Figure 3: Coexpression of ZAP70 suppresses chronic autonomous BCR-signaling and NFAT-activation.
(a-c) Analysis of BCR-mediated signaling in SYK−/− Ramos cells reconstituted with EV, SYK, ZAP70 or SYK + ZAP70. (a-b) Phosphorylation of BCR-activated substrates was measured by Western blot following stimulation with IgM (10 μg/mL). (c) Ca2+ flux following IgM stimulation was measured using Indo-1.(d) Basal calcium oscillations were measured in Ramos lines expressing a fluorescent calcium reporter (GCaMP6s). Images were taken of each well at 40 second intervals for one hour in a 37°C chamber. Represented are 40 individual cells analyzed per condition. (e-f) NFAT localization was analyzed in parental Ramos cells with ZAP70 overexpression following IgM stimulation: (e) Immunofluorescence staining for NFAT1 was performed by incubating slides with NFAT1-primary antibody overnight, followed by 1h incubation with AF-647 secondary antibodies at room temperature. Bar graph shows mean ± standard error of mean from three independent experiments analyzed using QuPath. Statistical significance was calculated using an unpaired Student’s t-test. (f) Western blotting of sub-cellular fractions (cytoplasmic/C vs. nuclear/N) in parental Ramos cells with ZAP70 overexpression.