat preimmune serum. (Lane 3) brain extract immunodepleted using anti-ZBP2 serum. Arrow indicates ZBP2. larly evident in neurons, consistent with observations of lo- calizing particles (Ainger et al., 1993; Zhang et al., 2001). ZBP2 and -actin mRNA are physically associated To determine whether the ZBP2 and -actin mRNA could be physically associated, we employed immunoprecipitation followed by RT-PCR assays to determine whether the immu- noprecipitated pellet with anti-ZBP2 antibodies contained -actin mRNA (Fig. 7). RNA was isolated from the immuno- precipitated pellet from brain extract using either anti-ZBP2 antibodies or rat preimmune serum, and then subjected to RT-PCR. A 398-bp fragment of the -actin mRNA 3 un- translated region (UTR) was chosen for PCR amplification. We used a plasmid without -actin cDNA as a negative con- trol (Fig. 7, lane 1), and a plasmid with -actin cDNA as a positive control (Fig. 7, lane 6). A fragment of the 3 UTR of -actin mRNA was amplified from the immunoprecipitations with ZBP2 antiserum or antibodies (Fig. 7, lanes 3 and 5). The immunoprecipitations with preimmune rat serum or IgG did not demonstrate any PCR-amplified DNA fragments (Fig. 7, lanes 2 and 4). This experiment indicated that -actin mRNA and ZBP2 were most likely physically associated with each other in chicken brain extracts. Portions of ZBP2 can determine nuclear and cytoplasmic distribution To determine whether ZBP2 shuttles between the nucleus and cytoplasm, we fused it to enhanced green fluorescence protein (EGFP) and analyzed the compartmentalization of the fluorescent protein in both fibroblasts and neurons. The various portions of ZBP2 that were fused to GFP included the 47-aa segment (red), the central four KH domains (blue), and Figure 5. Embryonic expression of ZBP2. Proteins were extracted from chicken embryo brains at various stages and run on 10% SDS-PAGE before Western blotting using ZBP2 and -actin antibodies. The relative ratios of ZBP2 per actin intensity were calculated for the stages indicated (in days).