Journal: Nature Communications

Article Title: Viral suppressors of the RIG-I-mediated interferon response are pre-packaged in influenza virions

doi: 10.1038/ncomms6645

Figure Lengend Snippet: ( a ) A549 cells were infected with the indicated viruses (multiplicity of infection (MOI)=5) and prepared for WB analysis 8 h.p.i. Enhanced type I IFN response following disruption of the ESIE-motif was analysed by probing for Y701-phosphorylation of STAT1 (full blot is shown in ). Shown are representative results from three independent experiments. ( b ) Polymerase activity was measured in A549 cells. Cells not expressing PB1 served as a negative control (Ctrl); bars show mean±s.d. of three independent experiments. ( c ) A549 cells treated with cycloheximide (CHX) were infected with the indicated viruses (MOI=5) and prepared for qRT–PCR analysis. Elevated mRNA levels of IFNβ were measured following disruption of the ESIE-motif 8 h.p.i.; bars show mean±s.d. of three independent experiments. CHX treatment was controlled by WB analysis of NS1 (full blot is shown in ). ( d – f ) BALB/c mice ( n =5 per group) were infected i.n. with 10 4 PFU and ( d ) examined daily for survival for 15 days p.i. ( e ) Virus titres of infected mouse lungs were determined 3 days p.i. ( f ) Elevated mRNA levels of IFNβ were measured 2 days p.i. Depicted are fold changes compared with PBS-inoculated mice. ( e , f ) Each dot represents an individual mouse; bars show mean. For statistically analysis ( b , c and f ), analysis of variance with Tukey’s test for multiple comparisons, ( d ) log-rank ( χ 2 ) test for statistical analysis of Kaplan–Meier survival data and ( e ) Student’s t -test were performed; * P <0.05, ** P <0.01, *** P <0.001, NS=not statistically significant.

Article Snippet: STAT1 phosphorylation was analysed with a phospho-specific anti-STAT1 [pY701] rabbit mAb (1:1,000; Cell Signaling; #9171).

Techniques: Infection, Disruption, Phospho-proteomics, Activity Assay, Expressing, Negative Control, Quantitative RT-PCR, Virus

Journal: Nature Communications

Article Title: Heterozygous missense variant of the proteasome subunit β-type 9 causes neonatal-onset autoinflammation and immunodeficiency

doi: 10.1038/s41467-021-27085-y

Figure Lengend Snippet: a, b Pernio-like violaceous rash in patient 1 at 7-month-old ( a ) and patient 2 at 1-month-old ( b ). c – e myositis by leg magnetic resonance imaging ( c ), pulmonary hypertension by echocardiogram( d ), and basal ganglia calcification by head computed tomography (CT) ( e ) in patient 1. f IFN scores of peripheral bloods of 11 healthy participants as controls, three Aicardi–Goutières syndrome (AGS) patients with IFIH1 variant and patient 2 at 84-day old. The score was regarded as positive if it exceeded +2 SD (5.04, dotted line) of the average (solid line) IFN score from healthy controls. Statistical analysis was performed with two-sided Mann–Whitney test. g The skin biopsy samples of a healthy participant as a control and patient 2 at 52-day-old stained with anti-p-STAT1 Ab. A scale bar, 100 μm. h The STAT1 phosphorylation assay with SV40-transformed dermal fibroblasts from healthy volunteers as controls and Patient 2. The expression levels of IFN-γ-induced p-STAT1 were enhanced in patient 2 fibroblasts compared to control fibroblasts.

Article Snippet: Expression of p-STAT1, STAT1, and β-actin was evaluated with anti-p-STAT1 (pY701) Ab (58D6, Cell Signaling 9167, 1:1000), rabbit polyclonal anti-STAT1 Abs (Stat1α (C-24), SANTA CRUZ sc-345, 1:1000) and anti-β-actin Ab (AC-74, Sigma-Aldrich A2228, AC-74, 1:2000), respectively.

Techniques: Magnetic Resonance Imaging, Computed Tomography, Variant Assay, MANN-WHITNEY, Control, Staining, Phospho-proteomics, Transformation Assay, Expressing