Characterization of the GANP protein.
(A) The GANP protein is detected as a 210-kd protein in cytoplasmic and nuclear fractions from WEHI-231 by Western blot analysis after immunoprecipitation with the anti-GANP 42-23 mAb. Anti-glutathione-S-transferase (GST) mAb was used as an isotype-matched control mAb. (B) Spleen B cells from normal BALB/c mice were stimulated with F(ab′)2 of goat anti-μ Ab (10 μg/mL) and anti-CD40 mAb (10 μg/mL) for 48 hours and stained with the anti-GANP 29-15 mAb. The upper panel shows the profile of the cytoplasmic staining analyzed by the flow cytometry (FACScan). After permeabilization, cells were stained with the 29-15 mAb followed by fluorescein isothiocyanate-conjugated anti-κ. Two-color staining with phycoerythrin-B220 mAb showed the increase of GANP in B220-positive B cells. The lower panel shows cytostaining on the smears with the 29-15 mAb in combination with ALP-antirat Ig. (C) Reverse transcriptase-polymerase chain reaction assay showed the up-regulation of ganp messenger RNA in B cells stimulated with antimouse μ Ab and anti-CD40 in vitro. HPRT was used as a control to confirm the amount of each template. (D) The cell lysate was prepared from unstimulated (left) or stimulated (right) cells and subjected to anti-GANP immunoprecipitation. In vitro kinase reaction was carried out for 10 minutes with the anti-GANP 42-23 immunoprecipitates in the presence of γ-adenosine triphosphate labeled with phosphorous 32. Phosphorylation on the proteins was detected by autoradiography after separation with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Arrow indicates the position of phosphorylated GANP.
