Journal: Iranian Journal of Basic Medical Sciences

Article Title: Quercetin of huoxuehuayu tongluo decoction and azithromycin combination therapy effectively improves rat tubal factor infertility by inhibiting inflammation

doi: 10.22038/IJBMS.2024.72049.15662

Figure Lengend Snippet: Basic information of primary antibodies in Western blot

Article Snippet: Anti-p-Tyk2 , bs-3437R , Rabbit , 1:1000 , Bioss , China.

Techniques: Western Blot

Journal: The Journal of Biological Chemistry

Article Title: SADS-CoV nsp1 inhibits the STAT1 phosphorylation by promoting K11/K48-linked polyubiquitination of JAK1 and blocks the STAT1 acetylation by degrading CBP

doi: 10.1016/j.jbc.2024.105779

Figure Lengend Snippet: Nsp1 induced JAK1 degradation. A and B , HEK-293T cells and LLC-PK1 cells were transfected with pCAGGS-3×Flag-nsp1. After 24 h, the HEK-293T cells were incubated with human IFN-β for 4 h, and the LLC-PK1 cells were stimulated by SeV for 8 h. JAK1, p-JAK1, TYK2, and p-TYK2 expression was detected by Western blotting. All protein levels were analyzed using ImageJ. Western blotting assay was repeated in two independent experiments. C , schematic presentation of the ISGF3 activation process created using BioRender.com . D , HEK-293T cells were transfected with pCAGGS-3×Flag-nsp1-mutant. After 24 h, HEK-293T cells were incubated with human IFN-β for 4 h. JAK1 and p-JAK1 expression was detected by Western blotting. All protein levels were analyzed using ImageJ. Western blotting assay was repeated in two independent experiments. The data are the means ± SD. The p -value was calculated using the t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. HEK, human embryonic kidney cell line; IFN-β, interferon-β; ISGF3, interferon-stimulated gene factor 3; JAK1, Janus kinase 1; nsp1, nonstructure protein 1; SeV, Sendai virus; TYK2, tyrosine kinase 2.

Article Snippet: This study used the following antibodies and reagents: mouse anti-FLAG-tag monoclonal antibody (mAb), mouse anti-HA-tag mAb, horseradish peroxidase (HRP)-conjugated goat anti-mouse (H+L), HRP-conjugated goat anti-rabbit (H+L), HRP-conjugated mouse anti-rabbit (L), HRP-conjugated goat anti-mouse (L), p-STAT1 rabbit mAb, STAT1 rabbit mAb, p-STAT2 rabbit mAb, STAT2 rabbit mAb, IRF9 rabbit mAb, p-JAK1 rabbit mAb, JAK1 rabbit mAb, TYK2 rabbit mAb, and p-TYK2 rabbit mAb (ABclonal); CREB-BP rabbit mAb (Affinity); beta-actin rabbit antibody (Proteintech); MG132 and Z-VAD-FMK (Beyotime); CHX710 (MedChemExpress); human IFN-β (InvivoGen); Lipofectamine 3000 transfection reagent (Sigma); and Dylight-conjugated 488 goat anti-mouse IgG (Abbkine).

Techniques: Transfection, Incubation, Expressing, Western Blot, Activation Assay, Mutagenesis, Virus

Journal: The Journal of Biological Chemistry

Article Title: SADS-CoV nsp1 inhibits the STAT1 phosphorylation by promoting K11/K48-linked polyubiquitination of JAK1 and blocks the STAT1 acetylation by degrading CBP

doi: 10.1016/j.jbc.2024.105779

Figure Lengend Snippet: Nsp1 inhibited STAT1 acetylation and dephosphorylation. A and B , HEK-293T cells were plated onto 6-well plates and transfected with pCAGGS-3×Flag-nsp1. Then, the cells were incubated with DMEM or MG132 (5 μM) for 6 h. The cells were collected and incubated with STAT1-tagged beads. The interaction between STAT1 and TCPTP/Ace was detected using Western blotting. C , HEK-293T cells were plated onto 6-well plates and transfected with pCAGGS-3×Flag-nsp1-mutant. The cells were incubated with DMEM for 6 h. Then, the cells were collected and incubated with STAT1-tagged beads. The interaction between STAT1 and TCPTP/Ace was detected using Western blotting. D , schematic presentation of the STAT1 phosphorylation-acetylation-dephosphorylation cycle created using BioRender.com . E and F , HEK-293T cells were plated in 60-mm glass-bottom dishes and transfected with pCAGGS-3×Flag-nsp1 (5000 ng per dish). The cells were collected after 24 or 36 h. Nuclear and cytoplasmic proteins were extracted using a protein extraction kit. Then, the CBP and STAT1 expression levels and STAT1 phosphorylation level were detected by Western blotting. CBP, CREB-binding protein; DMEM, Dulbecco’s modified Eagle’s medium; HEK, human embryonic kidney cell line; IFN-β, interferon-β; JAK1, Janus kinase 1; nsp1, nonstructure protein 1; STAT, signal transducer and activator of transcription; TCPTP, T cell protein tyrosine phosphatase; TYK2, tyrosine kinase 2.

