Figure 4. Nedd4-2 interacts with p-Syk and mediates its ubiquitination in mast cells.

(a) Immunoblot analysis of phosphorylated (p-) and total signalling Lyn, Syk, LAT1, ERK1/2 and NF-κB-p65 proteins in whole cell lysates prepared from IgE anti-DNP (SPE-7; 2 μg ml⁻¹) sensitized WT or Nedd-4-2^−/− FLMCs stimulated with DNP–HSA (20 ng ml⁻¹) for the indicated time points. Immunoblot analysis of Nedd4-2 immunoprecipitated (IP) with Syk (b) and p-Syk (c) and whole-cell lysates of FLMCs prepared and stimulated with DNP–HSA as in a. IP: MOCK indicates an IP control performed without antibody. Ubiquitylation of Syk (d) and p-Syk (e) using Agarose-Tandem Ubiquitin Binding Entities in cell extracts from WT and Nedd4-2^−/− FLMCs. Data are representative of the three (a–e) independent experiments performed, each of which gave similar results. (f) Representative confocal micrographs of intracellular calcium influx over indicated times in IgE anti-DNP (SPE-7, 2 μg ml⁻¹) sensitized WT and Nedd4-2^−/− FLMCs incubated with Fluo-3 (5 μM) for 30 min before addition of DNP–HSA (10 ng ml⁻¹). Scale bars, 20 μm. (g) Representative experiment of DNP induced-Ca²⁺ influx in WT versus Nedd4-2^−/− FLMCs (left panel) and quantified analyses of area under the Ca²⁺ influx curves (right panel). Arrow indicates time of DNP–HSA addition to the cells. Data (right panel, mean±s.e.m.) are pooled from the four independent experiments performed, each of which gave similar results. *P<0.05 for indicated comparison (one-way analysis of variance (ANOVA) with Bonferroni post test).