1 Introduction [1] [2] flΔneo/flΔneo [3] [4] [5] [5] [6–8] [6,7] [9–11] [11] [6,12] [6,13] [6,12] δ [13] [14] [15] [5] [16,17] C. elegans [16,17] [18,19] [20] [21] 2 Results and discussion 2.1 −/− flΔneo/flΔneo + flΔneo/flΔneo [4] −/− [22] Fig. 1 −/− Fig. 1 Fig. 1 α −/− −/− [23,24] [13] −/− Fig. 1 −/− −/− [4,25] Fig. 1 2.2 −/− Fig. 1 −/− −/− Fig. 2 Fig. 2 −/− Fig. 2 −/− Fig. 3 −/− Fig. 2 [17] −/− Fig. 3 +/+ −/− 3 Concluding remarks I [6,12] [21] −/− While loss of PDK1 does not prevent the activation of PKD, the issue of whether the efficiency of PKD activation is affected in PDK1-null cells requires further experimentation. While robust activation of PKD signalling is observed in PDK1-null ES cells and pre-T cells in response to phorbol esters or CD3 crosslinking we cannot exclude the possibility that PKD activation may be compromised in PDK1-null mature peripheral T cells during physiological peptide:MHC stimulation, however these experiments await the development of genetic models to test this. [4,25] −/− [26] 4 Materials and methods 4.1 Cell preparation and stimulation +/+ −/− [5] l 4.2 Mice −/− flΔneo/flΔneo Cre lck [2,4] −/− 4.3 Purification of thymocyte subpopulations Antibodies conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC) and biotin were obtained from PharMingen. Cells were stained for surface expression of the following markers: CD4, CD8, CD25, CD44, CD3ε, γδ, B220, and Thy1. The DN4 subpopulation were defined by their cell surface marker expression and subsequently sorted using a Vantage cell sorter (Becton Dickinson) or AutoMacs magnetic cell sorter (Miltenyi Biotec). 4.4 Cell lysis and Western blot analysis 6