Article Snippet: This study used the following antibodies and reagents: mouse anti-FLAG-tag monoclonal antibody (mAb), mouse anti-HA-tag mAb, horseradish peroxidase (HRP)-conjugated goat anti-mouse (H+L), HRP-conjugated goat anti-rabbit (H+L), HRP-conjugated mouse anti-rabbit (L), HRP-conjugated goat anti-mouse (L), p-STAT1 rabbit mAb, STAT1 rabbit mAb, p-STAT2 rabbit mAb, STAT2 rabbit mAb, IRF9 rabbit mAb, p-JAK1 rabbit mAb, JAK1 rabbit mAb, TYK2 rabbit mAb, and p-TYK2 rabbit mAb (ABclonal); CREB-BP rabbit mAb (Affinity); beta-actin rabbit antibody (Proteintech); MG132 and Z-VAD-FMK (Beyotime); CHX710 (MedChemExpress); human IFN-β (InvivoGen); Lipofectamine 3000 transfection reagent (Sigma); and Dylight-conjugated 488 goat anti-mouse IgG (Abbkine).

Techniques: De-Phosphorylation Assay, Transfection, Incubation, Western Blot, Mutagenesis, Phospho-proteomics, Protein Extraction, Expressing, Binding Assay, Modification

Journal: The Journal of Biological Chemistry

Article Title: SADS-CoV nsp1 inhibits the STAT1 phosphorylation by promoting K11/K48-linked polyubiquitination of JAK1 and blocks the STAT1 acetylation by degrading CBP

doi: 10.1016/j.jbc.2024.105779

Figure Lengend Snippet: Schematic diagram of SADS-CoV nsp1 blocking IFN production and response. SADS-CoV nsp1 inhibited TBK1 phosphorylation by preventing TBK1 ubiquitin modification, ultimately blocking IRF3 activation. SADS-CoV nsp1 blocked IFN transcriptional enhancer formation by inducing CBP degradation. SADS-CoV nsp1 promoted K11/K48-linked JAK1 polyubiquitination, then induced JAK1 degradation through the proteasome pathway. SADS-CoV inhibited STAT1 phosphorylation by inducing JAK1 degradation. SADS-CoV nsp1 inhibited STAT1 acetylation and dephosphorylation by inducing CBP degradation. This schematic diagram was created using BioRender.com . CBP, CREB-binding protein; IFN-β, interferon-β; IFNAR, interferon alpha and beta receptor subunit; IRF9, interferon regulatory factor-9; ISG, interferon-stimulated gene; ISGF3, interferon-stimulated gene factor 3; ISRE, interferon-stimulated response elements; JAK1, Janus kinase 1; nsp1, nonstructure protein 1; SADS-CoV, Swine Acute Diarrhea Syndrome Coronavirus; STAT, signal transducer and activator of transcription; TCPTP, T cell protein tyrosine phosphatase; TYK2, tyrosine kinase 2.

Article Snippet: This study used the following antibodies and reagents: mouse anti-FLAG-tag monoclonal antibody (mAb), mouse anti-HA-tag mAb, horseradish peroxidase (HRP)-conjugated goat anti-mouse (H+L), HRP-conjugated goat anti-rabbit (H+L), HRP-conjugated mouse anti-rabbit (L), HRP-conjugated goat anti-mouse (L), p-STAT1 rabbit mAb, STAT1 rabbit mAb, p-STAT2 rabbit mAb, STAT2 rabbit mAb, IRF9 rabbit mAb, p-JAK1 rabbit mAb, JAK1 rabbit mAb, TYK2 rabbit mAb, and p-TYK2 rabbit mAb (ABclonal); CREB-BP rabbit mAb (Affinity); beta-actin rabbit antibody (Proteintech); MG132 and Z-VAD-FMK (Beyotime); CHX710 (MedChemExpress); human IFN-β (InvivoGen); Lipofectamine 3000 transfection reagent (Sigma); and Dylight-conjugated 488 goat anti-mouse IgG (Abbkine).

