Fig. 2 Nuclear factor (NF)-κB signalling pathway in control and progranulin (PGRN)-deficient lymphoblasts. (A) Immortalized lymphoblasts from controls and carriers of the c.709–1G > A GRN mutation were transiently transfected with a NF-κB-luciferase reporter plasmid (3EnhancerConA). To evaluate the effectiveness of transfection, cells were co-transfected with a green fluorescent protein (GFP)-reporter plasmid. Twenty-four hours after nucleofection, cells were harvested and lysed to determine luciferase activity using the Luciferase Assay System (Promega). The graph corresponds to the mean ± standard error of the mean (SEM) of 3 independent experiments relativized by GFP. Statistical analysis was performed using the Student t test (t4 = 6.203, **p < 0.01 compared with control cells). (B, C and D) Immortalized lymphoblasts from controls and carriers of the c.709–1G > A GRN mutation, asymptomatic or patients with frontotemporal lobar degeneration (FTLD)-TDP, were seeded at an initial density of 1 × 106 × mL−1, and 24 hours later cells were collected and lysed to prepare whole cell extracts or to separate nuclear and cytosolic fragments. Levels of proteins were analyzed using Western blotting. (B) Representative immunoblots show the levels of pIkBα (left panel) and IkBα (right panel). Data represent means ± SEM of different experiments carried out with 8 controls and 12 GRN mutation carriers (6 asymptomatic and 6 FTLD). β-actin was used as loading control. Statistical analysis was performed using 1-way analysis of variance (ANOVA; IkBα: F2,26 = 9.7, pIkBα: F2,19 = 4.69, *p < 0.05 and **p < 0.01 compared with control cells). (C and D) Representative immunoblots showing the nuclear (left) and cytosolic (right) content of (C) p50 or (D) p65 NF-kB subunits. Lamin B1 and α-tubulin antibodies were used as loading and purity control of the nuclear and cytosolic fractions, respectively. Data represent means ± SEM of different experiments using 7 controls, 8 asymptomatic GRN mutation carriers and 6 patients with FTLD. Statistical analysis was performed using 1-way ANOVA (Nuclear p65: F2,13 = 5.676, p < 0.05; cytosolic p65: F2,13 = 13.32, p < 0.01; Nuclear p50: F2,37 = 8.715, p < 0.01; cytosolic p50: F2,37 = 18.00, p < 0.01 compared with control cells).