The data discussed in this article Ganetespib cancer have been deposited in the National Center for Biotechnology Information��s Gene Expression Omnibus and are accessible through GEO series accession number GSE 24736 ( http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=”type”:”entrez-geo”,”attrs”:”text”:”GSE24736″,”term_id”:”24736″GSE24736). Real-time quantitative RT-PCR. Mature naive B cells were enriched from the blood of healthy donors by negative selection using the Naive B Cell Isolation Kit II (Miltenyi), followed by the depletion of transitional/new emigrant B cells after PE anti�Chuman CD10 staining and anti-PE magnetic bead treatment (Miltenyi). Total RNA was extracted from mature naive B cells using the Absolutely RNA Microprep Kit, followed by cDNA synthesis with the Superscript II RT.

Real-time quantification was performed with an iCycler IQ5 thermal cycler (Bio-Rad) using Evagreen (Bio-Rad) and primers reported in Supplemental Table 53. Actin primers were a gift from O. Henegariu, Yale University. Quantification of the gene of interest was analyzed by the ��Ct method using ACTIN as the reference gene. Relative expression was calculated according the formula 2�C(CTgene�CCTactin). B cell activation. Naive B cells were plated at 150,000�C200,000 cells per well in a 96-well plate in RPMI 10% serum and various concentrations of polyclonal F(ab��)2 rabbit anti-human IgM (Jackson ImmunoResearch Laboratories Inc.) or multimeric soluble recombinant human CD40L (Alexis Biochemicals) for 48 hours. Flow cytometry analysis was performed using anti-CD25�CFITC, CD86-APC (BioLegend), CD69-PE, CD19-PECy7, CD95/Fas-APC (BD Biosciences �� Pharmingen).

Statistics. Differences were analyzed for statistical significance with 2-tailed unpaired Student��s t tests, using SigmaPlot software (Systat). A P value of less than 0.05 was considered significant. Supplementary Material Supplemental data: Click here to view.(3.6M, pdf) Acknowledgments We thank S. Rudchenko for cell sorting. This work was supported by grants T32 AI089704 (to L. Menard), AI061093, AI071087, and AI082713 from the NIH/National Institute of Allergy and Infectious Diseases and 33-2008-406 from the JDRF (to E. Meffre); by the JDRF�CCenter for Translational Research (to J.H. Buckner); and by NIH grant DK077905 (to C. Abraham). In addition, we thank the Benaroya Research Institute Translational Research Program and Diabetes Clinical Research Unit for subject recruitment.

Footnotes Conflict of interest: The authors have declared that no conflict of interest exists. Citation for this article: J Clin Invest. 2011;121(9):3635�C3644. doi:10.1172/JCI45790. David Saadoun��s present address is: Department of Internal Medicine and Laboratory I3 ��Immunology, Immunopathology, Immunotherapy,�� UMR CNRS 7211, INSERM U959, Groupe Hospitalier Brefeldin_A Piti��-Salpetri��re, Universit�� Pierre et Marie Curie, Paris, France. Isabelle Isnardi��s present address is: Novartis, Basel, Switzerland.