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Figure 4. Strong induction of p53 protein in human cells exposed to roscovitine. Top, whole cell extracts prepared from human control cells and cells exposed to 20 µmol/L roscovitine were separated on a 10% SDS gel (30 µg/lane). Transfer and immunoblotting conditions as in Fig. 2. Blots were incubated with monoclonal anti-p53 antibodies DO-1 and sequentially with anti-PARP-1, anti-PCNA and anti-actin antibodies. The partial lack of the 89-kDa band in the third lane is due to air bubble generation and inappropriate protein transfer. Lysate of human cervix carcinoma HTB-31 cells expressing high levels of mutant p53 was loaded in the last lane. Bottom, the concentration of total p53 in cell extracts prepared from control cells and cells treated with 20 µmol/L roscovitine for the indicated periods of time were determined by ELISA. For the standard curve, p53 protein within the range from 0 to 8 ng/mL was used. The assay was done in duplicate according to the manufacturer's procedure.
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