Figure 5
Manipulation of CC3 expression affects expression levels of DDB2 and p21CIP1. (A) Western blot analysis of U373 clones for expression levels of DDB2, p21CIP1 and GAPDH (loading control). Lysates were prepared from mock-treated cells and UV-treated (20 J/m2) cells at 2 hours after exposure. Cell lysates were electrophoresed and subjected to Western blotting with the antibodies to indicated proteins. (B). Western blot analysis of MCF10A and MCF7, control, and with silenced CC3, for expression levels of the indicated proteins. (C) Same as in (A) and (B) with HepG2 cells transduced with a control or CC3 expressing lentiviral vector. The protein bands were quantified after scanning with the Kodak Imaging Station 2000R with GAPDH signals used to normalize the data. (D) Subcellular localization of endogenous p21CIP1 in HepG2 cells with or without exogenously expressed CC3. Cells were stained for CC3 and p21CIP1 before and 2 hours after irradiation with UV (20 J/m2). The results are average of three experiments in which at least 300 cells were counted. (E). Localization of p21CIP1 in HepG2 cells before and 2 hours after irradiation with UV (20 J/m2). Experiments were done as in (D). (F) BrdU incorporation into MCF10A cells after exposure to 20 J/m2 of UV. Cells were allowed to recover for 30 minutes after exposure, pulsed with 30 minutes with BrdU and incubated for further 1.5 hours in fresh media. After fixation, cells were stained with the anti-BrdU FITC conjugated antibody.