50 The Journal of Cell Biology | Volume 156, Number 1, 2002 tive gel and visualized by autoradiography. To establish the specificity of RNA-protein interactions, competition assays were performed by preincu- bating the protein extract with unlabeled RNA competitors. UV crosslinking of RNA–protein complexes was performed by irradiat- ing the reactions on ice in a UV Chamber (GS gene linker; Bio-Rad Labora- tories) with 254-nm, 8-W UV bulbs for 10 min and resolved by 4% native gel. The UV crosslinked RNA–protein complexes, detected by autoradiog- raphy, were cut from the gel, mixed with SDS loading buffer and incu- bated with 10 U RNase T1 for 30 min at room temperature. The gel slices were loaded on a 10% SDS-polyacrylamide gel and electrophoresed to distinguish the RNA–protein complexes. Affinity purification of proteins that bind to RNA 2 ml of poly(U) agarose beads (type 6; Amersham Pharmacia Biotech) were suspended in RNA binding buffer (25 mM Tris HCl, pH 7.4, 100 mM KCl) and packed into a 10-ml column. About 1 mg in vitro synthesized poly(A) zipcode RNA was added to the column and cycled four times. The efficiency of RNA bound to poly(U) agarose beads was monitored by mea- suring the amount of [32P]-labeled RNA present in the RNA preparation. After binding, the beads were equilibrated with the extract buffer, mixed with 40 ml of brain protein extracts or 20 ml fibroblast extracts containing 50 U/ml RNasin (Promega), and incubated for 1 h at room temperature with gentle shaking. To lower nonspecific protein binding, yeast tRNA and heparin were added to 50 g/ml and 5 mg/ml to binding buffer, respec- tively. The beads were then centrifuged for 2 min at 1,000 g, resuspended in binding buffer, and repacked into a 10-ml column. The column was ex- tensively washed in 5 20 ml of binding buffer as follows: (1) binding buffer; (2) binding buffer 40 g/ml yeast RNA; (3) binding buffer 5 mg/ ml heparin; (4) binding buffer 0.1% Triton X-100; and (5) binding buffer only. Proteins retained on the RNA affinity column were step eluted with 20 mM Hepes buffer, pH 7.4, containing 0.5, 1, and 2 M KCl. The eluted proteins were analyzed by SDS-PAGE and band shift assay. Production of rat anti-ZBP2 antibodies The 2-M KCl elution fraction from zipcode affinity column was concen- trated with centricon-30 filter and eletrophoresed in 10% SDS-PAGE. After staining with Coomassie blue, the expected protein bands were cut from the gel, crushed, mixed with adjuvant, and injected into rats (Covance, Inc.) for antibody production. The titer of the antiserum was tested by im- munoblot. Immunoblotting Aliquots of proteins were resolved in 10% SDS-PAGE and transferred onto Zeta membranes by a semidry transferring blotter (Bio-Rad Laboratories). The membranes were blotted overnight with PBS containing 5% nonfat milk at 4 C and then incubated with antiserum against ZBP2 (1:2,000) in PBS containing 1% BSA for 2 h at room temperature. After washing three times with PBS/0.3% Tween-20, the membranes were incubated with horseradish peroxidase conjugated goat anti–rat antibodies (1:8,000), ZBP2 was detected with the ECL system (Amersham Pharmacia Biotech) or with DAB/H2O2. Immunoprecipitation and -actin mRNA detection 10 g purified antibodies or 5 l of antiserum against ZBP2 were incu- bated with 100 l of brain protein extract for 1 h at 4 C with gentle agita- tion. 30 l of protein G–coupled agarose beads (Pierce Chemical Co.) were added to the mixture and incubated for another 1 h at 4 C. After cen- trifugation for 5 min at 1,500 g, the supernatant was taken for immunoblot- ting and RNA band shift assays. For analysis of ZBP2, the pelleted agarose beads were extensively washed with PBS and the proteins bound to the beads were eluted with 100 l of ImmunoPure IgG Elution Buffer (Pierce Chemical Co.). For detecting -actin mRNA, the pelleted agarose beads were washed three times with DEPC-PBS, resuspended in 100 l DEPC water and boiled for 10 min. Total RNA was extracted from the superna- tant using TRIzol RNA isolation reagent (GIBCO BRL). RT-PCR for -actin mRNA was performed for 20 cycles with 1 l of RNA as template using ReadyToGo RT-PCR-beads (Amersham Pharmacia Biotech). The following primers were used for amplification: 5 CTGACACCTTCACCATTCCAG 3 and 5 ATTGCTGACAGGATGCAGAAG 3 . The expected PCR product is a 398-bp DNA fragment of -actin mRNA in 3 UTR at the positions of 1021–1419. Isolation and cloning of ZBP2 cDNA Two primers, zbp2–1 and zbp2–2, were designed and synthesized accord- ing to the two peptide sequences (ZBP2 amino acids 621–627 and 685– 709, respectively) and human KSRP cDNA sequence (zbp2–1 GCTTGG- GAGGAGTACTACAA, zbp2–2 AGGACCTGGGGTCTGTCCGTA): A 200- bp DNA fragment was amplified