FCRLA is an intracellular B cell protein that belongs to the FcR-like family. its absence/low level expression in plasmacytomas and CD38-positive PCs (15, 16). In this study we extended our FCRLA expression studies to mice and have analyzed constitutive expression in conventional mice and SPF mice, as well as changes in expression following activation and Mouse monoclonal to BID during an immune response. We found that mouse na?ve B cells express FCRLA at a low level. Significant up-regulation of FCRLA occurs in a small fraction of B cells (FCRLAbr) generated in response to antigenic challenge. The FCRLAbr cells can be further divided into two subsets, one with a high level of cytoplasmic Ig and the other with either low or undetectable cytoplasmic Ig. The phenotypic features of these cells only partially overlap with the typical characteristics of PCs and memory cells. Importantly, the FCRLAbr cells accumulate in the bone marrow of immunized mice, suggesting their possible involvement in long-term immunity. 2. Materials and Methods 2.1. Mice Conventional BALB/c mice were housed within the animal facility at the Institute of MK-8033 Cytology and Genetics (SB RAS, Novosibirsk, Russia). Unless otherwise stated, 8C10-wk-old female mice were used. Specific pathogen-free (SPF) female mice were purchased from the Animal Breeding Facility, Branch of Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry (Pushino, Russia). SPF CBA/J mice at 12 wk of age were used for mitogenic stimulation. To study FCRLA expression during an immune response or -probes under high stringency conditions following the Bio-Rad recommendations. 2.3. RT-PCR The mouse T cell line EL4, B cell lines A20 and M12, macrophage cell line J774, pro-B cell line L1210, melanoma MK-8033 B16, and plasmacytomas NS1 and NS0 were maintained in RPMI 1640 supplemented with 50 MK-8033 g/ml gentamicin, 2mM L-glutamine, and 10% FBS. Total RNA extracted from the cell lines was reverse transcribed with SuperScript II RNase H reverse transcriptase (Gibco-BRL, Grand Island, NY, USA) according to the manufacturers recommendations. The following gene-specific primer pair was used in the RT-PCR analysis of expression: forward, 5-ATGTCTGCCCTGCTGTGCTCC-3 and reverse, 5-GACCAGATGACCGAGGAGAGC-3. The samples underwent denaturation MK-8033 at 94C for 3 min followed by 30 cycles of amplification (94C for 30 s, 68for 30 s, 72C for 1 min). A positive control (the plasmid) and a negative control containing all the reagents except cDNA were included in every PCR analysis. The cDNA samples were additionally checked by applying RT-PCR analysis to -actin. Oligonucleotides used as primers for PCR amplification of a mouse -actin fragment were 5-CGCGAGAAGATGACCCAGATC-3 and 5-TTGCGATCCACATCTGCTGG-3. 2.4. Rabbit antiserum Recombinant mouse FCRLA protein was generated using an expression system. The pT7-ABPb and pT7-TZZb expression vectors were generously provided by Dr. S. Stahl (The Royal Institute of Technology, Stockholm, Sweden). The FCRLA fragment lacking the predicted leader peptide and the fourth domain was expressed as a part MK-8033 of the ABP- or TZZ-fused proteins. FCRLA-TZZ, which contains an protein A-derived Ig binding motif, was affinity purified using rabbit IgG coupled to Sepharose 4B (Pharmacia Biotech, Uppsala, Sweden). A rabbit was immunized with the purified protein (three injections 200 g each in complete Freunds adjuvant, Sigma-Aldrich). The immunoglobulin fraction of antiserum was prepared by precipitation with (NH4)2SO4 (40% saturation). 2.5. Western blotting Cells (5 104 per sample) were lysed for 5 min in a loading SDS buffer at 100C and subjected to reducing 12 % SDS-PAGE. After electrophoresis, the separated proteins were transferred to nitrocellulose membrane Hybond-C (Amersham Biosciences, Piscataway, NJ, USA). The membrane was immuno-stained using FCRLA-specific Ab and HRP conjugated anti-rabbit IgG. 2.6. Transfections 293T cells were transiently transfected with the pCI-neo-plasmid DNA using Unifectin 56 (IBCH, Moscow, Russia) according to the manufacturers protocol. After 72 hours,.