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. 2017 Feb 6;114(8):E1490–E1499. doi: 10.1073/pnas.1616420114

Fig. 6.

Fig. 6.

Reduced expression of Iqgap2 is sufficient to increase mast cell degranulation. (A) Expression of IQGAP family members Iqgap1 and Iqgap3 was measured by qRT-PCR in WT and KO mast cells. Each dot represents one experiment. Mean ± SEM; unpaired t test, two-tailed. (B) Mast cells were transiently transfected with a pool of siRNAs to knock down Iqgap2 expression. Forty-eight hours after transfection, total RNA was extracted and the extent of Iqgap2 down-modulation was assessed by qRT-PCR. Each dot represents one experiment. Mean ± SEM; paired t test, two-tailed (****P < 0.0001). (C) Same as B, except that transfected cells were stimulated with IgE and antigen, and their ability to degranulate was measured by the release of β-hexosaminidase enzyme. n = 4 independent experiments. Mean ± SEM; unpaired t test, two-tailed. (D) Cells were treated as in C, except that cytokine production was measured by intracellular staining. Each dot represents one experiment. Mean ± SEM; paired t test, two-tailed. (E) WT and KO mast cells were transduced with a lentiviral vector to stably express HA-NFAT1(4–460)-GFP. After selection, cells were stimulated with IgE and antigen for the indicated times, and nuclear translocation of HA-NFAT1(4–460)-GFP was followed over time using a fluorescence microscope. Representative of n = 8 independent experiments. (F) Same as B, except that protein extracts were prepared from transduced cells and the extent of HA-NFAT1(4–460)-GFP dephosphorylation upon stimulation was assessed by Western blot using an anti-HA antibody. (G) Nuclear translocation of endogenous NFAT1 upon stimulation of WT and KO cells with 0.5 μM ionomycin was visualized by immunofluorescence using an anti-NFAT1 antibody. Nuclei were counterstained with DAPI. (H) Calcium flux was measured by flow cytometry in WT and KO cells loaded with Fluo-4 AM and stimulated with IgE anti-DNP antibody and HSA-DNP antigen as well as ionomycin. Shown are the compiled data of n = 5 independent experiments. Mean ± SEM; two-way ANOVA. MFI, mean fluorescence intensity.