from a chicken embryo brain library (CLONTECH Laboratories, Inc.) and verified by sequencing. This fragment was used for screening the chicken embryo brain library. Two cDNA clones were isolated from the library screening. After sequence analysis, it was evident that both clones contained a 500-bp COOH-terminal coding sequence followed by 150 bp of 3 UTR. 5 RACE PCR was applied to ob- tain an NH2-terminal fragment of ZBP2 (CLONTECH Laboratories, Inc.) RACE 4 primers were used in the 5 RACE experiment (RACE6–1: CACGCGGCGTTGGGGTCGGCTGC, RACE7: TTATAGGCGGCCCA- CGCGGCGTTGG, RACEF1: GAACGCGTCCTTCCGGATCC, RACEF2: CCCAATTTTTGCCGCTATCTG). The sequences of DNA fragments ampli- fied by RACE were determined by automated DNA sequence analyses. The full-length gene was spliced together by PCR and confirmed by se- quencing. In situ hybridization and immunocytochemistry In situ hybridization was performed on fibroblasts as described by Kis- lauskis et al. (1993), and on neurons as described by Zhang et al. (1999). 9-d-old CEFs were cultured in MEM supplemented with 10% FCS. After growing on coverslips for 2 d, the cells were fixed in 4% paraformalde- hyde in PBS for 10 min and permeabilized with 0.5% Triton X-100 for an- other 10 min at room temperature. Distribution of ZBP2 in CEFs was visu- alized by incubation of rat antiserum against ZBP2 (1:800) followed by Cy3 or FITC conjugated rabbit anti–rat antibodies (1:500; Jackson Immu- noResearch Lab). Neurons were cultured in N2 conditioned media with 2% fetal bovine serum (FBS) for 2 or 4 d and fixed in 4% paraformalde- hyde in PBS for 15 min at room temperature. After permeabilization with 0.5% Triton X-100, the cells were incubated with rat antiserum against ZBP2 (1:750) followed by Cy3-labeled rabbit anti–rat antibodies (1:750). For in situ hybridization and immunochemistry, cells were first processed for hybridization of -actin mRNA, washed with PBS, and immunostained as previously described (Zhang et al., 1999). Cells were viewed with an Olympus B 60 with 60 Plan Apo 1.4NA objective. Images were ac- quired with a cooled CCD camera, operated by Espirit imaging software. GFP fusion protein constructs Human KSRP cDNA (the homologue of chicken ZBP2), a gift from D. Black (University of California at Los Angeles, Los Angeles, CA), was sub- cloned to pEGFP C1 vector (CLONTECH Laboratories, Inc.) at the EcoRI and Hind III sites. (pGFP-KSRP). All other EGFP fusion constructs are either full-length or truncated chicken ZBP2. The central domain fragment con- taining aa sequence 103–650, including the 47-aa sequence unique to ZBP2 and 4-KH domains, was amplified by PCR and cloned into pEGFP- C1 vector at EcoRI and Xbal sites to generate plasmid pGFP-CD. The 4-KH domains of ZBP2 were cloned into pEGFP-C1 vector at SalI and XbaI site to produce construct pGFP-KH. The 47-aa unique sequence was amplified by PCR and introduced into pEGFPC1 at EcoRI Hind III (site pGFP-IN). The COOH-terminal domain, including from amino acids 650 to the stop codon, were excised from the plasmid pBS-LS2 (isolated from library screening) using EcoRI and XhoI sites and cloned into pEGFPC1 vector, generating pGFP-CT. Full-length ZBP2 was cloned into pEGFPC2 (CLON- TECH Laboratories, Inc.) at EcoRI and Hind III sites to generate pGFP-FULL. The 47-aa unique sequence was deleted from full-length ZBP2 by two-step splicing PCR, and was cloned into pEGFPC2 vector by EcoRI and Hind III sites to generate pGFP- 47. All constructs mentioned above were verified by DNA sequencing. Transfection and imaging of transfected cells CEFs were transfected for 4–5 h with GFP–ZBP2 constructs using Qiagen’s Effectene Reagent according to manufacturer’s protocol. Cultured neurons were transfected with DOTAP as described (Zhang et al., 1999). GFP im- aging with or without FISH was performed (Zhang et al., 1999). For CEFs, the GFP and -actin mRNA signals were visualized by fluorescence mi- croscopy using an Olympus B 60 microscope with a 60 objective, n.a. 1.4. At least 50 transfected cells were counted per coverslip for the -actin mRNA localization. The EGFP/ZBP2 fusion expressed in neurons was identified using a fluo- rescence microscopy (Nikon Eclipse inverted microscope) equipped with 60 Plan-Neofluar objective, phase optics, 100 W mercury arc lamp and HiQ bandpass filters (ChromaTech). Fluorescence images were immedi- ately acquired in a constant exposure time (1 s) with a cooled CCD camera that was run by IP Lab computer software. The perimeter of each axonal dendrites (first 30 m from cell body) was traced using phase image. Re- gion of interest was transferred to the fluorescence image in the same cell, and fluorescence intensity of -actin mRNA was measured by IP Lab soft-