PMA treatment upregulates the levels of p27^Kip1 protein in U937 cells. (A) p27^Kip1 Northern blot. Total RNA was isolated from U937 cells treated with PMA for the indicated times and 20 μg/lane was probed with a ³²P-labeled p27^Kip1 cDNA. The same gel was stained with ethidium bromide and photographed to show equal loading of RNA. (B) PMA upregulates the total cellular levels of p27^Kip1 protein. Cell lysates (260 μg total protein) were resolved by SDS-PAGE and subjected to Western blotting with p27^Kip1 antibody (1:5,000 dilution). (C) Cyclin E-associated p27^Kip1 protein increases during PMA-induced differentiation of U937 cells. PMA-treated cell lysates (1.4 mg total protein) were immunoprecipitated with cyclin E monoclonal antibody agarose conjugate, and the associated proteins were subjected to Western blotting with a p27^Kip1 antibody (1:5,000 dilution).