Intracellular accumulation of TNFR1 protein in TRAPS. (A) Western blots of TNFR1 in cell lysates from the indicated cell types or organs from TRAPS-knockin mice harboring the indicated heterozygous (Het) or homozygous (Hom) TNFR1 mutations. WT C57BL/6 or TNFR1 knockout (KO) mice were used as controls. (B) Increased stability of TNFR1 protein in TRAPS MEF. Cells were treated with 10 μg/mL of cycloheximide for the indicated times, and TNFR1 levels were analyzed by Western blot. (C) Quantitation of Western blots of intracellular TNFR1 in PBMC from TRAPS patients with structural TNFR1 mutations (n = 23), from patients with R92Q or P46L functional polymorphisms (n = 10), and from healthy donor controls (WT; n = 9). Fold increase was calculated by dividing the density of TNFR1 bands from the patient relative to the average of control samples run on the same gel, after correction for loading with actin or HSC70. TNFR1 levels were significantly higher in TRAPS patients than in controls (P = 0.005, by unpaired two-tail Student's t test), whereas TNFR1 levels from patients with functional polymorphisms were not significantly different from controls. Inset shows an example of the primary Western blot data for three patients heterozygous for the indicated TNFR1 mutations and two healthy donor controls. (D) Resident peritoneal macrophages from mice of the indicated genotype were stimulated with 100 ng/mL murine TNF. IL-6, and MIP-2 were measured after 6 h. The data are averages ± SEM of at least three independent experiments, each with values normalized to the secretion of cytokines by WT cells in each experiment. IL-6 and MIP-2 levels produced by WT macrophages ranged from 933–3,367 pg/mL and 5,957–47,519 pg/mL, respectively. *, comparisons with WT cells with P < 0.05 by unpaired Student's t test.