Figure 4.

Functional testing of R75W CARD11. JPM50.6 cells were transfected with EV, WT or R75W CARD11-FLAG expression constructs, without (A) or with (B) a WT CARD11-V5 construct. After 24 hrs, transfectants were stimulated with anti-CD3/CD28 Abs or PMA/ionomycin for 24 hrs, and GFP expression was measured by flow cytometry. Histograms from one representative experiment (left panels) denote %GFP+ cells -/+ stimulation. Mean fluorescence intensity (MFI) of GFP+ cells was graphed for 3 independent experiments (middle panels); asterisks denote statistically significant decreases in GFP MFI for R75W relative to WT -/+ WT CARD11-V5 (paired t-test, p <0.04). CARD11 protein expression was measured in cell lysates by immunoblotting; β-actin serves as a loading control.