Techniques: Blocking Assay, Phospho-proteomics, Ubiquitin Proteomics, Modification, Activation Assay, De-Phosphorylation Assay, Binding Assay

Journal: Frontiers in Pharmacology

Article Title: Crosstalk between TBK1/IKKε and the type I interferon pathway contributes to tubulointerstitial inflammation and kidney tubular injury

doi: 10.3389/fphar.2022.987979

Figure Lengend Snippet: Type I interferons promote inflammation in renal tubular cells and kidney tissue. (A) Quantitative reverse transcription PCR (qRT-PCR) analysis of ISGs ( Oasl2, Usp18 ) in MCT cells stimulated with increasing concentrations of IFNβ for 6 and 24 h. Values for mRNA were normalized to GAPDH expression. Data are expressed as the Mean ± SEM of three independent experiments. * p ≤ 0.05 vs untreated control cells. (B) Time-course (minutes, m) for the activation of the TI-IFN pathway. Representative western blot of phosphorylated isoforms of STAT1, JAK1 and TYK2 in MCT cells stimulated with 1.0 mUI/ml IFNβ. Tubulin was used as loading control. (C,D) Activation of the NF-κB pathway. (C) Cells were stimulated as indicated in A and the mRNA expression of cytokines ( Cxcl10, Ccl2, IL-6 ) assessed by PCR. Data are Mean ± SEM of three independent experiments. * p ≤ 0.05 vs untreated control cells. (D) Representative western blot of phosphorylated IκBα (upper panel) and detection of nuclear p65 expression by immunofluorescence and confocal microscopy (green signal) (lower panel) in MCT cells stimulated with IFNβ for the indicated times (minutes, m). Original magnification ×400. Scale bar 10 µM. (E,F) Inhibition of the TI-IFN pathway by functional blockade of IFNAR (E) or pharmacological inhibition of TYK2/JAK1 (F) . MCT cells were preincubated with 10 μg/ml neutralizing anti-IFNAR antibody (IFNAR-Ab) or with 2.5 μM PF-06700,841 (PF) for 1 h before the addition of IFNβ for 6 h or 24 h Oasl2 mRNA expression was assessed by RT-PCR after 24 h whereas Cxcl10, Ccl2, and Il-6 gene expression were measured after 6 h. Results are shown as Mean ± SEM ( n = 3). * p ≤ 0.05 vs untreated control cells; # p ≤ 0.05 vs IFNβ treatment. (G,H) Activation and functional analysis of TBK1/IKKε signaling. (G) MCT cells were stimulated with 1.0 mUI/ml IFNβ for the indicated times (minutes, m). Activation of TBK/IKKε was detected by their phosphorylated isoforms by western blot (pTBK1, upper panel) or confocal microscopy (pIRF3, lower panel). Original magnification ×400. Scale bar 10 µM. (H) Transcriptional response in MCT cells pretreated for 1 h with 50 µM amlexanox (AMX) before 1.0 mUI/ml IFNβ addition was assessed by qRT-PCR following 6 h ( Cxcl10, Ccl2, Il-6 ) or 24 h ( Oasl2 ). Data are Mean ± SEM of three independent experiments. * p ≤ 0.05 vs untreated control cells; # p ≤ 0.05 vs IFNβ-treated cells. (I) Molecular routes activated by IFNβ in tubular cells. IFNβ activates the canonical TI-IFN pathway leading to ISG expression by binding to IFNAR and recruiting TYK2/JAK1/STAT1 signaling (blue pathway). Downstream IFNAR or TYK2/JAK1, IFNβ also promotes the synthesis of proinflammatory cytokines and ISGs by activating the NF-κB (red pathway) and IRF3 (green pathway) transcription factors, respectively. Inhibiting TI-IFN signaling at IFNAR (IFNAR-Ab) or TYK2/JAK1 (PF-06700,841: PF) downmodulates the direct (blue pathway) or indirect (green pathway) pathways driving cytokine and IFNα/β gene expression, consistent with crosstalk between the TI-IFN and NF-κB pathways (discontinued blue line). Likewise, interfering with TBK1/IKKε activation (AMX) decreases both ISG and cytokine gene expression, disclosing a second crosstalk between the TBK1/IKKε signaling node and NF-κB. (J-L) Murine model of systemic IFNβ injection for evaluation of the renal response. (J) Experimental design for IFNβ administration to mice. (K) Time-course of mRNA expression of canonical ISGs ( Oasl2, Usp18, Ifit1 ) and chemokines and cytokines ( Cxcl10, Ccl2, Il-6 ) in kidneys from control or IFNβ-injected mice. Bar charts represent the Mean ± SEM for each gene (n = 5-6 mice/group). * p ≤ 0.05 vs control mice. (L) Immunohistochemical analysis and quantification of markers for mononuclear phagocytes (F4/80) (upper panel) and lymphocytes (CD3) (lower panel) in kidneys from control and IFNβ-injected mice. Representative microphotographs of immune cells in kidney tissue from control and IFNβ-injected mice at 24 h. The number of cells per high power field (hpf) was quantified and results were expressed as Mean ± SEM. * p ≤ 0.05 vs control untreated mice (n = 4-6 mice/group). Original magnification ×200. Scale bar 100 µm.

