Characterization of hCDC5 antisera. (A) Immunoblots of whole-cell protein extracts from NIH 3T3 (lanes 1, 2, and 5) and HeLa (lanes 3, 4, and 6–8) cells probed with preimmune (lanes 1 and 3), immune (lanes 2 and 4), and affinity-purified (lanes 5 and 6) antisera raised against the C terminus of hCDC5 (hCDC5C), and preimmune IgG (lane 7) and affinity-purified antisera (lane 8) raised against the N terminus of hCDC5 (hCDC5N). (B and C) Analysis of hCDC5 produced in vitro. hCDC5 or 6XHis/Myc-tagged hCDC5 proteins were produced in vitro in the presence (B) or absence (C) of TRAN³⁵S-LABEL. In B, total products of the reactions (lanes 1 and 2), preimmune (lane 3) or anti-hCDC5C (lane 4) immunoprecipitates, and immunoprecipitates of the 6XHis/Myc-tagged hCDC5 with no primary antibody (lane 5) or 9E10 antibody (lane 6) were resolved by SDS/PAGE. Proteins were detected by fluorography. In C, an NIH 3T3 cell lysate (lane 1), hCDC5 produced in vitro (lane 2), and 6XHis/Myc-tagged hCDC5 produced in vitro (lane 3) were resolved by SDS/PAGE and immunoblotted with anti-hCDC5C serum. In both B and C, asterisks denote the 6XHis/Myc-tagged species of hCDC5. For all panels, numbers to the left indicate molecular mass (in kDa).

Subcellular localization of CDC5 throughout the cell cycle and in serum-deprived cells. (A) Phase contrast (a, d, g, and j) and fluorescence micrographs of NIH 3T3 cells stained with Hoechst (b, e, h, and k) and affinity-purified hCDC5C antiserum (c, f, i, and l). Cells in interphase (a–c), metaphase (d–f), anaphase (g–h), and telophase (j–l) are shown. (B) Localization of CDC5 in serum-deprived NIH 3T3 cells. Phase contrast (a) and fluorescence micrographs of NIH 3T3 cells deprived of serum for 24 h and stained with Hoechst (b) and affinity-purified hCDC5C antiserum (c). (C) Localization of CDC5 in nuclear and cytoplasmic fractions of asynchronously growing and serum-deprived NIH 3T3 cells. Equivalent amounts of nuclear and cytoplasmic fractions prepared from asynchronously growing NIH 3T3 cells or cells that were deprived of serum for 24 h were analyzed by immunoblotting by using either hCDC5C antiserum or a monoclonal anti-actin antibody. (Bars = 10 μm.)

CDC5 is a component of the nuclear speckles. (A) NIH 3T3 cells were costained with affinity-purified hCDC5C antiserum and a monoclonal antibody against SC35. Fluorescein and Texas Red-conjugated secondary antibodies were used to detect the distribution of CDC5 (a, d, and g) and SC35 (b, e, and h), respectively. Confocal images of cells in interphase (a–f) and telophase (g–i) were obtained and merged (c, f, and i). Regions of colocalization are yellow in merged images. (B) Asynchronously growing tsBN2 cells were shifted to the nonpermissive temperature and costained with affinity-purified hCDC5C antiserum (a) and a monoclonal antibody to SC35 (b). The confocal images were merged in c. (C) Myc-tagged Clk/Sty kinase was transfected into NIH 3T3 cells. Phase contrast images (a) of cells costained with anti-Myc epitope monoclonal antibodies (9E10; b) and affinity-purified hCDC5C antiserum (c) at 24 h after transfection. (Bars = 10 μm.)

hCDC5 interacts with core components of the splicing machinery and the spliceosome in vitro. (A) Immunoprecipitations (IP) from a nuclear splicing extract were performed with three monoclonal antibodies [irrelevant monoclonal antibody (AK105), anti-Sm (Y12), and anti-snRNA cap (anti-trimethylguanosine; α-m₃G)] and preimmune (PI) or immune (I) hCDC5C antiserum. Immunoprecipitates were blotted with hCDC5C antiserum. (B) hCDC5 or Sm proteins were immunoprecipitated from an in vitro splicing reaction containing labeled β-globin pre-mRNA at 45 or 90 min after addition of pre-mRNA, and 1/10 of the total reactions were run as standards. The identities of the RNA species are indicated to the left.

S. cerevisiae cells lacking CEF1 are defective in pre-mRNA splicing. Strains containing (CEF1) or lacking the endogenous copy of CEF1 (cef1Δ) harboring plasmid-borne CEF1 cDNA under the control of the GAL1 promoter were maintained in synthetic medium containing raffinose and galactose. CEF1 expression was repressed by shifting the cells to synthetic medium containing glucose (SD). Aliquots of cells were collected at the number of hours indicated after the shift into SD. Total RNA was also purified from temperature-sensitive mutants prp3–1, prp18 (ts503), and cdc28–1N shifted to the restrictive temperature (35.5°C) for the number of hours indicated. Total RNA (20 μg) was electrophoresed and blotted. (A) Northern blots probed with the ACT1 intron sequence or the RP51a ORF. (B) Northern blots probed with the labeled ORFs for DYN2 and DBP2. (C) Northern blots probed with the GLC7 ORF or TUB3 intron sequence. PC, precursor mRNA; M, mature mRNA.