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Supporting Information Figure 1. (A, B) Mice BMMCs were transiently transfected with SIRPα-siRNA as well as scramble-siRNA for 48 hours, the cells were collected and the expression level of SIRPα was detected by (A) immunoblotting and (B) flow cytometry. Data shown are from one experiment representative of four independent experiments. Isotype control, gray filled; SIRPα-si-KD, gray line; scramble, black line. GAPDH was detected as a loading control. (C, D) RBL-2H3 cells with SIRPα overexpression were established and examined by (C) immunoblotting and (D) flow cytometry. Data shown are from one experiment representative of three independent experiments. Isotype control, gray filled; RBL-OV, gray line; RBL-VT, black line. GAPDH was detected as a loading control.

Supporting Information Figure 2. SIRPα negatively regulates allergen-induced cytoskeletal rearrangement and calcium release in RBL-2H3 cells. (A) SIRPα-OV and –VT RBL-2H3 cells were sensitized with 0.4 μg/ml anti-DNP IgE and stimulated with 20 ng/ml DNP-BSA (left) or PMA plus ionomycin (right). Cell degranulation was detected by β-hexosaminidase release. Data are shown as mean ± SEM of three samples pooled from four independent experiments. (B) Polymeric microtubule formation was analyzed in activated SIRPα-OV and -VT RBL-2H3 cells. Images are shown from one experiment representative of three experiments. The relative ratio (intensity of poly-MT vs intensity of total tubulin in unstimulated cells) is shown as mean ± SEM of the same experiments. (C) SIRPα-OV and -VT RBL2H3 cells were sensitized with IgE (0.4 μg/ml) and stained with Fluo-3 AM. The kinetics of calcium flux was detected as the dynamic changes of green fluorescence intensity and monitored by confocal microscopy Zess LSM510META (magnification 60×; scale bar 10μm). The results are analyzed by Mann-Whitney test. *p<0.05, n.s. no significant difference.

Supporting Information Figure 3. (A-C) SIRPα-VT, -OV RBL-2H3 cells were activated, (A) IL-4, (B) IL-13 and (C) TNFα mRNA levels were detected by real-time PCR. Data are shown as means ± SEM of four individual experiments and analyzed by Mann-Whitney test. *p<0.05.

Supporting Information Figure 4. Overexpression of SIRPα inhibits NF-κB and NFAT activation in response to FcεRI-aggregation. (A, B) RBL2H3-VT and –OV cells were transfected with 200 ng/ml (A) NF-κB or (B) NFAT reporter plasmids together with the control plasmid pRL-TK (2 ng/ml) for 24 hours. Cells were activated for the indicated times and the relative luciferase units (RLU) were measured. Data are shown as mean ±SEM of four samples pooled from three individual experiments. Statistical significance determined by Mann-Whitney test. *p < 0.05; **p < 0.01.

Supporting Information Figure 5. (A) SIRPα-si-KD and si-scramble BMMCs were activated and immunoprecipitated by the antibody against phosphotyrosine (4G10). The FcεRI γ subunit was detected. Data shown are from one experiment representative of three independent experiments. (B) The activity of Lyn kinase (Tyr396) in activated SIRPα-si-KD and si-scramble BMMCs was detected by immunoblotting. The relative intensity is shown as the mean values of three independent experiments. (C) Immunoprecipitation assay was performed on BMMCs by anti-SIRPα anibody. The interaction of SIRPα with IKKβ and SHP2 was detected by immunoblotting. Data shown are from one experiment representative of three experiments. (D) BMMCs were transiently transfected with SHP2-siRNA and scramble for at least 36 hours and then activated by IgE-DNP, the cells were collected at the indicated times and the phosphorylation bands of IKKβ and IκBα were detected by immunoblotting. Data shown are from one experiment representative of three independent experiments. GAPDH was detected as a loading control.

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