Regulation of PTPN22 expression in normal and CLL B cells. (A) Western blot analysis of cellular extracts obtained from CLL cells immediately on isolation or after 24 hours in culture without or with the caspase inhibitor Z-VAD, the calpain inhibitor calpeptin (Calp.), or the proteasome inhibitor MG132. Because MG132 induces apoptosis in CLL cells, this inhibitor was used both alone and in combination with Z-VAD. Normalized PTPN22 levels are expressed as fold change relative to the level in freshly isolated CLL cells. (B) Expression of PTPN22 and SHP-1 was evaluated in freshly isolated CLL cells, CLL cells cultured for 24 hours in the absence of any stimuli (unst.) or CLL cells cultured for 24 hours with sol-aIgM, imm-aIgM, CD40L + enhancer, CpG, M2-10B4 bone marrow stromal cells, B-cell activating factor, vascular endothelial growth factor, or TPA. Actin was used as a loading control. Shown are 2 representative experiments of 8 performed with sol-IgM, imm-IgM, CpG, stromal cells, and TPA, and 4 performed with CD40L, vascular endothelial growth factor, and TPA. Changes in PTPN22 expression during culture with sol-aIgM, imm-aIgM, CpG stromal cells, and TPA are shown separately for the 5 samples with high PTPN22 levels in freshly isolated CLL cells (top panel) and the 3 samples with low/absent PTPN22 expression (bottom panel). Values in graphs represent mean ± SD. The BJAB cell line was used as an internal standard. (C) PTPN22 expression in normal T and B cells after 24 hours in culture with the indicated stimuli. Ion. indicates ionomycin. (D) Effects of ruboxistaurin and sotrastaurin on PTPN22 and SHP-1 expression in CLL cells cultured for 24 hours with or without stromal cells.