Journal: eLife

Article Title: Defects in translation-dependent quality control pathways lead to convergent molecular and neurodevelopmental pathology

doi: 10.7554/eLife.66904

Figure Lengend Snippet:

Article Snippet: After blocking in 5% nonfat dry milk (Cell Signaling, #9999S), blots were probed with primary antibodies at 4°C overnight: rabbit anti-phospho-eIF2α S51 (Cell Signaling, #9721, 1:1000), rabbit anti-eIF2α (Cell Signaling, #9722, 1:2000), rabbit anti-Hbs1l (Proteintech, 10359–1-AP, 1;1000), rabbit anti-Pelo (Proteintech, 10582–1-AP, 1:2000), rabbit anti-GAPDH (Cell Signaling, #2118, 1:10,000), rabbit anti-phospho-p70S6K T389 (Cell Signaling, #9234, 1:1000), rabbit anti-phospho-p70S6K (Cell Signaling, #2708, 1:1000), mouse anti-vinculin (Sigma, V-9131, 1:20,000).

Techniques: RNAscope, Multiplex Assay, SYBR Green Assay, Sequencing, Quantitative RT-PCR, Software

Journal: Molecular Medicine Reports

Article Title: Influenza virus non-structural protein 1 inhibits the production of interferon β of alveolar epithelial cells upon the infection of influenza A H1N1

doi: 10.3892/mmr.2017.7138

Figure Lengend Snippet: Primer sequences for quantitative real-time PCR.

Article Snippet: FITC-conjugated anti-IFNAR2 antibodies and isotype antibodies were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Sequencing

Journal: Molecular Medicine Reports

Article Title: Influenza virus non-structural protein 1 inhibits the production of interferon β of alveolar epithelial cells upon the infection of influenza A H1N1

doi: 10.3892/mmr.2017.7138

Figure Lengend Snippet: NS1 downregulates the expression of receptors for IFNβ-IFNAR1 and IFNAR2. (A and B) The expression of Ifnar1 and Ifnar2 was assessed by real-time q-PCR. NS1-expressing A549 cells produced lower amounts of mRNAs of IFNAR1 and IFNAR2 (P<0.001, n=4). (C) The protein levels of IFNAR1 and IFNAR2 were measured by western blot analysis. NS1 inhibited the upregulation of IFNAR1 and IFNAR2 of A549 cells upon H1N1 infection. (D) The surface expression of IFNAR1 and IFNAR2 on infected A549 cells were checked by flow cytometry. NS1-expressing A549 cells (blue curves) had decreased levels of IFN receptors in A549 cells infected with H1N1. (E) The proportion of IFNAR1-expressing cells of infected A549 cells with or without NS1 expression measured by flow cytometry, P<0.001, n=4. (F) The percentage of IFNAR2-expressing cells infected with H1N1, P<0.001, n=4. *P<0.05; ***P<0.001. NS1, non-structural protein 1; IFN, interferon; HA, haemagglutination tests; n.s., non significant.

Article Snippet: FITC-conjugated anti-IFNAR2 antibodies and isotype antibodies were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Expressing, Produced, Western Blot, Infection, Flow Cytometry

Journal: Nature structural & molecular biology

Article Title: STAT2 is an essential adaptor in USP18-mediated suppression of type I interferon signaling

doi: 10.1038/nsmb.3378

Figure Lengend Snippet: (a) U6A cells, stably transduced with control (−) or C-terminally Myc-tagged STAT2 (STAT2-Myc), were infected with MIP control (−) or MIP-USP18 (+) retrovirus. With or without IFNα (1000 U/ml) treatment for 15 minutes cell lysates were collected and analyzed by Western blotting with the indicated antibodies. (b–d) U6A cells, stably transduced to express either full-length STAT2 (STAT2-Myc), or the indicated STAT2 deletion mutant (b, STAT2ΔCC-Myc; c, STAT2ΔDB-Myc; d, STAT2ΔCC/DB-Myc) with or without IFNα (1000 U/ml) treatment for 15 minutes cell lysates were collected and analyzed by Western blotting with the indicated antibodies. (e) Histograms showing the surface expression of IFNAR1 and IFNAR2 following infection with indicated constructs in U6A cell lines.

Article Snippet: Anti-IFNAR2 FITC was purchased from Sino Biological Inc. Anti-USP18 antibody is described as before .

Techniques: Stable Transfection, Transduction, Infection, Western Blot, Mutagenesis, Expressing, Construct

Journal: Nature structural & molecular biology

Article Title: STAT2 is an essential adaptor in USP18-mediated suppression of type I interferon signaling

doi: 10.1038/nsmb.3378

Figure Lengend Snippet: (a) IB analysis of WCL and anti-FLAG IP derived from U6A cells 24 hours after co-transfection with plasmids encoding USP18, FLAG-IFNAR2, and increasing amounts of STAT2-Myc expression construct. The relative USP18 binding to IFNAR2 from three independent experiments was quantified and plotted as the ratio of IFNAR2-bound USP18 to total USP18 (right panel). Data are normalized to the maximum binding (lane 4). (b) Recruitment of USP18 and STAT2 to micropatterned IFNAR2 in STAT2-deficient U6A cells as illustrated in the cartoon. U6A cells transfected with HaloTag-mTagBFP-IFNAR2 and mEGFP-USP18 (USP18 –STAT2) (green channel, left image) and U6A cells transfected with HaloTag-mTagBFP-IFNAR2, mEGFP-USP18 and STAT2-TagRFP-T (USP18 +STAT2) (green channel, right image). Representative images of 17 cells analyzed in two independent experiments. (c) Recruitment of USP18 to immobilized IFNAR2 (R2) into micropatterns was quantified in U6A cells by determining the contrast of the fluorescence intensities inside and outside the patterns. For comparison, constitutive binding of STAT2 (positive control) and cytosolic mEGFP (negative control) to micropatterned full length IFNAR2 expressed in U6A cells was quantified. n: number of cells analyzed in two independent experiments. Significance was quantified using the two-sample Kolmogorov-Smirnov test. *** P < 0.001, n.s., not significant. (d) The C-terminal STAT2 interacting region of IFNAR2 is necessary for recruiting USP18. Quantification of recruitment of mEGFP-USP18 co-expressed with STAT2-TagRFP-T to immobilized HaloTag-IFNAR2 or C-terminally truncated HaloTag-IFNAR2 (R2Δ375) in Hela cells. As negative controls, HeLa cells were transfected with cytosolic mEGFP or TagRFP-T, respectively. n: number of cells analyzed in two independent experiments. Significance was quantified using the two-sample Kolmogorov-Smirnov test. *** P < 0.001, n.s. not significant.

Article Snippet: Anti-IFNAR2 FITC was purchased from Sino Biological Inc. Anti-USP18 antibody is described as before .

Techniques: Derivative Assay, Cotransfection, Expressing, Construct, Binding Assay, Transfection, Fluorescence, Positive Control, Negative Control