Figure 2. Human SHARPIN deficiency impairs canonical NF-κB mediated signaling.

(a) LUBAC subunit expression in PBMCs from SHARPIN-deficient patient (P1) and an unrelated healthy donor. (b) LUBAC subunit expression in fibroblasts from three unrelated healthy donors, SHARPIN (P1)- and HOIL1-deficient patients, and in P1 fibroblasts complemented with wild type SHARPIN. (a-b) Note that the expected truncated SHARPIN protein (18kDa: arrowhead) was not observed. * indicates non-specific bands. (c) Western blot of immunoprecipitated extracts from EBV-immortalized lymphoblastic cells of P1 and a healthy donor. (d) Immunoprecipitation of LUBAC in HEK293T. V5-HOIP and FLAG-baits (SHARPIN or GFP) were transiently overexpressed in HEK293T, and immunoprecipitation using anti-FLAG beads was performed. The eluates were subjected to western blotting. (e) TNFR1-signaling complex (TNFR1-SC) formation in fibroblasts from P1 and a healthy donor. Fibroblasts were stimulated with modified tandem affinity purification (moTAP)-tagged TNF (1 μg/ml) for the indicated times. TNFR1-SC was purified with anti-FLAG immunoprecipitation, and analyzed by western blotting. (f-g) P1’s (f) EBV-immortalized lymphoblastic cells and (g) fibroblasts showed attenuated induction of NF-κB after TNF stimulation (20 ng/ml). These data are representative of three (a, b, e, g) or two (c, d, f) independent experiments.