primary antibody cgas  (Proteintech)

Journal: Journal of Human Immunity

Article Title: Recurrent severe viral infection in a child with inherited complete TBK1 deficiency

doi: 10.70962/jhi.20250058

Figure Lengend Snippet: Homozygosity for a LOF TBK1 mutation in the patient. (A) Family pedigree showing segregation of the TBK1 mutation. PCR products were amplified from genomic DNA extracted from the granulocytes of P1 and both parents and subjected to Sanger sequencing. (B) TBK1 mRNA levels (upper panel) were determined by RT-qPCR on HEK293T cells 24 and 48 h after transfection with empty vector (EV), WT, and mutant TBK1 constructs. Western blot analysis was performed to assess the levels of protein for TBK1 (lower panel), autophosphorylated TBK1 (p-TBK1, Ser172), IRF3, and autophosphorylated IRF3 (p-IRF3, Ser396) in HEK293T cells at 24 and 48 h after transfection with EV, N-terminally flag-tagged WT, and mutant TBK1 constructs. The results shown are representative of three independent experiments. (C) Pulse-chase analysis of WT and mutant TBK1 protein stability. HEK293T cells were transfected with flag-tagged WT or mutant TBK1 expression plasmids for 24 h. Cells were then treated with cycloheximide (CHX; 100 ng/ml) for the indicated time points to inhibit protein synthesis, followed by western blot analysis (bottom). TBK1 protein levels were quantified by densitometry, normalized to GAPDH, and plotted over time (top). Data shown are representative of three independent experiments. The data shown are the means ± SEM of three independent experiments. P values were obtained by one-way ANOVA with Tukey’s multiple comparisons tests, and the P values for the 4-, 8-, and 12-h time points are indicated for the comparison of P1’s cells with control cells. ns; P > 0.05; *P < 0.05; and **P < 0.01. (D) ISRE, IRF3, and NF-κB promoter-driven luciferase reporter assays were performed on HEK293T cells 24 h after transfection with ISRE, IRF3, or NF-κB reporter plasmids along with EV, WT, and mutant TBK1 constructs. Luciferase activity was measured to assess TBK1-mediated activation. The results shown are representative of three independent experiments. The data shown are the means ± SEM of three experiments with three biological replicates, P values were obtained by one-way ANOVA with Tukey’s multiple comparisons tests. ns; P > 0.05, ****P < 0.0001. Source data are available for this figure: .

Article Snippet: RT-qPCR was performed with Applied Biosystems TaqMan assays for TBK1 (HS00179410_m1), IFNB1 (Hs01077958_s1), IFNL1 (Hs00601677_g1), IFIT1 (Hs00356631_g1), IL6 (Hs00174131_m1), and the β-glucuronidase (GUS; #4310888E) housekeeping gene for normalization.

Techniques: Mutagenesis, Amplification, Sequencing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Construct, Western Blot, Pulse Chase, Expressing, Comparison, Control, Luciferase, Activity Assay, Activation Assay

Journal: Journal of Human Immunity

Article Title: Recurrent severe viral infection in a child with inherited complete TBK1 deficiency

doi: 10.70962/jhi.20250058

Figure Lengend Snippet: Impaired induction of IFNs via TBK1-mediated pathways in the patient’s SV40-fibroblasts. (A) TBK1 mRNA levels (left panel) were measured by RT-qPCR in fibroblasts (SV40-fibroblasts) from healthy controls (C1, C2), P1, and two other TBK1-deficient patients (WT/G159A, W619*/W619*), and an immunoblot analysis of endogenous TBK1 protein levels (right panel) was performed with antibodies against the N-terminus and C-terminus of TBK1. The results shown are representative of three independent experiments. (B) SV40-fibroblasts from healthy controls (C1, C2), P1, two other TBK1 patients (WT/G159A, W619*/W619*), and a TLR3 −/− HSE patient were left unstimulated (NS) or were stimulated with poly (I:C) alone, Lipofectamine alone (Lipo), or both (poly (I:C)+Lipo), for 6 h. The relative expression levels of IFNB1 , IFNL1 , IFIT1 , and IL6 were measured by RT-qPCR. The results shown are from three independent experiments. (C) IFNB mRNA levels were measured by RT-qPCR in SV40-F from healthy controls ( n = 3), P1, and HSE patients with TBK1-deficient (WT/G159A) and AR IFNAR1 deficiencies. Cells were either left uninfected (NS) or infected with HSV-1 (KOS strain, MOI = 1) for 24 h. Data represent the means of three independent experiments. (D) SV40-F from healthy controls (Ctrls, n = 3), P1, and HSE patients with TBK1-deficient (WT/G159A) and AR IFNAR1 deficiencies were either left untreated or pretreated with IFN-β for 24 h, followed by infection with HSV-1 (MOI = 0.001). Viral replication was assessed at the indicated time points post-infection using the TCID 50 virus titration method. Data represent means ± SEM from three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test; P values at 48 h after infection compare P1’s cells to controls. ***P < 0.0001. Source data are available for this figure: .

Article Snippet: RT-qPCR was performed with Applied Biosystems TaqMan assays for TBK1 (HS00179410_m1), IFNB1 (Hs01077958_s1), IFNL1 (Hs00601677_g1), IFIT1 (Hs00356631_g1), IL6 (Hs00174131_m1), and the β-glucuronidase (GUS; #4310888E) housekeeping gene for normalization.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Infection, Virus, Titration