Fig 2: LynA persists in primed cells treated with 3-IB-PP1.(A) Immunoblots from unprimed and IFN-?-primed CskAS BMDM lysates show levels of LynA and corresponding Erk phosphorylation during 3-IB-PP1 treatment. (B) Kinetics of LynA degradation relative to the maximum value within each experiment (primed cells, t = 0). (Quantified by densitometry. Error bars reflect the standard deviation over four independent experiments. The p-value is derived from a ratio paired, two-tailed t test.) See Figure 11—figure supplement 1 for independently normalized comparison of Lyn degradation in primed and unprimed cells.DOI: http://dx.doi.org/10.7554/eLife.09183.026

Fig 3: ß-glucan decreases Fc?RIIA effective 2D affinity for hIgG in a LacCer C24- and Lyn- and SHP-1-dependent manner(A) DMF-differentiated HL60 cells expressing Fc?RIIA (HL60–2A) treated with ß-glucan exhibited decreased affinity for hIgG-coated RBCs in the presence of LacCer C24 compared with LacCer C16 or vehicle. Non-specific binding as measured with RBCs without hIgG was negligible.(B) Lyn knockdown HL60–2A cells (Lyn-1) showed increased affinity for hIgG-coated RBCs compared to scRNA control in the presence of ß-glucan but not in dextran.(C) SHP-1 knockdown HL60–2A cells showed similar results as Lyn-1 knockdown cells, with the addition of ß-glucan leading to increased 2D affinity of knockdown cells for IgG. Conversely, there was a significant drop in affinity upon SHP-1 knockdown in the presence of dextran control.All measurements in (B) and (C) were in the presence of LacCer C24. Non-specific binding controls without hIgG are shown. AcKa values represent ligand-receptor density normalized adhesion frequencies. Data are average ± SD, and individual values are plotted. *p < 0.05, **p < 0.01, ***p < 0.001 using an unpaired Student’s t test.

Fig 4: Cell proliferation in response to downregulation of specific SFKsThe histograms show cell proliferation of melanoma cells infected with shRNA targeting SRC, LYN, YES or FYN compared to none-infected cells (None) and control shRNA (Control) (left panels A–D). The shRNA knockdown was validated by western probing for the expression of the SFK employing the respective antibodies (right panels A–D). SFK: SRC Family Kinase.

Fig 5: Regulation of DHA on CD19/Fc?RIIb-Lyn-SHP-1 pathway and effect of intracellular Ca2+ flux in ST486 cells. (A) The cells were treated as above, and then stimulated with anti-human IgM (20 µg/ml) for 1 h. The protein expression levels of CD32b (Fc?RIIb), GAPDH, Lyn, phospho-CD19 and SHP-1 were detected by western blot. This part of the experiment was repeated three times. (B) The cells were incubated in RPMI 1640 medium containing 10% fetal bovine serum and Fluo-4 AM (5 µmol), incubated at 37°C for 1 h, washed 3 times, and continuously monitored by flow cytometry. Anti-human IgM (20 µg/ml) was added at 50 s, add CaCl2 to the final concentration of 2 mM at 400 s, and the intracellular Ca2+ flux was continuously detected to 800 s (C–F) With GAPDH as the internal reference, the relative expression of each protein was expressed as the protein/GAPDH. The experiments were repeated three times and analyzed using one-way ANOVA. *p < 0.05 and **p < 0.01 vs. the Mut group; # p < 0.05 vs. the MTX group.