Phosphorylation status of pleckstrin. The following cells were analyzed: Mutu-I(c59) (lane 1), Mutu-III(c62) (lane 2), BL29 (lane 3), IARC167 (lane 4), E-C3 (lane 5), EE346-G7 (lane 6), and dG75 (lane 7). (A) Immunoprecipitation: 10⁷ exponentially growing cells were washed twice in phosphate/carbonate free Minimum Essential Medium (GIBCO-BRL, Gaithersburg, MD) and preincubated in 1 mL of medium for 1.5 hours at 37°C in a 5% CO₂ atmosphere. For labeling, 200 μCi of Phosphorus-32 (orthophosphoric acid in water, DuPont [Boston, MA], 5 mCi/mL) was added and cells were incubated for 2 hours at 37°C. Cells were washed twice in phosphate-buffered saline and lysed in 400 μL of RIPA buffer (supplemented with 1 mmol/L phenylmethylsulfonyl fluoride, 10 μg/mL of aprotein and 10 μg/mL leupeptin) for 30 minutes on ice. After high-speed centrifugation the supernatant was preabsorbed with protein A-sepharose and half of the supernatant was incubated with (+ lanes) or without (− lanes) 1.5 μL of polyclonal antipleckstrin rabbit antiserum for 5 hours at 4°C. The immunocomplexes were collected by incubation with protein A-sepharose followed by centrifugation. The pellets were washed 3 times with RIPA buffer, then subjected 10% SDS-PAGE. The dried gels were exposed to a Kodak Storage Phosphor Screen (Eastman Kodak, Rochester, NY) at room temperature for 3 days and visualized on a PhosphorImager system (Molecular Dynamics, Sunnyvale, CA). (B) Immunoblot: An aliquot of the ³²P-labeled cell lysates was subjected to a 10% SDS-PAGE, transfered to nitrocellulose, and pleckstrin protein was detected by an antipleckstrin rabbit serum. Signals were quantitated on a Densitometer system (Molecular Dynamics). (C) Histogram of pleckstrin expression. The relative level of pleckstrin protein and radiolabeled pleckstrin was determined and the ratio of radiolabeled pleckstrin divided by total pleckstrin protein was calculated. Isogenic cell pairs are grouped.