Article Snippet: The following primary antibodies were used to detect specific proteins of interest: rabbit polyclonal anti-p-STAT1 (Tyr701) (Invitrogen, 44-376G), p-TYK2 (pTyr1054) (Origene, TA333304), and p-IKKε (Ser172) (Sigma Aldrich, 06-1340); rabbit monoclonal anti-p-TBK1/NAK (S172) (D52C2) XP ® (Cell Signaling Technology, 1,675,483), TBK1/NAK (E8I3G) (Cell Signaling Technology, 38,066), IKKε (D61F9) XP ® (Cell Signalling Technology, 3416), pIRF3 (Ser396) (4D4G) (Cell Signalling Technology, 4,947) and IRF-3 (D83B9) (Cell Signalling Technology, 4,302); monoclonal mouse anti-pIKBα (Santa Cruz, sc-8404).

Techniques: Reverse Transcription, Quantitative RT-PCR, Expressing, Control, Activation Assay, Western Blot, Immunofluorescence, Confocal Microscopy, Inhibition, Functional Assay, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Binding Assay, Injection, Immunohistochemical staining

Journal: Frontiers in Pharmacology

Article Title: Crosstalk between TBK1/IKKε and the type I interferon pathway contributes to tubulointerstitial inflammation and kidney tubular injury

doi: 10.3389/fphar.2022.987979

Figure Lengend Snippet: TWEAK and LPS activate the TBK1/IKKε and TI-IFN pathways in cultured kidney tubular cells and in vivo in the kidneys. (A,B) TWEAK and LPS activated the TBK1/IKKε/IRF3 pathway as assessed by phosphorylated TBK1 and IKKε detected by western blot (A) and phosphorylated IRF3 detected by western blot (B, upper panel) or immunofluorescence (B, lower panel) as compared with vehicle-treated control cells (Cont). Tubular MCT cells were stimulated with 100 ng/ml TWEAK or 1 μg/ml LPS for 15-60 min (m). Figures are representative of experiments repeated at least thrice. Original magnification ×400 in B (lower panel). Scale bar 10 µM. (C–E) TWEAK and LPS activated the TI-IFN pathway. (C) Quantitative RT-PCR for mRNA expression of Ifna , Ifnb1 , and Ifnar1/2 in MCT cells stimulated with TWEAK or LPS for 6 and 24 h, and TWEAK-dependent IFNβ secretion assessed in cell culture supernatants (24 h) by ELISA (right upper panel). Results are expressed as the Mean ± SEM of at least three individual experiments. * p ≤ 0.05 vs control untreated cells . (D) Representative western blots of increased levels of phosphorylated STAT1 (pSTAT1) and TYK2 (pTYK2) in total protein extracts from MCT cells stimulated with 100 ng/ml TWEAK or 1 μg/ml LPS. Stimulation times are expressed in hours (H) . (E) Extended gene expression study of samples analyzed in panel C shows that TWEAK and LPS also upregulated ISG genes ( Ifit1, Usp18, Oasl2, Cxcl10 ). Results are the Mean ± SEM. * p ≤ 0.05 vs control untreated cells. (F) The contribution of the autocrine/paracrine recruitment of the TI-IFN pathway to TWEAK- and LPS-elicited responses in MCT cells was studied by blocking IFNAR with 10 μg/ml neutralizing anti-IFNAR antibody before stimulation with 100 ng/ml TWEAK or 1 μg/ml LPS. Gene expression was evaluated by q-RT-PCR after 6 h ( Cxcl10, Ccl2 ) or 24 h ( Oasl2 ). Results are expressed as Mean ± SEM of four or five experiments. * p ≤ 0.05 vs control untreated cells, # p ≤ 0.05 vs TWEAK- or LPS-stimulated cells. (G) Pharmacological inactivation of canonical IKKs limits TBK1/IKK-dependent ISG transcription in TWEAK- or LPS-stimulated MCT cells. Cultured MCT cells were pretreated for 1 h with chemical inhibitors of IKKα/β, namely 10 µM parthenolide (Parth) or 2.5 µM IKK16, before the addition of TWEAK or LPS for 24 h. Gene expression of ISGs ( Usp18, Oasl2 ) was assessed by q-RT-PCR. Bar chart represents the Mean ± SEM. * p ≤ 0.05 vs control vehicle-stimulated cells and # p ≤ 0.05 vs TWEAK- or LPS-stimulated cells ( n = 3/4).

