Figure 9. Immunoprecipitation assay of total proteins prepared from primary mouse keratinocyte cultures with the indicated antibodies.

Primary mouse keratinocytes cultured for five days were treated with Latrunculin or with Latrunculin+Rottlerin for 12 h. Then the keratinocytes were collected for protein preparations. Protein samples were immunoprecipitated with six antibodies as indicated, respectively. (A). Proteins immunoprecipitated by antibodies against adducin and p-adducin were analyzed by immunoblotting using adducin and p-adducin antibodies as indicated. (B). Proteins immunoprecipitated with antibodies against PKCδ and p-PKCδ were analyzed by immunoblotting using adducin and p-adducin antibodies as indicated. (C). Proteins immunoprecipitated by antibodies against spectrin and actin were analyzed by immunoblotting using adducin and p-adducin antibodies as indicated. Note: IFNγ antibody was used as a control antibody for the IP experiments. As shown in Fig. 6, the Spectrin, PKCδ and P-PKCδ antibodies did not across react with the immunoprecipitates by IFNγ antibody. Neither adducin nor phosphorylated adducin antibody had a cross reaction with IFNγ antibody (data not shown).