Fig. 7. CK2-dependent phosphorylation of ankyrin regulates its ability to associate with spectrin in vitro. Erythroid cells were lysed in isotonic buffer containing 1% Triton X-100, and an -spectrin immunoprecipitate was prepared from the resulting detergent insoluble fraction and washed into low salt buffer. The immunoprecipitate was then incubated with low salt buffer. The immunoprecipitate was then incubated with low salt buffer (lane 1) or with ankyrin that had been immunopurified from control cells (lane 4), from cells treated with calyculin A (lane 2), from cells treated with calyculin A plus TBB (lane 3) or from cells treated with TBB alone (lane 5). Following extensive washing in low salt buffer, the immunoprecipitates were split in half and processed for immunoblotting with ankyrin-specific or -spectrin-specific antibodies. The dash to the left of the ankyrin immunoblot indicates the basally phosphorylated 225 kDa ankyrin isoform.