Article Snippet: The following primary antibodies were used to detect specific proteins of interest: rabbit polyclonal anti-p-STAT1 (Tyr701) (Invitrogen, 44-376G), p-TYK2 (pTyr1054) (Origene, TA333304), and p-IKKε (Ser172) (Sigma Aldrich, 06-1340); rabbit monoclonal anti-p-TBK1/NAK (S172) (D52C2) XP ® (Cell Signaling Technology, 1,675,483), TBK1/NAK (E8I3G) (Cell Signaling Technology, 38,066), IKKε (D61F9) XP ® (Cell Signalling Technology, 3416), pIRF3 (Ser396) (4D4G) (Cell Signalling Technology, 4,947) and IRF-3 (D83B9) (Cell Signalling Technology, 4,302); monoclonal mouse anti-pIKBα (Santa Cruz, sc-8404).

Techniques: Cell Culture, In Vivo, Western Blot, Immunofluorescence, Control, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Gene Expression, Blocking Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Membrane (M) and Spike (S) Proteins Antagonize Host Type I Interferon Response

doi: 10.3389/fcimb.2021.766922

Figure Lengend Snippet: M protein inhibits the interaction between KPNA6 and IRF3. (A) Partial results of the IP-MS assay using Flag-tagged M protein as a bait. HEK293T cells were co-transfected with plasmid encoding Flag-tagged SARS-CoV-2 M protein. At 24 hpt., cells were treated with SeV for 12 h Co-IP was performed by incubating the cell lysates with anti-Flag magnetic beads overnight, and eluted proteins were subjected to western blotting verification to confirm successful IP of viral proteins. The elution mixture was processed for protein identification by Mass Spectrometry. The results were analyzed by Proteome Discoverer 2.2 software. (B) Co-IP of M and IPed KPNA proteins. HEK293T cells were co-transfected with Flag-tagged SARS-CoV-2 M plasmid and each Myc-tagged KPNA2/KPNA3/KPNA4/KPNA6 plasmid. At 36 hpt., Co-IP was performed by incubating the lysates with the anti-Flag antibody magnetic beads overnight. After extensive washing, the eluate was analyzed by western blotting with indicated antibodies. (C) Co-IP of IRF3 and KPNA2 or KPNA6. HEK293T cells were co-transfected with HA-tagged IRF3 plasmids and Myc-tagged KPNA2 or KPNA6 plasmids or empty plasmids. At 36 hpt., Co-IP was performed by incubating the lysates with the anti-HA antibody overnight before the addition of magnetic beads. (D) HEK293T cells were transfected with plasmids encoding IRF3-HA and KPNA2-Myc, together with or without M-Flag. The cell lysate was subjected to a Co-IP assay using anti-HA or anti-Myc. (E) HEK293T cells were transfected with plasmids encoding IRF3-HA and KPNA6-Myc, together with or without M-Flag. Then the cell lysate was subjected to a Co-IP assay using anti-HA or anti-Myc. (F) Interaction between endogenous IRF3 and KPNA6 in the presence of M protein. HEK293T cells were transfected with M-Flag for 24 h and then treated with SeV for another 12 h, the cells were harvested and subjected to a Co-IP assay using IgG or IRF3 antibody. (G) HEK293T cells were transfected with M-Flag for 24 h and treated with SeV for 12 h. IRF3 in the nuclear fractions or the cytoplasmic was determined by immunoblotting analyses. GAPDH and Lamin B1 served as cytoplasmic and nuclear protein controls, respectively. (H) Nuclear translocation of IRF3. HeLa cells were transfected with M-Flag or empty vector. At 24 hpt., cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking with PBS containing 2% fetal bovine serum (FBS), the cells were probed with primary antibodies (anti-Flag and anti-IRF3) and secondary antibodies (anti-Alexa Fluor 488 and anti-Alexa Fluor 648). Scale bar, 10 µm. Representative blots and fluorescence pictures of 3 independent experiments were shown. The percentages of IRF3 in the nucleus (out of total IRF3 signal) were quantified by ImageJ software. ***P<0.001, t -test.

