Figure 1. Expression of PKD isoforms in the thymus.

(a) Characterization of anti-phospho-PKD1/2/3 Abs (anti-pPKDs). Reactivities of anti-pPKDs were evaluated by western blot analysis of lysates from HEK293 cells overexpressing WT PKD2 and mutants. Note that overexpressed PKD2 is constitutively phosphorylated. Anti-PKD1/2/3 (anti-PKDs) blotting was also performed as a control. (b) Preselection OT-I DP thymocytes, that are obtained from TAP-deficient mice transferred with OT-I Tg BM cells, were stimulated with a variety of OVA peptides (10 μM) for the indicated times and phosphorylation of PKD and Erk was analysed. PKD, Erk and β-actin were used as loading controls. (c) RT-PCR analysis of expression of PKD isoforms in the thymus, spleen, ovary and lung from C57BL/6 mice. mRNA expression of β-actin was analysed as a control. Threefold serial dilutions of cDNAs were used as templates. (d) RT-PCR analysis of PKD isoforms expressions in various cell subsets sorted by flow cytometry from WT thymocytes. Results are presented as expression relative to β-actin. ND, not detected. (e) mRNA copy number was evaluated by molecular indexing assay. The amount of RNA used in the experiment is indicated on the x axis. Data are representative of two independent experiments (a–c). Data are presented as mean±s.d. of triplicate assays and representative of two independent experiments (d,e).