Fig. 8. pRb does not colocalize with Mcm proteins within licensed chromatin in permeabilized cells. An aliquot of the same CldU-labeled W138 asynchronous cell culture used for the results shown in Fig. 7 was extracted for 3 minutes on ice with 0.5% Triton X100 in cytoskeleton (CSK) buffer. The cells were then fixed with formaldehyde and stained with anti-pRb rabbit polyclonal antibodies, anti-Mcm7 mouse monoclonal antibodies and anti-BrdU rat monoclonal antibodies as described in Fig. 7. The cells were classified into G1, early-S, middle-S and late-S according to the presence of detergent-resistant Mcm7 chromatin association, the presence or absence of CldU staining and the type of CldU replication pattern. DNA was stained with DAPI. Images corresponding to double- and triple-labeling protocols were pseudocolored as indicated within each panel. Sites that co-localize appear yellow for red/green, light blue or blue-green for blue/green, and magenta for blue/red merged images, respectively. Identical results were obtained with anti-Mcm2 and anti-Mcm3 antibodies. Compared to Fig. 7, longer exposure times were used to capture the pRb images due to the weaker fluorescent signal as a result of the release of a fraction of nuclear pRb by the nonionic detergent extraction procedure.