Article Snippet: The following antibodies were used: rabbit anti-DYKDDDDK tag (ABclonal, Cat # AE005), rabbit anti-HA (CST, Cat # 3724S), rabbit anti-IRF3 (ABclonal, Cat # D199862-0100), rabbit anti-p-IRF3 (CST, Cat # 4947S), rabbit anti-TBK1 (4A Biotech co.Ltd, Cat# 4ab032308cs), rabbit anti-p-TBK1 (Absin, Cat # abs140019), rabbit anti-KPNA6 (ImmunoWay, Cat # YN3049), rabbit anti-STAT2 (CST, Cat # 72604S), rabbit anti-p-STAT2 (CST, Cat # 88410S), rabbit anti-p-STAT1 (CST, Cat # 9167S), rabbit anti-p-TYK2 (Absin, Cat # abs131318), rabbit anti-TYK2 (Absin, Cat # abs131318a), rabbit anti-Myc (Proteintech, Cat # I6286-I-AP), anti-rabbit IgG HRP-linked antibody (CST, Cat # 7074), mouse anti-DYKDDDDK tag (Bioworld, Cat # AP0007), mouse anti-GAPDH (Proteintech, Cat # 60004-I-Ig), mouse anti-VSV-G (Abgent, Cat # AP1016a) and HRP goat anti-mouse IgG (BioLegend, Cat # 405306).

Techniques: Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Magnetic Beads, Western Blot, Mass Spectrometry, Software, Translocation Assay, Blocking Assay, Fluorescence

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Membrane (M) and Spike (S) Proteins Antagonize Host Type I Interferon Response

doi: 10.3389/fcimb.2021.766922

Figure Lengend Snippet: A subset of viral proteins antagonize IFN-β production. (A) HEK293T cells cultured in 24-well plates (1 × 10 5 cells per well) were transfected with Flag-N1 empty vector (200 ng) or the SARS-CoV-2 protein plasmids (200 ng). At 24 h after transfection, cells were stimulated by SeV (MOI=1), and at 12 h after stimulation, the cells were harvested for RNA extraction and subsequent RT-qPCR analysis to assess the expression of IFN-β or ISG (IFIT1). (B) The Flag-N1 empty vector and the SARS-CoV-2 protein plasmids (100 ng) were transfected with the indicated combinations of plasmids expressing RIG-IN (10 ng), MAVS (10 ng), TBK1 (100 ng), and IRF3-5D (10 ng) into HEK293T cells cultured in 96-well plates (0.5 × 10 5 cells per well). The IFN-β-Luc (50 ng) plasmids were co-transfected to assess the activation of IFN promoter and the pRL-TK (5 ng) was transfected as an internal control. Dual luciferase assays were performed 36 hpt. Results were shown as Mean ± SD. Statistical significance was assessed via comparison to the Flag-N1 control using one-way ANOVA with Dunnett’s correction, **p < 0.01, ***p < 0.001. ns, not significant. The data shown are representative of 3 independent experiments. (C) Phosphorylation of IRF3 and TBK1. HEK293T cells were transfected with viral protein-encoding plasmids (1 µg), treated with SeV for 12 h, and analyzed for phosphorylated IRF3 (anti-p-IRF3 at S396), total IRF3 (anti-IRF3), phosphorylated TBK1 (anti-p-TBK1 at S172), total TBK1 (anti-TBK1), and GAPDH (anti-GAPDH) by western blotting. Representative blots of three independent experiments are shown. (D) Summary of the antagonism of IFN-I production. The potential inhibitory steps are indicated for individual viral proteins.

