Inhibition of NFAT pathway in primary murine B cells leads to reduced IRF4 expression and IL-10 levels correlating with changes in B cell surface phenotype.

<p>(A–E) Primary murine B cells were isolated as indicated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003858#s4" target="_blank">materials and methods</a>, transduced with retroviruses expressing either M2 or M2stop. At the time of transduction, half of the cells received 500 ng/mL CsA and the other half was left untreated. (A) The cells were analyzed for Thy1.1 expression by flow cytometry on days 2 to 5 post-transduction. Cells were first gated on live cells and then gated on Thy1.1 and plotted as frequency of cells that were positive for Thy1.1. (B) Supernatants were collected from the cells used in (A) and analyzed for IL-10 levels by ELISA. (C and D) Whole cell lysates from cells in replicate wells as in (A) were harvested for analyzed for IRF4, NFATc1, NFATc2 and β-actin expression by immunoblotting. 10 µg protein was loaded per well. (E) Gated live cells from the indicated conditions were analyzed for their expression of the surface markers B220, CD138, MHC-IA^b, IgD, CD23 and CD25. Representative histograms are from day 4 post-transduction depicting one of triplicate samples per condition.