Immunofluorescent localization and immunochemical determination of cyclophilin-A with specific rabbit antisera.
We raised rabbit antisera against homogeneous bovine cyclophilin A (CypA) and we report their use for its immunofluorescent and immunochemical detection without resorting to cyclosporine binding. Indirect immunofluorescence demonstrated that in tissue culture cells CypA is present in the cytoplasm diffusely and also associated with vesicles and the Golgi apparatus. In mitotic cells CypA is increased in amount and redistributed away from cytoplasmic organelles. High levels of CypA were demonstrated in murine splenic erythroblasts and myeloblasts, but they became undetectable during differentiation to mature erythrocytes and granulocytes. Large, often granular, lymphocytes stained very intensely, but small lymphocytes demonstrated variable staining. Dot blot immunoassays demonstrated that murine tissues contain similar amounts of CypA. During CsA treatment murine liver can increase its CypA content much more than spleen. In summary, we demonstrated that cells known to be resistant to the effects of CsA have high levels of CypA. Also tissues that are resistant to CsA can increase their levels more than sensitive tissues upon CsA exposure. Taken together these results suggest that CypA plays a role in cell cycle progression and that sensitivity to CsA may not be simply a reflection of the baseline CypA levels, but may also be affected by the regulation of these levels. Further work is needed in order to delineate the role of CypA in the cell cycle and its relation to the action of CsA.
Sarris
A H
AH
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510.
Harding
M W
MW
Jiang
T R
TR
Aftab
D
D
Handschumacher
R E
RE
D000591:Amino Acid Isomerases / Q000032:analysis*; D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D002352:Carrier Proteins / Q000032:analysis*; D002478:Cells, Cultured; D016572:Cyclosporine / Q000494:pharmacology; D005260:Female; D005455:Fluorescent Antibody Technique; D007120:Immunochemistry; D019696:Peptidylprolyl Isomerase; D011817:Rabbits
D002352:Carrier Proteins; D016572:Cyclosporine; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase