BMMCs were sensitized with either anti-DNP IgE (10 µg/ml) or rat anti-mouse FcγRII/III 2.4G2 (10 µg/ml) and stimulated with DNP-HSA (100ng/ml) (IgE XL) or goat anti-rat IgG (10 µg/ml) (2.4G2 XL) respectively for the indicated times. (a) Fyn immunoprecipitation and western blotting with anti-Src kinase activation loop antibody (pY416). Total Fyn expression is shown as a loading control. The image is representative of 3 independent experiments. Data shown are mean ± SEM. The fold induction was quantified by densitometry analysis of band intensity with Image J software. The mean fold change is relative to the control cells receiving media alone as stimulus. (b) Western blot for activated Lyn, detected with anti-phosphotyrosyl Lyn antibody. Total Lyn expression represents the loading control. The image is representative of 3 independent experiments. (c) Fyn was immunoprecipitated and activated Fyn was detected by an anti-Src kinase activated loop antibody (pY416) after 2.4G2 XL. Total Fyn expression is shown as a loading control. (d) Representative immunoblot of the phosphorylation kinetics of Lyn, Akt, Erk, p38 and JNK after 2.4G2 XL. Total protein expression is shown as loading control. (e) Phosphorylation kinetics of activated Lyn, Akt, Erk, p38 and JNK upon 2.4G2 XL. The densitometry analysis of band intensity was calculated using the Image J software. Fold change is relative to the control cells incubated in media alone. The graph summarizes 3 independent experiments. Data shown are mean ± SEM.

Fyn or Lyn kinases were knocked down by siRNA in BMMCs, which were then stimulated with 2.4G2 XL for the indicated times. (a) Representative image of Lyn, Fyn, phospho-Akt, phospho-JNK, phospho-p38 immunoblot after SDS-page western blot. Total Fyn, Lyn, Akt, JNK, p38 and GAPDH lanes are shown as loading controls. (b) Phosphorylation kinetics of activated Akt, p38 and JNK upon 2.4G2 XL. The densitometry analysis of band intensity was calculated using the Image J software. Fold change is relative to the control cells incubated in media alone.

(a, b, c) WT and Fyn KO BMMC were activated with 2.4G2 XL as described in Figure 1 for 1 hour to measure early phase mediators (Histamine) or 18 hours for cytokine release, analyzed by ELISA. (d, e) WT and Fyn KO bone marrow-derived macrophages were activated for 18 hours, and cell supernatant was analyzed by ELISA. (f, g, h) WT and Fyn KO bone marrow-derived basophils were activated for 18 hours and cell supernatant analyzed by ELISA. Data shown are mean ± SEM of at least 3 independent experiments done in triplicate. *p<0.05; **p<0.001; ***p<0.0001 based on Student’s t test Fyn WT to Fyn KO cells.

(a, b, c) WT and Lyn KO BMMC were activated with 2.4G2 XL as described in Figure 1 for 1 hour to measure early phase mediators (Leukotriene C4) or 18 hours for cytokine release, and analyzed by ELISA. (d, e) WT and Lyn KO bone marrow-derived macrophages were activated for 18 hours, and cell supernatant was analyzed by ELISA. (f, h) WT and Lyn KO bone marrow-derived basophils were activated for 18 hours as described above, and cell supernatant was analyzed by ELISA. Data shown are mean ± SEM of at least 3 independent experiments done in triplicate. *p<0.05; **p<0.001; ***p<0.0001 based on Student’s t test WT to Lyn KO cells.

(a,b) PSA was induced with 500µg of 2.4G2 injected i.v.. Changes in the core body temperature were measured by rectal probe [Fyn WT (PBS, n=4; 2.4G2, n=6), Fyn KO (PBS, n=3; 2.4G2, n=5), Lyn WT (PBS, n=3; 2.4G2, n=3), Lyn KO (PBS, n=3; 2.4G2, n=4)]. Data shown represent the mean ± SE. (c) Blood basophils (IgE+CD49b+), (d) monocytes (CD11b+Gr1−) and (e) neutrophils (CD11b+Gr1+) counts for Fyn (WT, n=8; KO, n=8) and Lyn (WT, n=8; KO, n=8) mice. Data shown represent the mean ± SEM.

(a, b) Circulating histamine levels 2 hours after PSA induction [Fyn WT (PBS, n=4; 2.4G2, n=6), Fyn KO (PBS, n=3; 2.4G2, n=5), Lyn WT (PBS, n=3; 2.4G2, n=3), Lyn KO (PBS, n=3; 2.4G2, n=4)]. Data shown represent the mean ± SE. (c) Fyn KO (n=4) and Lyn KO (n=4) mice or control littermates (Fyn, n=4; Lyn, n=4) were injected i.v. with 5 mg of histamine and core body temperature was measured by rectal microprobe. Data shown represent the mean ± SEM.

(a) PSA was induced in mast cell-deficient mice (Wsh−/−, n=4) as well as their WT controls (C57BL6, n=5) by intravenous injection of 500µg of 2.4G2. The core body temperature was monitored by rectal microprobe. Data shown are the mean ± SEM. Two hours after the induction of PSA in (a), blood was collected from mice, serum prepared and the amount of circulating histamine was assessed by ELISA (b). Data shown are the mean ± SE.

(a) PSA was induced by intravenously delivery of 700 ng PAF in Fyn KO (n=3) and Lyn KO (n=4) as well as their littermate controls (Fyn, n=4; Lyn, n=4). Core body temperature was measured by rectal microprobe. Data shown represent the mean ± SE. (b) Enzymatic activity of serum levels of PAF acetylhydrolase (PAFAH) in Fyn and Lyn KO mice and their control littermates (n≥ 9 per group). Data shown represent the mean ± SEM.