Figure 3

A – Affinity chromatography of mitotic PP1 on GST-Rb-Sepharose column. Mitotic Hela cells extract (40 mg) was applied to a 1 ml GST-Rb-Sepharose column (detailed in the Methods section). The collected fractions were assayed for PP1 activity and pooled as indicated. One aliquot of the pool was concentrated by precipitation in the presence of 7% TCA and subjected to electrophoresis and immunoblotting to detect PP1 isoforms (inset). – B – Gel-filtration of the PP1 eluted from the affinity column. Following concentration, one half of the activity pool was applied to an FPLC Superose 12 HR 10/30 gel filtration column. 0.2 ml fractions were collected, after discarding the first 6 ml, and assayed for PP1 activity. – C – Gel-filtration of the trypsin-treated PP1 pool. The remaining half of the concentrated activity pool was subjected to limited tryptic-proteolysis prior to gel filtration. Molecular weight markers: catalase (250 k), BSA (68 k), ovalbumin (43 k), carbonic anhydrase (29 k). The data presented are representative of several experiments (see Results).