FIG. 4.

(A) K-bZIP influence on cell cycle division. For determination of cell growth rate, 5 × 10⁴ cells of Flag-293-K-bZIPwt 1, Flag-293-K-bZIPwt 2, Flag-K-bZIPΔBR, and Flag-293-vector were seeded in 60-mm plates and cultured in 10% fetal bovine serum-DMEM. Cell numbers were counted at the indicated time points postseeding. Triplicate plates were prepared for each cell clone. (B) Inhibition of CDK2 kinase activity in Flag-293-K-bZIPwt cell lines. CDK2 kinase activity was determined by in vitro kinase assay after immunoprecipitation (IP) of CDK2 from stable cell lines. Histone 1 was used as a substrate for the assay. The amounts of the CDK2 were measured by immunoblotting. W.B., Western blotting. (C) Upregulation of p21 in Flag-293-K-bZIPwt and Flag-K-bZIPΔLZ cell lines. Protein expression levels of p21 in stably K-bZIP-expressing cell lines were measured. Total cell lysates were prepared from the stable cell lines. Actin served as an internal control for the amounts of protein on the membrane. (D) Upregulation of p21 in transiently transfected HeLa cells. HeLa cells were transfected pFlag-empty vector or pFlag-K-bZIPwt. At 72 h posttransfection, total cell lysates were obtained, and 50 μg of protein was loaded in each lane and probed with anti-p21 antibody. Actin served as an internal control for the amount of protein on the membrane.