Figure 6

Mechanism of downregulation of PKC-α by PknG. (A) Cloning of pknG in pIRES2-EGFP vector; M, DNA ladder; 1, pIRES2-EGFP-pknG undigested; 2, pIRES2-EGFP undigested; 3, pIRES2-EGFP digested with BamHI; 4, pIRES2-EGFP-pknG digested with HindIII; 5, pIRES2-EGFP-pknG digested with BamHI, right oriented recombinants will produce 1.6 kb fragment; (B) and (C) pIRES2-EGFP-pknG was transfected in THP-1 cells and after 48 h cells were lysed and immunoblotted with anti-serum against PknG and with PKC-α antibodies, lane 1 macrophages transfected with vector alone and lane 2 transfected with pIRES2-EGFP-pknG. (D) 5 μg PknG was incubated with immunoprecipitated PKC-α in kinase buffer for 30 min in presence of [γ32P]-ATP then resolved by 8% SDS-PAGE and exposed to X-Ray film., lane 1 PknG alone; lane 2 PKC-α and PknG, lane 3 PKC-α and Histone-4 and lane 4 PknG and Histone-4. (E) THP-1 cell lysate was immunoprecipitated with either antibodies against PKC-α or PKC-δ using protein G Sepharose. The immunoprecipitated proteins were incubated with 5 μg purified PknG for 1 h and immunoblotted with PKC-α and PKC-δ antibodies. (F) Macrophage cell lysate (50 μg) was incubated with 5 μg purified PknG or buffer alone for indicated times and immunoblotted with PKC-α antibodies. (G) THP-1 cell lysate was immunoprecipitated with antibodies against PKC-α and immunoprecipitates were treated with 5 μg of purified PknG for the time points as indicated and then immunoblotted with PKC-α antibody. The experiments were repeated at least 3 times.