Amino acid sequences of human (h) Cybr and murine (m) GRASP aligned using the CLUSTALW program. Identical amino acids are indicated in the consensus sequence. Fuchsia denotes PDZ domains, and yellow denotes coiled-coil domains.

Distribution of Cybr mRNA in human cells and tissues. (A and B) Blots with poly(A)⁺ RNA from the indicated tissues were hybridized with ³²P-labeled Cybr cDNA (Upper). After stripping, filters were hybridized with β-actin cDNA. (C) Each lane contained 20 μg of total RNA from cells incubated for 6 h without additions or with IL-2, IL-12, or IL-2 plus IL-12 (2/12). Some cells were incubated with cycloheximide (CHX) for 30 min before the addition of the indicated cytokines. (D) Each lane contained 20 μg of total RNA from Th1 or Th2 lymphocytes untreated or incubated for 6 h with IL-2 and IL-12 (2/12) or phorbol 12-myristate 13-acetate and ionomycin (P/I) as indicated. Blots were hybridized with the Cybr cDNA (Upper), and then stripped and hybridized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (Lower). The experiments in C and D were replicated at least three times.

Coimmunoprecipitation of overexpressed Cybr and cytohesin-1 mutants. (A) Diagram of cytohesin-1 mutants. (B) 293T cells were transfected with 2 μg of X-press vector containing Cybr cDNA (Cy) and empty Flag vector or 2 or 6 μg of Flag vector containing cytohesin-1 cDNA (C-1) at different ratios (Cy/C-1 = 1:0, 1:1, and 1:3). Proteins were immunoprecipitated (IP) from samples of cell lysates with anti-X-press antibody, separated by SDS/PAGE, and immunoblotted with anti-Flag (Top) or anti-X-press (Middle) antibodies. Proteins from samples of whole-cell lysates (WCL) were separated also in the same gel as the immunoprecipitates and reacted with anti-Flag antibody (Bottom). (C) 293T cells were transfected with 2 μg of X-press vector containing Cybr cDNA and 6 μg of empty Flag vector (EV) or vector containing intact cytohesin-1, mutant 44–398, or mutant 44–258. Proteins immunoprecipitated from cell lysates with an anti-Flag antibody were separated by SDS/PAGE before immunoblotting with anti-X-press (Upper) or anti-Flag (Lower) antibodies. Lane 1 contains 100 μg of whole cell lysate (WCL) from 293T cells transfected with 4 μg of intact Cybr. The arrows indicate the Ig heavy chain (IgH) and Cybr. The experiments in B and C were replicated at least five times.

Coimmunoprecipitation of overexpressed cytohesin and Cybr mutants. (A) Diagram of Cybr mutants. (B) 293T cells were transfected with 6 μg of Flag vector containing cytohesin-1 cDNA and 6 μg of empty X-press vector (EV) or vector containing intact Cybr or mutant 78–359, 166–359, 1–217, or 1–178. Proteins immunoprecipitated from cell lysates with anti-Flag antibody were separated by SDS/PAGE before immunoblotting with anti-X-press (Upper) or anti-Flag (Lower) antibodies. The arrows indicate the Ig heavy chain (IgH) and Cybr or Cybr mutants. (C) 293T cell were transfected with 6 μg of Flag vector containing cytohesin-1 cDNA and 2 μg of empty X-press vector or vector containing Cybr mutant 166–359, 1–217, 1–198 or 1–178. Proteins immunoprecipitated from cell lysates with an anti-X-press antibody were separated by SDS/PAGE before immunoblotting with anti-Flag (Upper) or anti-X-press (Lower) antibodies. The arrow indicates Cybr mutants. (D) 293T cells were transfected with 6 μg of Flag-tagged cytohesin-1 mutant 1–55 cDNA and 6 μg of empty X-press vector or vector containing Cybr mutant 166–359, 1–217, 1–198, or 1–178. Proteins from samples of cell lysates were immunoprecipitated with anti-Flag antibody separated by SDS/PAGE and immunoblotted with anti-X-press (Upper), anti-Flag (Lower) antibodies. The arrows indicate Ig light chain (IgL) and Cybr mutants. The experiments in B–D were replicated at least five times.

Effect of Cybr on guanosine 5′-O-(thiotriphosphate) (GTPγS) binding by ARF1; modification of cytohesin-1 GEP activity. (A) ARF1 (50 pmol) was incubated without additions (○), or with 11 pmol of cytohesin-1 (⋄), 2 pmol of His-6-Cybr (●), or both cytohesin-1 and Cybr (♦) for the indicated times at 30°C before measurement of bound [³⁵S]guanosine 5′-O-(thiotriphosphate). (B) ARF1 (50 pmol) was incubated with 11 pmol of cytohesin-1 and the indicated amounts of GST-Cybr (♦) or GST (⋄) for 20 min at 30°C. As controls, ARF1 without cytohesin-1 was incubated with GST-Cybr (●) or GST (▴), and GST-Cybr was incubated alone (○). The data are means ± SD of values from triplicate assays. The findings were replicated at least three times with different preparations of recombinant proteins.