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The impact of SYK inhibition on <t>NLRP3</t> activation. Cells were treated with VLPs (20 µg/mL) and mAbs (7.5 µg/mL) for 24 h, and 1 µM of SYK inhibitor R406 or NLRP3 inhibitor <t>MCC950</t> was added 1 h before treatment. (A) Western blot data show the expression of the indicated protein in microglia lysates, and the images are representative of five to six experiments. (B) Quantification of NLRP3 expression from Western blot data, normalised to a loading control, N = 5–6. (C) Quantification of ASC speck formation in primary microglia, N = 6. (D, E) IL‐1β secretion and (F) TNF‐α secretion were tested by ELISA in microglia supernatants, (D) N = 4, (E) N = 4–6, (F) N = 8–9. (G) Fluorescent microscopy images of the immunostained NLRP3 (cyan) and ASC specks (yellow), NLRP3 was stained with primary Ab anti‐NLRP and secondary Ab—AlexaFluor 488, ASC specks were stained with primary Ab anti‐ASC‐PE. Representative images are shown. The scale bars indicate 50 µm in large images and 20 µm in magnified images. In (B–F), data are represented using a box plot with dots showing the number of individual experiments. Significance was established using one‐way ANOVA followed by Tukey's test * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
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The impact of SYK inhibition on NLRP3 activation. Cells were treated with VLPs (20 µg/mL) and mAbs (7.5 µg/mL) for 24 h, and 1 µM of SYK inhibitor R406 or NLRP3 inhibitor MCC950 was added 1 h before treatment. (A) Western blot data show the expression of the indicated protein in microglia lysates, and the images are representative of five to six experiments. (B) Quantification of NLRP3 expression from Western blot data, normalised to a loading control, N = 5–6. (C) Quantification of ASC speck formation in primary microglia, N = 6. (D, E) IL‐1β secretion and (F) TNF‐α secretion were tested by ELISA in microglia supernatants, (D) N = 4, (E) N = 4–6, (F) N = 8–9. (G) Fluorescent microscopy images of the immunostained NLRP3 (cyan) and ASC specks (yellow), NLRP3 was stained with primary Ab anti‐NLRP and secondary Ab—AlexaFluor 488, ASC specks were stained with primary Ab anti‐ASC‐PE. Representative images are shown. The scale bars indicate 50 µm in large images and 20 µm in magnified images. In (B–F), data are represented using a box plot with dots showing the number of individual experiments. Significance was established using one‐way ANOVA followed by Tukey's test * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Journal: European Journal of Immunology

Article Title: SYK Signalling in NLRP3 Inflammasome‐Mediated Response of Murine Microglia Activated by Immune Complexes Formed of Viral Proteins and Specific IgG

doi: 10.1002/eji.70199

Figure Lengend Snippet: The impact of SYK inhibition on NLRP3 activation. Cells were treated with VLPs (20 µg/mL) and mAbs (7.5 µg/mL) for 24 h, and 1 µM of SYK inhibitor R406 or NLRP3 inhibitor MCC950 was added 1 h before treatment. (A) Western blot data show the expression of the indicated protein in microglia lysates, and the images are representative of five to six experiments. (B) Quantification of NLRP3 expression from Western blot data, normalised to a loading control, N = 5–6. (C) Quantification of ASC speck formation in primary microglia, N = 6. (D, E) IL‐1β secretion and (F) TNF‐α secretion were tested by ELISA in microglia supernatants, (D) N = 4, (E) N = 4–6, (F) N = 8–9. (G) Fluorescent microscopy images of the immunostained NLRP3 (cyan) and ASC specks (yellow), NLRP3 was stained with primary Ab anti‐NLRP and secondary Ab—AlexaFluor 488, ASC specks were stained with primary Ab anti‐ASC‐PE. Representative images are shown. The scale bars indicate 50 µm in large images and 20 µm in magnified images. In (B–F), data are represented using a box plot with dots showing the number of individual experiments. Significance was established using one‐way ANOVA followed by Tukey's test * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: SYK kinase inhibitor R406 (#inh‐r406), NLRP3 inhibitor MCC950 (#inh‐mcc), and Zymosan (#tlrl‐zyn) were from Invivogen.

Techniques: Inhibition, Activation Assay, Western Blot, Expressing, Control, Enzyme-linked Immunosorbent Assay, Microscopy, Staining