Researchers discover new autoinflammatory disease, suggest target for potential treatments NIAIDNews NatureComms
To detect SKAP2 phosphorylation, HEK293FT cells transfected in 12-well plates with constructs encoding SKAP2 andmutants as described above were lysed in 250 μl lysis buffer containing 1% n-Dodecyl-β-D-maltoside, 30 mM Tris-HCl pH 7.4, 120 mM NaCl, 2 mM KCl, 2 mM EDTA, 10% glycerol, cOmplete™ EDTA-free Protease Inhibitor Cocktail , and PhosSTOP™ phosphatase inhibitor cocktail . Lysates were cleared by centrifugation at 21,000 ×for 10 min at 4 °C.
Patient 3: Serum levels of E-selectin, L-selectin, ICAM-1, and lipocalin were detected by ELISA in the samples of Patient 3 and 21 healthy controls. The absorbance was measured by ELx800UV absorbance microplate reader . Serum IL-6 level was determined by routine in-house method , and the data were acquired using Immulite 2000 XPi .
Normalization procedure: In order to plot patients 1, 2, and 3’s serum markers IL-6 , ICAM-1, L-selectin, E-selectin and lipocalin on the same graphs, we normalized data using the 2.5th and the 97.5th percentiles for the healthy controls for the respective assays that were used. Normalized values depicted in the graphs correspond to the analyte concentration minus 2.5%ile of HC, divided by the 97.5%ile minus 2.
$${{{{{{\rm{Normalized}}}}}}\; {{{{{\rm{value}}}}}}}=\frac{{{{{{\rm{analyte}}}}}}\; {{{{{\rm{conc}}}}}}.-\,2.5\%{{{{{\rm{ile}}}}}}\; {{{{{\rm{of}}}}}}\; {{{{{\rm{HC}}}}}}}{97.5\%{{{{{\rm{ile}}}}}}-2.5\%{{{{{\rm{ile}}}}}}\; {{{{{\rm{of}}}}}}\; {{{{{\rm{HC}}}}}}}$$Whole blood samples from healthy controls and patients were collected into EDTA tubes .
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