Fig. 2. Forskolin mediates a transient dephosphorylation of pRB. Reh cells were treated with forskolin (100 µM) for the hours indicated. Total cell lysate (50 µg) from treated and untreated cells were subjected to immunoblot analysis as described in Materials and Methods (A), using monoclonal antibodies towards pRB. (B) Anti-RB immunoblotting of lysates from resting T-lymphocytes (lane 1), Reh-control lysate (lane 2), forskolin-treated Reh-cells for 2 hours (lane 3), PHA/ionomycin-activated T-lymphocytes for 50 hours (lane 4) and anti-Fas antibody (CH-11)-treated jurkat wt cells for 4 hours (lane 5). (C) An immunoblot with forskolin-treated Reh-lysates, as described above, was detected with the different anti-pRB-phosphorylated antibodies, as indicated in the figure. Total expression of pRB was detected on the same blot as a control for equal loading of protein in each lane. One representative experiment of five is shown.