Characterization of cytohesin-1 monoclonal antibodies: expression in neutrophils and during granulocytic maturation of HL-60 cells.
ADP-ribosylation factors (Arf) are small GTP-binding proteins involved in vesicular transport and the activation of phospholipase D ( PLD). The conversion of Arf-GDP to Arf-GTP is promoted in vivo by guanine nucleotide exchange factors such as ARNO or cytohesin-1. In order to examine the expression of ARNO and cytohesin-1 in human granulocytes, we generated specific polyclonal and monoclonal antibodies (mAbs). We also overexpressed GFP-ARNO and GFP-cytohesin-1 in RBL-2H3 cells to characterize the specificity and the ability of cytohesin-1 mAbs to immunoprecipitate cytohesin-1. Among the hybridomas secreting cytohesin-1 mAbs, only the clones 2E11, 1E4, 3C8, 6F5, 4C7, 7A3 and 8F7 were found to be specific for cytohesin-1. Furthermore, mAb 2E11 immunoprecipitated GFP-cytohesin-1 but not GFP-ARNO under native conditions. In contrast, mAbs 5D8, 4C3, 2G8, 6G11, 4C3, 6D4, 7B4 and 6F8 detected both cytohesin-1 and ARNO as monitored by immunoblotting. Although mAb 6G11 detected both proteins, this antibody immunoprecipitated GFP-ARNO but not GFP-cytohesin-1 under native conditions. Another antibody, mAb 10A12, also selectively immunoprecipitated GFP-ARNO under native conditions, but the epitope recognized by this mAb is unlikely to be linear as no signal was obtained by immunoblotting. Immunoprecipitation with a cytohesin-1 polyclonal antibody and blotting with cytohesin-1 specific mAbs revealed that cytohesin-1 is highly expressed in neutrophils. Cytohesin-1 can be detected in HL-60 cells but the endogenous protein levels were low in undifferentiated cells. Using the specific cytohesin-1 mAb 2E11 we observed a marked increase in levels of cytohesin-1 expression during dibutyryl-cyclic AMP-induced granulocytic differentiation of HL-60 cells. These data suggest that cytohesin-1, which may have important functions in neutrophil physiology, can be useful as a potential marker for granulocytic differentiation.[1]References
- Characterization of cytohesin-1 monoclonal antibodies: expression in neutrophils and during granulocytic maturation of HL-60 cells. Garceau, V., Houle, M.G., Chouinard, F., Gagnon, S., Harbour, D., Naccache, P.H., Bourgoin, S.G. J. Immunol. Methods (2001) [Pubmed]
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