Announcement

**sharon sanders** · August 1, 2006, 11:40 PM

Figure 1

Fig. 1. Role of PKC in Th2 differentiation and cytokine synthesis. (a) Secretion of IFN- in Th1 cells or IL-5, IL-4, IL-13, and IL-10 in Th2 cells in WT or PKC?/? (KO) mice stimulated with anti-CD3 was determined by ELISA. (b) IL-4 intracellular staining of Th2 cells in WT or PKC?/? (KO) mice either untreated or treated with anti-CD3 alone or in combination with anti-CD28. Inset numbers represent the percentage of IL-4-positive cells. The results in a are the mean ? SD of three independent experiments with incubations in triplicate. The results in b are representative of another two experiments. ***, P < 0.001.

**sharon sanders** · August 1, 2006, 11:41 PM

Figure 2

Fig. 2. Th2 transcription factors in PKC?/? cells. (a) Intracellular staining of GATA3 in Th0, Th1, and Th2 cells in WT or PKC?/? (KO) mice. The solid line represents GATA3 staining; the dashed line corresponds to the mouse IgG1 isotype control. Inset numbers represent the percentage of GATA3-positive cells. (b and c) Western blot analysis of nuclear levels of GATA3, Stat6, RelA, c-Maf, and NFATc1 in cell cultures as above (b) or in Th2 cells stimulated with anti-CD3 plus anti-CD28 (c). Anti-proliferating cell nuclear antigen was used as a loading control in b and c. The results are representative of three independent experiments.

**sharon sanders** · August 1, 2006, 11:44 PM

Figure 3

Fig. 3. Biochemical signaling pathways in PKC?/? cells. (a) Western blot analysis of PKC and PKC/ in lung from WT and PKC?/? (KO) mice. (b) Extracts from CD4+ T cells incubated under Th0, Th1, or Th2 polarizing conditions were analyzed by immunoblotting for PKC, GATA3, and T-bet expression and by PKC kinase activity (KA). (c) WT Th2-polarized T cells were incubated with or without anti-CD3 in the presence or absence of neutralizing anti-IL-4 or anti-IL-5 antibodies, and PKC kinase activity (KA) and PKC levels were determined. (d) PKC kinase activity (KA) and PKC levels were measured in Th2-polarized cells stimulated with IL-4 for different times. (e) Na?ve CD4+ T cells were stimulated or not stimulated with IL-4 or IL-2 for 10 min; afterward, phospho-Stat6, phospho-Jak1, and phospho-Stat5 levels, as well as levels of Stat6, Jak1, and Stat5, were determined by immunoblotting. (f) Western blot analysis of phospho-ERK, ERK, and NFATc1 in na?ve CD4+ T cells stimulated with anti-CD3 plus anti-CD28. (g) NF-B activation was analyzed by EMSA in nuclear extracts prepared from na?ve CD4+ T cells stimulated with anti-CD3 plus anti-CD28. (h) IB kinase activity was determined in the above extracts. The results are representative of three independent experiments. (i) Cytokine production of IL-4 and IL-5 analyzed by ELISA in na?ve CD4+ T cells, either WT or PKC?/? (KO), stimulated with anti-CD3 plus anti-CD28. The results are the mean ? SD of three independent experiments with incubations in triplicate.

**sharon sanders** · August 1, 2006, 11:46 PM

Figure 4

Fig. 4. PKC is required for Stat6 nuclear translocation. Th2 polarized WT or PKC?/? (KO) cells were restimulated or left unstimulated with anti-CD3 or anti-CD3 plus anti-CD28 in the absence (a) or presence (b) of IL-4, and nuclear translocation of Stat6 was determined by confocal analysis. Stat6 (green) or TO-PRO nuclear (blue) staining was visualized. The results are representative of three independent experiments.

**sharon sanders** · August 1, 2006, 11:48 PM

Figure 5

Fig. 5. Role of PKC in OVA-induced allergic airway disease. (a?d) OVA-induced allergic airway inflammation is impaired in PKC?/? mice. Total cell numbers and eosinophils in BAL fluids were determined (a). Lung sections were prepared and stained with H&E (b) or with anti-CD11b (c). Levels of IL-4, IL-5, IL-13, and eotaxin in the BAL fluids were measured by ELISA (d). (e) Adoptively transferred Th2 WT into PKC?/? (KO) cells can generate airway inflammation. Th2 WT and PKC?/? (KO) cells were generated by using mice sensitized with OVA and alum. Th2 cells were collected and injected into either WT or PKC?/? (KO) recipient mice. The mice were exposed to aerosolized OVA, and lungs were prepared for histology and stained with H&E. Data shown are representative of two experiments that each time involved six WT and six PKC?/? (KO) mice. a and d are the mean ? SD; n = 6 for each genotype. **, P < 0.01; ***, P < 0.001.

Announcement

Control of T helper 2 cell function & allergic airway inflammation by PKC

Control of T helper 2 cell function & allergic airway inflammation by PKC

Comment

Comment

Comment

Comment

Comment