Relative levels of intercellular and intracellular cyclins. (A) A representative example of the immunoblotting analysis showing the relative levels of mitotic cyclins in HeLa and RPE1 cells. Different concentrations of plasmids expressing AID-cyclin A, B1, or B2 were transfected into HeLa cells to serve as standards. Lysates from asynchronous (Asy) and G₂ HeLa and RPE1 cells were loaded. The samples were analyzed with antibodies against mAID or individual cyclins. (B) RPE1 cells contain relatively lower concentrations of B-type cyclins compared to HeLa cells. HeLa and RPE1 cells from asynchronous (Asy) and RO3306-treated (G₂) populations were analyzed with flow cytometry or immunoblotting. (C) Comparison of the expression of mitotic cyclins in HeLa and RPE1 cells. The relative expression of cyclin A, B1, and B2 in asynchronous (Asy) and G₂ lysates was determined using the respective AID-cyclin standards. The expression level of each cyclin was normalized to that cyclin in asynchronous HeLa cells. Mean of two independent experiments. (D) B-type cyclins are more abundant than cyclin A. The relative expression of cyclin A, B1, and B2 in asynchronous (Asy) and G₂ lysates was determined using the respective AID-cyclin standards. The relative levels between each AID-cyclin were first determined using an antibody against AID. The expression level of endogenous cyclins was normalized to that of cyclin B1 in asynchronous HeLa (upper panel) or RPE1 (lower panel). Mean of two independent experiments.