p21 and p27 expression in PB cells. (A) Autoradiographs of RT-PCR products. Normal BM MNCs (lane 1), PB MNCs (lane 2), lymphocytes (lane 3), and monocytes (lane 4) were analyzed for p21 and p27 mRNA expression by RT-PCR. Arrows indicate the PCR products corresponding to p21, p27, and β-actin. (B) Western blot analysis. Each lane was loaded with 30 μg of total protein from MEG-01s cells harvested 48 hours after 10 nmol/L TPA addition (positive control, lane 1), PB MNCs (lane 2), lymphocytes (lane 3), and monocytes (lane 4). Western blot analyses with anti-p21 (6B6) and anti-p27 (G173-524) antibodies were performed as described in Materials and Methods. Data were confirmed in 3 more independent experiments. Cells were immunostained using anti-p21 antibody, 6B6 (C through G), and anti-p27 antibody, F-8 (H through L). Immunostaining was performed as described in Materials and Methods using the APAAP method. Nuclei were counterstained with hematoxylin. Positive cells showed red nuclear staining. (C and H) MEG-01s cells harvested 24 hours after the addition of 10 nmol/L TPA (positive control), (D and I) PB MNCs, (E and J) lymphocytes, (F and K) monocytes, and (G and L) granulocytes (original magnifications: [C] and [H], ×50; [D] through [G] and [I] through [L], ×200).