Figure 1.

TPA-dependency and isoform specificity of PKCε-Stat3 nteraction. A: PKCε over-expressing transgenic mice (line 215) (TG) and their wildtype littermates (WT) (2 mice/group) were treated topically with 5 nmol of TPA or vehicle acetone in 0.2 ml. Mice were sacrificed at 1, 3, and 5 h post-treatment and total epidermal lysates were prepared. Epidermal protein (25 μg) was subjected to SDS-PAGE and then blotted with antibodies specific for PKCε, Stat3, pStat3Tyr705, and pStat3Ser727. Bi,ii: Quantification of Western blot A (normalized to total Stat3)(Mean ± SE). C: Total epidermal lysate prepared at 3 h post-TPA treatment (Figure 1A) was pooled. Total epidermal cell lysate (100 μg) was immunoprecipitated using Stat3 or PKCε antibody. The immunoprecipiate was then subjected to SDS-PAGE and blotted with antibodies to PKCε, Stat3, pStat3Tyr705, pStat3Ser727. Di,ii: Quantitation of Western blots shown in (C). E: Total epidermal lysates were prepared at 0,1, 3, and 5 h post-TPA treatment (A) was pooled. Total epidermal cell Iysate (100 μg) was immunoprecipitated using Stat3 or PKCε antibody and blotted for PKCε and Stat3. Fi,ii: Quantitation of Western blots shown in (E). G: Total epidermal cell lysate (100 μg) prepared from untreated and 3 h post-TPA treated transgenic mice (A) was pooled and then immunoprecipitated using Stat3 antibody. Stat3-immunoprecipitated proteins were subjected to SDS–PAGE and immunoblotted with the indicated PKC isoform (α, β, δ, ε, γ, η, ι, λ) antibody. Input is 25 mg of epidermal lysate. Positive controls were purchased from Santa Cruz Biotechnologies (Santa Cruz CA), PKCα, βI, and δ were Jurkat T-lymphocyte whole cell lysate, PKCλ and ζ were A431 epidermoid carcinoma whole cell lysate, PKCβII and μ were K562 leukemia whole cell lysate, PKCε was IMR-32 neuroblastoma whole cell lysate, PKCγ was 293T human embryonic kidney whole cell lysate, PKCη was mouse lung tissue whole cell lysate, and PKCθ was Molt4 leukemia whole cell lysate. U: vehicle treated, T: TPA treated.