Figure 4.

β_1C expression is accompanied by increased p27^kip1 protein levels in NRP152 and CHO cells. (a) NRP152-β_1C (lane 2) or NRP152-β_1C-mock (lane 1) stable cell transfectants were cultured for 72 h in the absence of tetracycline, detached, and NRP152-β_1C was seeded on tissue culture dishes coated with TS2/16, whereas NRP152-mock transfectants were seeded on tissue culture dishes coated with Ha2/5 for 1 h at 37°C, washed three times with serum-free medium, and cultured for 20 h in growth medium. Cells were then lysed and p27^kip1 expression levels were evaluated by immunoblotting using 0.8 μg/ml MAB to p27^kip1 (top). (b and c) CHO-β_1C (b, lanes 1 and 2; c) or CHO-β_1C-mock (b, lanes 3 and 4) stable cell transfectants were cultured for 72 h in b and for the indicated times in c, either in the absence (b, lanes 2 and 4; c) or in the presence (b, lanes 1 and 3) of 1 μg/ml tetracycline. In these experiments cells were not detached and allowed to reattach to MAB to β₁ integrins, but were lysed in the tissue culture plate, and p27^kip1 expression levels were evaluated as described in a. The experiments were repeated three times using two different β_1C clones with similar results. Group differences were compared using Student's t test. The differences in p27^kip1 expression levels in CHO-β_1C, but not in CHO-mock transfectants in the presence or in the absence of tetracycline, are statistically significant (P = 0.03). Control for protein loading was provided by MAB to tubulin (a–c, bottom). Proteins were viewed by ECL. Time refers to the length of time in absence of tetracycline.