Journal: International journal of molecular sciences

Article Title: SARS-CoV-2 ORF7a Protein Impedes Type I Interferon-Activated JAK/STAT Signaling by Interacting with HNRNPA2B1.

doi: 10.3390/ijms26125536

Figure Lengend Snippet: Figure 5. Heterogeneous nuclear ribonucleoprotein A2B1 (HNRNPA2B1) interacts with ORF7a and is related to the inhibition of STAT2 phosphorylation by ORF7a. (A) ORF7a interacts with HNRNPA2B1 rather than STAT2. Cells were transfected with either EV or ORF7a plasmid (equipped with a Flag tag). After 24 h, the cells were stimulated with IFNα for an additional 0.5 h and then harvested for co-immunoprecipitation (co-IP) with anti-Flag IP resin. The input and IP products were analyzed by immunoblotting (IB) with anti-STAT2, anti-HNRNPA2B1, anti-Flag, and anti-tubulin antibodies. (B) The knockdown effect of si-HNRNPA2B1 on HNRNPA2B1 expression. Cells were transfected with non-targeting control siRNA (NC) or si-HNRNPA2B1 (si-A2B1) (100 nM). Twenty-four hours later, the cells were harvested for immunoblotting with anti-HNRNPA2B1 and anti-tubulin antibodies.

Article Snippet: The following primary antibodies were used: STAT1 (Cat# 14994T, CST, Danver, MA, USA, 1:1000), phospho-STAT1 (STAT1-y701) (Cat# ab109457, Abcam, Cambridge, UK, 1:1000), STAT2 (Cat# ab32367, Abcam, Cambridge, UK, 1:5000), phospho-STAT2 (STAT2-y690) (Cat# ab191601, Abcam, Cambridge, UK, 1:1000), β-tubulin (Cat# HC101, TransGen Biotech, Beijing, China, 1:1000), Histone H2A (Cat# 7631, CST, Danver, MA, USA, 1:1000), JAK1 (Cat# ab133666, Abcam, Cambridge, UK, 1:1000), phosphorylated JAK1 (JAK1-P) (Cat# ab138005, Abcam, Cambridge, UK, 1:1000), JAK2 (Cat# ab108596, Abcam, Cambridge, UK, 1:5000), phosphorylated JAK2 (JAK2-P) (Cat# WL02997, Wanlei bio, Shenyang, China, 1:500), TYK2 (Cat# A2128, Abclonal, Wuhan, China, 1:500), phosphorylated TYK2 (TYK2-P) (Cat# bs-3437R, Bioss Inc., Woburn, MA, USA, 1:1000), HNRNPA2B1 (Cat# AY2564, Abways, Beijing, China, 1:500), and Flag (Cat# 66008, Proteintech, Chicago, IL, USA, 1:5000).

Techniques: Inhibition, Phospho-proteomics, Transfection, Plasmid Preparation, FLAG-tag, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Knockdown, Expressing, Control

Journal: International journal of molecular sciences

Article Title: SARS-CoV-2 ORF7a Protein Impedes Type I Interferon-Activated JAK/STAT Signaling by Interacting with HNRNPA2B1.

doi: 10.3390/ijms26125536

Figure Lengend Snippet: Figure 7. The C-terminal domain of ORF7a is related to the restraint of STAT2 phosphorylation. Cells were transfected with EV, ORF7a FL, or ORF7a truncation constructs. Twenty-four hours later, the cells were exposed to IFNα (0.5 h) and then harvested to determine the levels of STAT1-y701 (A) and STAT2-y690 (B). (C,D) Densitometry analysis showing the relative fold changes in STAT1-y701 (C) and STAT2-y690 (D) compared to the levels in IFNα-stimulated EV control cells after normalization to tubulin. Data are presented as means ± SEM from three independent experiments. Statistical significance was determined by one-way ANOVA. * p < 0.05, ** p < 0.01 vs. EV + IFNα group; # p < 0.05, ## p < 0.01 vs. FL + IFNα group. (E) Determining the interaction between HNRNPA2B1 and ORF7a D3 or D4. Cells were transfected with EV, ORF7a D3, or D4 plasmid. After 24 h, the cells were stimulated with IFNα for an additional 0.5 h and then harvested for co-IP with anti-Flag IP resin. The input and IP products were analyzed by immunoblotting (IB) with anti-HNRNPA2B1, anti-Flag, and anti-tubulin antibodies. Representative images from three independent experiments are shown.

Article Snippet: The following primary antibodies were used: STAT1 (Cat# 14994T, CST, Danver, MA, USA, 1:1000), phospho-STAT1 (STAT1-y701) (Cat# ab109457, Abcam, Cambridge, UK, 1:1000), STAT2 (Cat# ab32367, Abcam, Cambridge, UK, 1:5000), phospho-STAT2 (STAT2-y690) (Cat# ab191601, Abcam, Cambridge, UK, 1:1000), β-tubulin (Cat# HC101, TransGen Biotech, Beijing, China, 1:1000), Histone H2A (Cat# 7631, CST, Danver, MA, USA, 1:1000), JAK1 (Cat# ab133666, Abcam, Cambridge, UK, 1:1000), phosphorylated JAK1 (JAK1-P) (Cat# ab138005, Abcam, Cambridge, UK, 1:1000), JAK2 (Cat# ab108596, Abcam, Cambridge, UK, 1:5000), phosphorylated JAK2 (JAK2-P) (Cat# WL02997, Wanlei bio, Shenyang, China, 1:500), TYK2 (Cat# A2128, Abclonal, Wuhan, China, 1:500), phosphorylated TYK2 (TYK2-P) (Cat# bs-3437R, Bioss Inc., Woburn, MA, USA, 1:1000), HNRNPA2B1 (Cat# AY2564, Abways, Beijing, China, 1:500), and Flag (Cat# 66008, Proteintech, Chicago, IL, USA, 1:5000).

