FIGURE 3.

The interaction of PKCδ with IL-32β and its involvement in IL-10 production. A, IL-32β-interacting PKC isoforms were screened by immunoprecipitation. HEK293 cells were co-transfected with 6×Myc-tagged IL-32β and each of 5×FLAG-tagged PKC isoforms (α, δ, ϵ, and θ). After overnight incubation, PMA (20 nm) treatment was performed for 90 min. Immunoprecipitation (IP) was carried out with 1 μg of Myc tag antibody. The expression level of each gene was determined by Western blotting (WB) with 30 μg of whole cell lysates (WCL). B, after co-transfection of HEK293 cells with IL-32β and PKCδ, cells were treated with 10 μm rottlerin (Rott) or Gö6976 (6976), a classical PKC inhibitor, for 1 h before PMA (20 nm) treatment. Immunoprecipitation was performed with 3 μg of PKCδ antibody. U937 cells were treated with increasing doses of rottlerin 1 h before the PMA (10 nm) treatment; thereafter, the cells were incubated for 24 h. C–E, culture media were collected for IL-10 (C), TNF-α (D), and IL-8 (E) ELISA. Nontreated cells were included as a control (con). All values are mean ± S.E. (n = 3). *, p < 0.05; 0 versus 1 μm rottlerin, **, p < 0.02; 1 versus 10 μm rottlerin (B), ***, p < 0.01; 0 versus 10 μm rottlerin (C).