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Effect of mutations on phosphorylation and electrophoretic mobility. PHAS-I proteins were expressed in HEK293 cells, which were incubated with 32Pi and insulin (see protocol 2 in Materials and Methods). The proteins were immunoprecipitated, subjected to SDS-PAGE, and transferred to an Immobilon membrane. (A) Phosphorimage showing 32P-labeled PHAS-I proteins. (B) PHAS-I immunoblot of the membrane. (C) Coomassie blue stain of recombinant PHAS-I proteins purified from bacteria.
