Figure 4.

Analysis of the MHC-PKC interactions with Dd14–3-3 and 14–3-3ζ. (A) Ax2 cells were developed and lysed by sonication, and the soluble and insoluble fractions were separated. MHC-PKC null cells were developed, and a whole cell extracts were prepared. The MHC-PKC was immunoprecipitated from these fractions and subjected to Western blot analysis using 14–3-3 antibodies as described in MATERIALS AND METHODS. (B) Ax2 and MHC-PKC null cells were developed and lysed by sonication, and the MHC-PKC was extracted from the insoluble fraction as described in MATERIALS AND METHODS. The solubilized MHC-PKC was incubated with 10 μM 14–3-3ζ and the mixture was immunoprecipitated with MHC-PKC antibodies and analyzed by Western blot analysis with 14–3-3 antibody as described in MATERIALS AND METHODS. (C) Ax2 cell lysates were incubated with 100 μM 259-Raf peptide, immunoprecipitated with MHC-PKC antibodies, and analyzed using Western blot analysis with 14–3-3 antibody as described in MATERIALS AND METHODS.