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Figure 2


Fig. 2. Coimmunoprecipitation of DLC1 with 14-3-3 proteins. (A) HEK293T cells were transiently transfected with Myc-tagged DLC1 and empty vector (–), EE-tagged 14-3-3{gamma} or 14-3-3{zeta} or HA-tagged 14-3-3{tau} expression vectors. 14-3-3 isoforms were immunoprecipitated from whole-cell extracts with EE- and HA-specific antibodies, respectively, and immune complexes were separated by SDS-PAGE. Coprecipitated DLC1 was detected by western blotting with Myc-specific antibody (top panels). Immunoprecipitation of 14-3-3 isoforms was verified by probing the membrane with EE- and HA-specific antibodies, respectively (middle panels), and expression of DLC1 was verified by immunoblotting of whole-cell extracts with Myc-specific antibody (bottom panels). (B) HEK293T cells were transiently transfected with expression vectors encoding EE-tagged 14-3-3{gamma} or 14-3-3{zeta}, or HA-tagged 14-3-3{tau}, and empty vector or Myc-tagged DLC1. DLC1 was immunoprecipitated from whole-cell extracts with Myc-specific antibody and immune complexes were separated by SDS-PAGE. Coprecipitated 14-3-3 isoforms were detected by western blotting with EE- and HA-specific antibodies, respectively (middle panels, indicated with arrowheads). IgG chains are indicated with asterisks. Immunoprecipitation of DLC1 was verified with Myc-specific antibody (top panels), and expression of 14-3-3 isoforms was verified by immunoblotting of whole-cell extracts with EE- and HA-specific antibodies (bottom panels). (C) HEK293T cells transiently expressing Myc-tagged DLC1 were lysed and 14-3-3 proteins were immunoprecipitated from whole-cell extracts with 14-3-3-specific rabbit pAb. An unrelated rabbit pAb was used as a control (con IgG). Immune complexes were separated by SDS-PAGE. Coprecipitated DLC1 was detected by western blotting with Myc-specific antibody (top panel). Immunoprecipitation of 14-3-3 proteins were verified by probing the membrane with 14-3-3-specific mouse mAb (middle panel). Expression of DLC1 was verified by immunoblotting of whole-cell extracts with Myc-specific antibody (bottom panel).