Journal: JCI Insight
Article Title: Serine/threonine phosphatase PP2A is essential for optimal B cell function
Figure Lengend Snippet: Increased PP2A expression and enhanced function in activated B cells or B cells from lupus-prone mice and SLE patients. ( A ) Western blot analysis on the expression of both scaffold (PP2A A ) and catalytic (PP2A C ) subunits in splenic B cells isolated from the indicated mice. Quantification of Western blots ( n = 3 per experiment, a representative experiment and pooled densitometry of the 3 experiments are shown). ( B ) Germinal center B cells (GC, CD19 + FAS + GL7 + ), plasma cells (PC, CD19 + IgG + CD138 hi ), marginal zone B cells (MZ, CD19 + CD21 + CD23 lo ), and follicular B cells (FO, CD19 + CD21 lo CD23 hi ) were FACS sorted for qPCR. Dot plots show the expression of PP2A A , and elevated expression was observed in GC and PC compared with MZ and FO. ( C ) Flow cytometry analysis of the expression of PP2Ac (indicated by mean florescence intensity, MFI) in total B cells from patients with SLE and matched healthy controls. ( D ) Dot plots show the increased MFI of PP2Ac in DN B cells (CD19 + IgD – CD27 – ), memory B cells (CD19 + IgD – CD27 + ), and plasma B cells (CD19 + IgG + CD138 + ) compared with naive B cells (CD19 + IgD + CD27 – ). ( E–H ) CD19 + human B cells were enriched by magnetic beads and cultured with the indicated stimuli for the indicated time. ( E ) Dot plots show the expression of PP2Aa in B cells stimulated with either CpG or anti-CD40 compared with unstimulated B cells 3 hours after stimulation. ( F ) Dot plots show the expression of PP2Aa in B cells stimulated with either CpG or anti-CD40 compared with unstimulated B cells 3 hours after stimulation. ( G ) Western blot analysis of the expression of PP2Aa and PP2Ac in human B cells stimulated with either CpG or anti-CD40 for 48 hours. ( H ) Dot plots show the quantification of PP2Aa and expression PP2Ac in human B cells stimulated with either CpG or anti-CD40 compared with control unstimulated B cells 3 hours after stimulation. ( I and J ) PP2A phosphatase activity was quantified using a kit from R D. The activity of PP2A is presented as the rate of phosphate release (pmol × 10 2 ). ( I ) Bar graph shows the PP2A phosphatase activity in ex vivo splenic B cells from lupus-prone Mrl.lpr mice compared with matching control Mrl.mpj mice (data pooled from 3 independent experiments, paired t test, mean ± SEM). ( J ) Splenic B cells were enriched from C57BL/6J WT mice by MACS and stimulated with the indicated reagents for several time periods; kinetic curves show the PP2A phosphatase activity ( n = 3 for 3 independent experiments, 2-way ANOVA analysis, mean ± SEM).
Article Snippet: PP2A phosphatase assay.B cells were isolated from mouse spleens, and PP2A phosphatase assay was performed as manufactory’s instructions (MilliporeSigma, catalog 17-313).
Techniques: Expressing, Mouse Assay, Western Blot, Isolation, FACS, Real-time Polymerase Chain Reaction, Flow Cytometry, Magnetic Beads, Cell Culture, Activity Assay, Ex Vivo, Magnetic Cell Separation