Journal: Molecular Biology of the Cell
Article Title: Differential mast cell outcomes are sensitive to FcεRI-Syk binding kinetics
Figure Lengend Snippet: Key mast cell outcomes are impaired in Syk mNG -Y130E cells. (A) Degranulation measured by relative β-hexosaminidase released after 30 min of incubation with 0.1 µg/ml DNP-BSA in Syk-KO cells reconstituted with Syk mNG -WT, Syk mNG -Y130E, or Syk mNG -Y130F. (B) Comparison of cytokine concentration in cell media of Syk-KO cells reconstituted with Syk mNG -WT, Syk mNG -Y130E, or Syk mNG -Y130F before and after 3-h stimulation with 0.1 µg/ml DNP-BSA. Results were repeated at least three times for all four cytokines. Bar plots represent mean and SD of technical replicates in one representative sample preparation. (C) The formation of actin plaques at the basolateral surface (left) and ruffling on the apical surface (right) in response to 0.1 µg/ml DNP-BSA treatment in both Syk mNG -WT and Syk mNG -Y130E reconstituted Syk-KO cells. Filamentous actin labeled with phalloidin-AF647 (red). Scale bar: 2 µm. (D, E) Heat maps of relative changes in intracellular calcium concentration upon either addition of a (D) low dose (0.001 µg/ml) or (E) high dose (1 µg/ml) of DNP-BSA. Each row represents the ratio of Fura-2 emission using 350-nm/380-nm excitation for a single cell over time. Ratio color bar scale range, one- to twofold increase: blue–red. (F) Relative increase in Fura-2 ratio per cell after addition. (G) Time between antigen addition and response for each cell. (F,G) Stimulated with 1 µg/ml DNP-BSA and calculated as described in Materials and Methods .
Article Snippet: DNP-BSA containing ∼15–25 DNP per BSA was purchased from Thermo Fisher Scientific (A23018), and DNP-lysine was purchased from Sigma (Nε-DNP-l -lysine hydrochloride, D0380).
Techniques: Incubation, Concentration Assay, Sample Prep, Labeling