Effects of LY on the expression and phosphorylation status of cell-cycle regulatory proteins. (a) Northern blot analysis of LY-treated HDFs. Total RNA was isolated from HDFs treated with 2 μM LY or 0.2 μg/ml adriamycin for the indicated periods; 7.5 μg of total RNA was loaded per lane. p21^{WAF1/CIP1/SDI} and, as a control, ELF1α mRNA were detected with ³²P-labeled probes. (b) Western blot analysis of HDFs treated with LY. Lysates were obtained from HDFs treated with LY for the indicated periods; 20 μg of protein was loaded per lane. After separation on 4–20% gradient gels and transfer to PVDF membranes, proteins were detected with antibodies against human p21, p53, p16, and — as a loading control — α-tubulin. (c) pRb phosphorylation status after treatment with LY. After addition of LY for the indicated periods, cell lysates were prepared and proteins were separated on a 7–12% gradient denaturing PAGE gel. For details, see the Methods; ppRb, hyperphosphorylated pRb; pRb, hypophosphorylated pRb.