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: Fig. 1 Clinical figures and IFN signature of patients. 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

Journal: PLoS Pathogens

Article Title: Down regulation of macrophage IFNGR1 exacerbates systemic L . monocytogenes infection

doi: 10.1371/journal.ppat.1006388

Figure Lengend Snippet: Naïve peritoneal cells were isolated from WT C57Bl/6 (Blue) and fGR1+ (Red) mice. A) FLAG expression was analyzed on both macrophage (CD90.2 -, CD11b hi , F480 hi ) (Left) and T cell (CD90.2+) (Right) populations by flow cytometry. B) Surface expression of IFNGR1 on unstimulated naïve peritoneal macrophages from both mouse genotypes. C) Naïve peritoneal macrophages from C57Bl/6, fGR1 x GR1 KO, or B6. ifngr1 -/- (GR1 KO) mice were stimulated with increasing concentrations of IFNγ for 30 min. Phosphorylation of Y701 STAT1 was detected by immunoblot. Graph depicts density of pSTAT1 bands normalized to the loading control β-actin, Arbitrary Units (AU). (n = 3 independent experiments).

Article Snippet: Blots were probed for pY701 STAT1 (58D6, Cell Signaling) or Total STAT1 (91-C, Cell Signaling) with β-Actin (8H10D10, Cell signaling) as a loading control on each blot.

Techniques: Isolation, Expressing, Flow Cytometry, Western Blot

Journal: PLoS Pathogens

Article Title: Down regulation of macrophage IFNGR1 exacerbates systemic L . monocytogenes infection

doi: 10.1371/journal.ppat.1006388

Figure Lengend Snippet: Peritoneal macrophages from naïve fGR1 and WT mice were A) stimulated for 30 min with increasing concentrations of IFNγ, B) 100 U/mL of IFNγ for 5, 30, 60 min and pY701 STAT1 determined by western blots. pSTAT1 blots were normalized to β-actin, Arbitrary Units (AU). C) Naïve peritoneal macrophages (CD90.2 -, CD11b hi , F480 hi ) from IFNAR KO (green), C57Bl/6 (blue), and fGR1 (red) were stimulated with 100 U/mL of IFNγ for 24 hrs and MHC II up regulation evaluated by flow cytometry. MFIs were normalized to their respective unstimulated controls and pooled from multiple experiments (n = 3 independent experiments, 1–3 mice per experiment).

Article Snippet: Blots were probed for pY701 STAT1 (58D6, Cell Signaling) or Total STAT1 (91-C, Cell Signaling) with β-Actin (8H10D10, Cell signaling) as a loading control on each blot.

Techniques: Western Blot, Flow Cytometry

Journal: PLoS Pathogens

Article Title: Down regulation of macrophage IFNGR1 exacerbates systemic L . monocytogenes infection

doi: 10.1371/journal.ppat.1006388

Figure Lengend Snippet: Naïve peritoneal macrophages were treated with 100 U/mL of IFNβ A) for 6 hrs to determine surface IFNGR1 expression; B) 5, 30, 60 min to evaluate STAT1 pY701 by western blot. pSTAT1 bands were normalized to β-actin loading control, Arbitrary Units (AU); C) for 24 hrs and MHC I expression determined by flow cytometry. D) Macrophages were pre-treated with 100 U/mL of IFNβ for 6 hrs before stimulation with 100 U/mL of IFNγ for 24 hrs and MHC II up regulation determined. All MFIs were normalized to their respective unstimulated controls and pooled from multiple experiments (n = 3 independent experiments, 1–3 mice per experiment, *Two-Tailed t-test).

Article Snippet: Blots were probed for pY701 STAT1 (58D6, Cell Signaling) or Total STAT1 (91-C, Cell Signaling) with β-Actin (8H10D10, Cell signaling) as a loading control on each blot.

Techniques: Expressing, Western Blot, Flow Cytometry, Two Tailed Test