A – Gel filtration of the PP1-RbCT complex. PP1 catalytic subunit (as in Fig. 4) was incubated with the carboxyl terminal region of Rb (obtained from GST-RbCT by thrombin-proteolysis) in 1:1 molar ratio at 4° for 30 min and applied to a Sephacryl S400 HR gel filtration column (further described in the Methods section). The column fractions were assayed for PP1 activity (filled circles). PP1 (open circles) or RbCT were also run separately on the column (see also part B of the figure). The molecular weight markers were as in Fig 3, with the addition of ferritin (440 k) and PP1 (35 k). – B – Immunological detection of PP1 and RbCT eluted from the gel filtration columns. Either PP1, or the PP1-RbCT complex or RbCT were subjected to gel filtration (see part A; here indicated as PP1, PP1+RbCT and RbCT columns). The fractions obtained from the columns were concentrated by precipitation and analyzed by electrophoresis and immunoblotting to detect PP1 (PP1 blot, using a mixture of the three isoform-specific antibodies) or RbCT (Rb blot). The data presented are representative of two independent experiments.