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. Author manuscript; available in PMC: 2021 Sep 29.
Published in final edited form as: Nat Genet. 2021 Mar 29;53(4):500–510. doi: 10.1038/s41588-021-00803-4

Extended Data Fig. 5. SYK response to stimulation in intestinal epithelial cells.

Extended Data Fig. 5

(a) Western blot analysis of SYK and pSYK (Y525/526) in SW480 cells transfected with plasmid expressing p.S550Y SYK or WT SYK. (b) Time-course of SYK phosphorylation upon zymosan (200 μg/mL) stimulation by Western blot analysis of SYK and pSYK (Y525/526) in SW480 cells for the indicated time (NT = untreated cells). (c) Western blot analysis of SYK, pSYK (Y525/526) and pSYK (323) in SW480 cells transfected with plasmid expressing p.S550Y SYK or WT SYK left either untreated (–) or stimulated with 20 μg/mL or 100 μg/mL zymosan. (d) Western blot analysis of SYK and pSYK (Y525/526) SW480 cells transfected with plasmid expressing p.S550Y SYK or WT SYK left either untreated (NT) or stimulated with curdlan (100 μg/mL) for the indicated time. (e and f) Western blot analysis of SYK and pSYK (Y525/526) in SW480 (e) or in SW480 cells transfected with plasmid expressing p.S550Y SYK (f), upon stimulation with zymosan (200 μg/mL) for 1 hour, in the presence or absence of SYK inhibitor (5 μM, Calbiochem, #574711) added 1 hour prior to zymosan stimulation. (g) IL-8, CXCL1, GM-CSF and CCL2 secretion by 20 hours zymosan-stimulated SW480 cells expressing WT SYK or p.S550Y SYK. SYK inhibitor (5 μM, Calbiochem, #574711) was added 1 hour prior to zymosan stimulation and analysis performed by ELISA. Median and interquartile range and minimum to maximum range; n of independent experiments/n of cell culture replicates: IL-8: (3/3–10); CXCL1, GM-CSF, CCL2: (2–3/2–7); Mann-Whitney test. (h) IL-8, CXCL1, GM-CSF and CCL2 secretion by SW480 cells expressing WT SYK or p.S550Y SYK cells incubated with/without R406 (5 μM) for 60 mins prior to the addition of curdlan and analyzed by ELISA 20 hours following stimulation. Median and interquartile range and minimum to maximum range; n of independent experiments/n of cell culture replicates: 3/3–6; Mann-Whitney test.