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. 1999 Feb 1;103(3):321–329. doi: 10.1172/JCI4585

Figure 5.

Figure 5

Cyclin A–associated kinase activity is inhibited in CHO-β1C cell transfectants. (a) CHO cells were transiently transfected using pBJ1-β1C (lanes 1 and 3) or pBJ1-β1A (lanes 2 and 4). Total cell lysates, obtained as described in Methods, were immunoprecipitated using 1 μg/ml rabbit affinity-purified antibodies to cyclin A (lanes 1 and 2) or to cyclin E (lanes 3 and 4), and the associated kinase activity was assayed in vitro using histone H1 as a substrate. Phosphorylated histone H1 was observed by autoradiography. The experiments were repeated two to five times with consistent results. (b) β1C expression does not affect cyclin E or cyclin A protein levels. Total cell lysates, obtained as described in a, were immunoblotted with 1 μg/ml rabbit affinity-purified antibodies to cyclin A (top) or to cyclin E (middle). Control for protein loading was provided by MAB to p130Cas (bottom). The experiments were repeated at least twice with consistent results. (c–e) CHO-β1C (lanes 1 and 2) or CHO-mock (lanes 3 and 4) stable cell transfectants were cultured in the absence of tetracycline for the indicated times. In c, total cell lysates were immunoprecipitated using rabbit antiserum to cyclin A, and the associated kinase activity was assayed as described above. The experiments were repeated twice using two different β1C clones with similar results. (d) Expression of β1C is accompanied by increased p27kip1 association with cyclin A. Total cell lysates were immunoprecipitated using rabbit antiserum to cyclin A, and the associated p27kip1 was detected by immunoblotting. (e) Total cell lysates were immunoblotted with rabbit affinity-purified antibody to cyclin A, as described above. In d and e, the experiments were repeated three times using two different β1C clones with similar results. In b, d, and e, proteins were viewed by ECL. Group differences were compared using Student's t test. In a and c, the differences between cyclin A-CDK activity in CHO-β1C versus CHO-β1A transfectants are statistically significant (in a, P = 0.0001; in c, P = 0.0069).