CD45 is a receptor-like tyrosine phosphatase that positively regulates BCR signaling by dephosphorylating the inhibitory tyrosine of the Src family kinases (SFKs). demonstrates that relaxing negative regulation of BCR signaling rather than enhancing positive regulation is critical for driving autoimmunity in this system. mice were generated directly on the C57BL/6 genetic background during N-ethyl-N-nitrosourea mutagenesis screen conducted at Australian National University (32). mice were backcrossed to C57BL/6 at least 6 generations. H/H (HE) mice and CD45?/? mice were previously described (19, 34). CD45 E613R and Lyn?/? mice were previously described (9, 28). All knockout and transgenic strains were fully backcrossed to C57BL/6 genetic background. Mice were used at 5C9 weeks of age for Perampanel all functional and biochemical experiments. All mice were housed in a specific pathogen free facility at UCSF according to the University Animal Care Committee and National Institutes of Health (NIH) guidelines. Antibodies and Reagents Antibodies were obtained as follows: murine CD3, CD4, CD5, CD8, CD19, CD21, CD22, CD23, CD44, panCD45, B220, CD45.1, AA4.1, IgM, IgD antibodies conjugated to FITC, PE, PerCP-Cy5.5, PE-Cy5.5, PE-Cy7, Pacific Blue, APC, or Alexa 647 (eBiosciences or BD Biosciences); pErk1/2 T202/Y204 (197G2) antibody and phospho-S6 ribosomal protein (S235/236) Al-488 Rabbit polyclonal to ACTR1A (2F9) antibody for intracellular staining; pSrc Y416, pLyn Y507, pSHP-1 Y564, pSHIP1 Y1020, SHP-1 and Lyn antibodies (Cell Signaling); SHIP1, Erk 1 and 2 antibodies (Santa Cruz Biotechnology); pLck Y505 and CD45 antibodies (BD Transduction); Lck (1F6), pY (4G10) and Zeta (6B10) antibodies were prepared in our laboratory; unconjugated CD3 (2C11) antibody (Harlan); goat anti-armenian hamster IgG(H+L) antibody, goat anti-mouse IgM, and goat anti-rabbit IgG antibody conjugated to either PE or APC (Jackson Immunoresearch). Flow Cytometry and data analysis Cells were stained with indicated antibodies and analyzed on FACSCalibur, or Fortessa (Becton Dickson) as previously described (29). Data analysis was performed using FlowJo (v8.8.4) software (Treestar Incorporated, Ashland, OR). Statistical analysis and graphs were generated using Prism v4c (GraphPad Software, Inc). Perampanel Densitometry measurements were performed using Kodak Image station/Molecular Imaging software. Lymph node T and B cell stimulation, and intracellular phospho-Erk and phospho-S6 staining Performed as Perampanel previously described (35). Phospho-S6staining performed using same protocol as phospho-Erk except phospho-S6 antibody is directly conjugated to fluorophore. Phospho-Erk assay also carried out after 15 minute preincubation with U0126 Mek1/2 inhibitor (Cell Signaling) at 10nM concentration in order to confirm specificity of antibody. Calcium measurements Assays were performed as previously described(35) except Indo-1 dye (Invitrogen) was Perampanel used to load cells, and a UV laser on the BD Fortessa was used for detection. Prior to stimulation and analysis, lymphocytes were surface stained for expression of CD23 and AA4.1 to identify B cell subsets. Stimulation was carried out using varying doses of anti-IgM Fab2. Immunoprecipitation and Immunoblotting Performed as previously described (21). Purified cell populations of splenic B cells and CD4+ T cells were obtained by sorting with MACS kits as per manufacturers instructions (Miltenyi). Blot densitometry was performed using Kodak Imagestation software. BAFF ELISA Kit obtained from R&D biosystems and used per manufacturers instructions assessed as previously described (36) Results Differential regulation of the SFKs Lck and Fyn by the CD45 E613R allele in T cells SFK phosphorylation and antigen receptor signaling are tightly regulated by CD45 in T cells. We sought to identify a biochemical signature of hyper-morphic and hypo-morphic CD45 function in T cells against which to compare the E613R allele of CD45. To do so, we took advantage of an allelic series of mice in which CD45 expression is varied across a broad range. The series includes mice harboring one.