PErk than cells with normal BCR (19). We have measured pErk by flow cytometry soon after treating immature B cells3?3Igi gene-targeted mice develop B cells that express a BCR certain for the MHC class I H-2Kb antigen. In this model, B cells are A when building on a H-2b genetic background, whereas they may be NA when on a H-2d genetic background (30, 35). Creating three?3 B cells undergo comprehensive receptor editing in H-2b mice and produce a mature B-cell population largely devoid of 3?3 antibodies (31, 35). Crossing 3?3Igi,H-2b mice to Rag1deficient animals outcomes in mice in which B cells are unable to carry out receptor editing and, therefore, only express the autoreactiveE2798 | pnas.org/cgi/doi/10.1073/pnas.Fig. 1. Basal pErk1/2 levels are decreased in autoreactive immature B cells and correlate with sIgM. (A) Surface IgM expression on bone marrow immature B cells analyzed ex vivo from three?3Igi nonautoreactive (NA), Rag1-/- autoreactive (A,Rag1), and nonautoreactive BCR-low (NA-low) mice. Cells have been gated as B220+IgM+IgD? Shaded histograms are B220?non-B cells. Much more than three independent experiments are represented. (B) Representative mean fluorescence intensity (MFI) of intracellular pErk measured by flow cytometry in bone marrow three?3Igi NA immature B cells stimulated for 5 min at 37 with anti-IgM F(ab)two or F(ab)two manage antibodies (in the absence of pervanadate). Cells were gated as B220+IgD? The gray dashed line would be the MFI with the pErk isotype manage antibody. (C ) Phospho-Erk in B220+IgM+IgD?immature B cells treated with pervanadate for 5 min at 37 . Shaded histograms show isotype control antibody. Three independent experiments are represented. (D) Relative pErk analyzed with the MSD ELISA platform in cell lysate of immature B cells sorted from bone marrow. Cells were left FGF-9 Protein medchemexpress untreated (Ideal) or treated with pervanadate (Left). Bar graphs represent average (+SD) pErk1/2 levels normalized to total Erk1/2 and compared with those in NA cells set arbitrarily to one hundred. P 0.05, n = 3 from three independent experiments. (E) IgM (Upper) and pErk (Reduce) levels in B220+IgM+IgD?pervanadate-treated cells from MD4 and MD4 ?ML5 mice. Shaded histograms are B220?cells (Upper) and MD4 cells stained with an isotype control antibody (Lower). Data are representative of two mice per strain. (F) Average MFI of pErk1/2 relative to defined IgM MFIs measured by flow cytometry in pervanadate-treated B220+IgD?bone marrow cells of C1QA Protein Purity & Documentation wildtype mice; n = 3. (G) Representative wild-type bone marrow B220+ cells analyzed for the expression of CD21 and IgM. The arrow indicates the degree of IgM at which differentiation of immature B cells (i.e., CD21 expression) starts.Teodorovic et al.using the tyrosine phosphatase inhibitor pervanadate for 5 min, as its detection within the absence of pervanadate (by flow or Western blot) proved inconsistent in our hands (19) (Fig. S1A). The effect of pervanadate in B cells is for the most component dependent on BCR expression and its ligand-independent activity (36, 37). Hence, we identify the pErk detected in immature B cells as basal, although the absolute level measured just after pervanadate remedy is inflated. Importantly, this basal degree of active Erk is markedly reduced than that acutely induced by BCR engagement and detected inside the absence of pervanadate (Fig. 1B and Fig. S1B). Antigen-induced BCR signaling, such as Erk activation, is identified to be somewhat short lived because it is rapidly reduced by the activity of phosphatases and other unfavorable f.