Techniques: Phospho-proteomics, Transfection, Construct, Control, Plasmid Preparation, Co-Immunoprecipitation Assay, Western Blot

Journal: Acta Neuropathologica

Article Title: Congenic expression of poly-GA but not poly-PR in mice triggers selective neuron loss and interferon responses found in C9orf72 ALS

doi: 10.1007/s00401-020-02176-0

Figure Lengend Snippet: Poly-GA, but not poly-PR, triggers microglial pro-inflammatory responses characteristic of disease-associated microglia, aging, and interferon that are similar to C9orf72 ALS patients. a Gene ontology analysis of genes differentially expressed between ALS cases without (“ALS”) or with C9orf72 (“C9”) mutation compared to controls (“Ctrl”). Terms with > 5 genes that are concordantly regulated in GA-Nes (GA con) or affected PR-Nes (PR con) mice are also analyzed. Concordantly regulated genes were defined by comparing averaged significant up/down-regulation from all analyzed regions in GA-Nes or affected PR-Nes mice and ALS patients. Number of genes in each group from left to right: 5810, 4742, 660, 5351, 1279, 689, 45, 999, 33, 111. SC spinal cord, CBL cerebellum. Full data is available in Table S4 and S5. b Network of the immune-related genes concordantly regulated in GA-Nes mice and C9orf72 -positive vs C9orf72 -negative ALS patients. c Correlation analysis of gene expression in isolated microglia from GA-Nes mice or whole tissue from different brain regions in GA-Nes- and PR-Nes-affected (affect) and asymptomatic (asym) mice with differentially expressed genes in microglial subpopulations identified by single-cell RNAseq in Aβ and SOD1 mouse models , p25 neurodegeneration mice and wildtype mice during development (cluster 1–9), injury and aging . Pearson’s correlation coefficient indicated by color and circle size. Non-significant correlations are crossed out. Size of the published genes sets from top to bottom: 1482, 4589, 730, 955, 271, 278, 332, 483, 295, 678, 452, 118, 158, 40, 83, 52, 66, 183, 192, 188, 22, 12, 130, 36. Strongest positive correlation with GA-Nes microglia is found in interferon-response clusters injury 2 and p25 cluster 6. d Immunoblots from whole neocortex of total and phosphorylated Stat effector proteins of interferon signaling in end-stage GA-Nes (+) and age-matched Nes controls (−). Calnexin is used as a loading control. e Relative phosphorylation levels normalized to total protein levels increase in end-stage GA-Nes only for Stat3 compared to age-matched Nes controls [Two-way ANOVA (effector × genotype) F (2,24) = 3.88, p = 0.035 η 2 = 0.323. Sidak post hoc pStat1 Nes (62.4 ± 24.0) vs GA-Nes (52 ± 31.1) ns p = 0.858, pStat2 Nes (20 ± 10) vs GA-Nes (14 ± 8.9) ns p = 0.998 , pStat3 Nes (38.6 ± 17.3) vs GA-Nes (82.6 ± 24.9) * p = 0.017], n = 5/group. f ELISA for IFNγ in whole neocortex homogenate is significantly increased in end-stage GA-Nes mice. [Mann–Whitney Nes (361 ± 60.7) vs GA-Nes (515 ± 49.3) ** p = 0.029, 97% CI 62.3–310] n = 4/group. g IFNγ in whole neocortex homogenate is not significantly changed in affected PR-Nes mice compared to Nes littermate controls. [ t (10) = 1.661, Nes (478 ± 94.9) vs PR-Nes affected (389 ± 91), p = 0.128 η 2 = 0.216] n = 6/group. Power to detect η 2 > 0.5 = 83.7%

Article Snippet: We performed western blotting using the following antibodies: GFP (632381, Takara 1:1000), Stat1 (#9172, Cell Signaling 1:1000), Tubb3 (TUJ1, Biolegend 1:1000), Dlg4 (PSD-95) (K28/74, Neuromab 1:1000), Syp (Sy38, Millipore 1:1000), Arc (sc-17839, Santa Cruz 1:200), pStat1 Tyr701 (#7649, Cell Signaling 1:500), Stat2 (D9J7L, Cell Signaling 1:1k), pStat2 Y690 (ab53132, Abcam 1:500), Stat3 (#79D7, Cell Signaling 1:1000), p-Stat3 Tyr705 (#9145, Cell Signaling 1:1000), calnexin (SPA-860F, Enzo 1:7000).

Techniques: Mutagenesis, Expressing, Isolation, Western Blot, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY