Kinase activities associated with CD44 are localized in low-density plasma membrane domains. (A) One milliliter of total Brij-58 lysate from 10 ×10⁶ PBMC was precleared with Pansorbin and immunoprecipitated on protein A/G beads coated with indicated antibodies or a control MoAb (against hapten TNP) and blocked subsequently with FCS. After washing, the beads were assayed for the associated kinase activity at 30°C for 15 minutes. The reaction was stopped by boiling in sample buffer, and the phosphorylated proteins separated by SDS-PAGE were detected by autoradiography. (B) After gradient centrifugation of the Brij-58 extract as in Fig 1, the top (3 through 5) and bottom (9 through 11) fractions were pooled separately. The bottom pool containing 80% of the cellular proteins was diluted four times to equalize the protein concentration. Immunoprecipitation with indicated antibodies from the pooled top or bottom fractions and kinase reaction were performed as in (A). The autoradiograms were obtained after overnight exposure at room temperature. Similar results were obtained with PHA blasts (not shown). Kinase activities associated with CD44 are better preserved in whole lysates or gradient top fractions when Brij-58 was used; experiments with TX-100–lysed samples required a much longer exposure time, but showed an identical phosphoprotein profile (not shown).