For amplification of BAFF-R, 5-ACTGCGTGTCCTGTGAGCTCTT-3 and 5-TCTGGGCCAGCTGTCTTGGT-3 were used as forward and reverse primers, and cycled 35 occasions at 94C 1 min, 55C 1 min, and 72C 1 min. in tradition medium at 100:1 volume. Freshly isolated T2 B cells were transduced with lentivirus as follows. Sorted T2 B cells were suspended in 250 l of growth medium comprising polybrene (Sigma-Aldrich) in the concentration of 16 g/ml in 24-well tradition dish, followed by addition of 250 l medium containing lentivirus. Then the tradition plates were centrifuged at 30C, 2,500 rpm for 90 min. After 48C72 h tradition, transduction efficiencies were monitored by EGFP manifestation. For transduction using lentiviral vectors, 60% of the cells were expressing EGFP regularly after 48 h when examined by infecting wild-type B cells. Passive Transfer of Bone Marrow Cells. For passive transfer of bone marrow cells, recipients were -irradiated in the dose of 10 Gy, followed by i.v. injection of 106 donor bone marrow cells. For analysis of cell autonomous defect of PLC-2?/? bone marrow cells, RAG-2?/? mice were injected i.v. with 5 106 bone marrow cells from wild-type or PLC-2?/? mice. RT-PCR. Manifestation levels of BAFF receptor (BAFF-R) or PLC-2 were analyzed by semi-quantitative RT-PCR. In brief, total RNA was extracted from 106 cells using Trizol (Invitrogen). Obtained RNA was reverse transcribed using random hexamer (TAKARA BIO) with SuperScriptII (Invitrogen) according to the manufacturer’s training. For amplification of BAFF-R, 5-ACTGCGTGTCCTGTGAGCTCTT-3 and 5-TCTGGGCCAGCTGTCTTGGT-3 were used as ahead and reverse primers, and cycled 35 occasions at 94C 1 min, 55C 1 min, and 72C 1 min. For amplification of mouse or rat PLC-2, 5-AGGCCAGTGCTGACCAGCTG-3 and 5-CTGTGAGCCGCAGCTCGAA-3 were used as ahead and reverse primers, and cycled 35 occasions at 94C 1 min, 55C 1 min, and 72C 1 min. To avoid detection of incomplete PLC-2 mRNA indicated in cells from PLC-2?/? mice, ahead primer was designed within the erased exon (18). For amplification of bcl-2, 5-TGCACCTGACGCCCTTCAC-3 and Morphothiadin 5-TAGCTGATTCGACCATTTGCCTGA-3 were used Morphothiadin as ahead and reverse primers, and cycled 35 occasions at 94C 1 min, 55C 1 min, and 72C 1 min. GAPDH was amplified as an internal control using 5-CCATCACCATCTTCCAGGAG-3 and 5-CCTGCTTCACCACCTTCTTG-3 as ahead and Morphothiadin reverse primers. 5-Bromo-2-deoxyuridine Labeling. 5-Bromo-2-deoxyuridine (BrdU) labeling was performed as reported previously (23). In brief, mice were fed with BrdU (Wako Pure Chemical Industries) in the drinking water at concentration of 1 1 mg/ml for 1 wk. Spleen cells were depleted of non-B cells as explained previously (18). After staining with biotinylated anti-CD21 combined with streptavidin-CyChrome and PE-conjugated anti-HSA, BrdU labeled T1, T2, and adult B cells were recognized using BrdU Circulation Kit (BD Biosciences) according to the manufacturer’s training. Calcium Measurement and Protein Analysis. Calcium mobilization was measured as explained previously (18) with several modifications. In brief, 5 107 spleen cells were loaded with 1.2 M Indo 1-AM (Sigma-Aldrich) at 37C for 45 min. Cells were washed and stained with FITC-conjugated anti-CD21, PE-conjugated anti-HSA, and CyChrome-conjugated anti-B220. Then 106 stained cells were stimulated with 2 PI4KA g/ml recombinant human being BAFF or 15 g/ml anti-IgM F(abdominal)2. Continuous monitoring of fluorescence from your cell suspension was performed using BD-LSR (Becton Dickinson). Stimulated splenic B cells (3 107) were solubilized and immunoprecipitated with anti-PLC-2 (Santa Cruz Biotechnology, Inc.) mainly because explained previously (18). Immunoprecipitates were separated by 7.5% SDS-PAGE, blotted and recognized by anti-phosphotyrosine Ab, 4G10 (Upstate) using the enhanced chemiluminescence system (Amersham Biosciences). Preparation of Nuclear Components. Stimulated cells (5 106) were washed with snow chilly PBS and cell pellet was suspended in 800 l hypotonic buffer (10 mM HEPES, pH 7.9, 10 mM Morphothiadin KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 5 g/ml antipain, 5 g/ml aprotinin, 5 g/ml leupeptin, 0.5 g/ml pepstatin, and 0.5 mM PMSF) for 10 min on ice followed by addition of 50 l of 10% NP-40. After vortexing for 20 s, nuclei were prepared by centrifugation for 1 min at 15,000 rpm. Then the Morphothiadin nuclear pellet was suspended in 50 l of high buffer (20 mM HEPES, pH 7.9, 0.4 M.