Regulation of SLY1 phosphorylation in thymocytes. A. Phosphorylation of SLY1 at serine27 in peripheral splenocytes. CD90+ MACS-isolated T cells were stimulated with αCD3 (10 μg/ml) for the indicated time and analysed by Western blotting. B. Thymocytes were left unstimulated or incubated for 20' with 20 ng/ml PDBU. Where indicated, cells had been pretreated with Rapamycin (20 nM) or Ly294002 (10 μM) for 1 hour. C. DN3 and DN4 thymocytes were single-cell-sorted according to the expression of CD90, CD25 and CD44, lysed and analysed by Western blotting. D. RAG1-/- or RAG1+/+ DN3 thymocytes were plated over night on OP9 cells. DN thymocytes were then sorted by single-cell flow cytometry, lysed and analysed by Western blotting.