Figure 3

LMP2A induces oscillatory Ca²⁺ waves caused by IP3 production. (A) DT40 cells that were left untreated (LMP2A -4-HT) or induced to express LMP2A for 12 h (LMP2A +4-HT) were subjected to IP3 analysis using a competitive binding assay with ³H-labeled IP3. Tamoxifen-treated parental cells (DT40 +4-HT) and cells that had been BCR-stimulated for three minutes (α-IgM) served as negative and positive control, respectively. Error bars represent standard deviation of four independent measurements. (B) DT40 B cells that were rendered positive for LMP2A expression by treatment with 4-HT for 14 h (black line) and LMP2A-negative DT40 cells treated with 4-HT for the same time (grey line) were loaded with Fura-2-AM. Subsequently, single cell [Ca²⁺] analysis giving the ratio of fluorescence intensities, F340/F380, was performed by microscopic imaging for 5 min. One representative record of 50 is shown for each sample. (C) LMP2A-negative DT40 cells were stimulated with anti-IgM antibodies (indicated by an arrow) and changes in intracellular Ca²⁺ concentrations were monitored for 30 min at the single cell level. (D) Fura-2-AM-loaded DT40 cells that were induced to express chimeric LMP72 (black line) or 4-HT treated LMP72-negative parental cells (grey line) were monitored for Ca²⁺ flux at the single cell level as described in B.