Article Snippet: The following antibodies were used: rabbit anti-DYKDDDDK tag (ABclonal, Cat # AE005), rabbit anti-HA (CST, Cat # 3724S), rabbit anti-IRF3 (ABclonal, Cat # D199862-0100), rabbit anti-p-IRF3 (CST, Cat # 4947S), rabbit anti-TBK1 (4A Biotech co.Ltd, Cat# 4ab032308cs), rabbit anti-p-TBK1 (Absin, Cat # abs140019), rabbit anti-KPNA6 (ImmunoWay, Cat # YN3049), rabbit anti-STAT2 (CST, Cat # 72604S), rabbit anti-p-STAT2 (CST, Cat # 88410S), rabbit anti-p-STAT1 (CST, Cat # 9167S), rabbit anti-p-TYK2 (Absin, Cat # abs131318), rabbit anti-TYK2 (Absin, Cat # abs131318a), rabbit anti-Myc (Proteintech, Cat # I6286-I-AP), anti-rabbit IgG HRP-linked antibody (CST, Cat # 7074), mouse anti-DYKDDDDK tag (Bioworld, Cat # AP0007), mouse anti-GAPDH (Proteintech, Cat # 60004-I-Ig), mouse anti-VSV-G (Abgent, Cat # AP1016a) and HRP goat anti-mouse IgG (BioLegend, Cat # 405306).

Techniques: Cell Culture, Transfection, Plasmid Preparation, RNA Extraction, Quantitative RT-PCR, Expressing, Activation Assay, Luciferase, Western Blot

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Membrane (M) and Spike (S) Proteins Antagonize Host Type I Interferon Response

doi: 10.3389/fcimb.2021.766922

Figure Lengend Snippet: M protein inhibits the interaction between KPNA6 and IRF3. (A) Partial results of the IP-MS assay using Flag-tagged M protein as a bait. HEK293T cells were co-transfected with plasmid encoding Flag-tagged SARS-CoV-2 M protein. At 24 hpt., cells were treated with SeV for 12 h Co-IP was performed by incubating the cell lysates with anti-Flag magnetic beads overnight, and eluted proteins were subjected to western blotting verification to confirm successful IP of viral proteins. The elution mixture was processed for protein identification by Mass Spectrometry. The results were analyzed by Proteome Discoverer 2.2 software. (B) Co-IP of M and IPed KPNA proteins. HEK293T cells were co-transfected with Flag-tagged SARS-CoV-2 M plasmid and each Myc-tagged KPNA2/KPNA3/KPNA4/KPNA6 plasmid. At 36 hpt., Co-IP was performed by incubating the lysates with the anti-Flag antibody magnetic beads overnight. After extensive washing, the eluate was analyzed by western blotting with indicated antibodies. (C) Co-IP of IRF3 and KPNA2 or KPNA6. HEK293T cells were co-transfected with HA-tagged IRF3 plasmids and Myc-tagged KPNA2 or KPNA6 plasmids or empty plasmids. At 36 hpt., Co-IP was performed by incubating the lysates with the anti-HA antibody overnight before the addition of magnetic beads. (D) HEK293T cells were transfected with plasmids encoding IRF3-HA and KPNA2-Myc, together with or without M-Flag. The cell lysate was subjected to a Co-IP assay using anti-HA or anti-Myc. (E) HEK293T cells were transfected with plasmids encoding IRF3-HA and KPNA6-Myc, together with or without M-Flag. Then the cell lysate was subjected to a Co-IP assay using anti-HA or anti-Myc. (F) Interaction between endogenous IRF3 and KPNA6 in the presence of M protein. HEK293T cells were transfected with M-Flag for 24 h and then treated with SeV for another 12 h, the cells were harvested and subjected to a Co-IP assay using IgG or IRF3 antibody. (G) HEK293T cells were transfected with M-Flag for 24 h and treated with SeV for 12 h. IRF3 in the nuclear fractions or the cytoplasmic was determined by immunoblotting analyses. GAPDH and Lamin B1 served as cytoplasmic and nuclear protein controls, respectively. (H) Nuclear translocation of IRF3. HeLa cells were transfected with M-Flag or empty vector. At 24 hpt., cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking with PBS containing 2% fetal bovine serum (FBS), the cells were probed with primary antibodies (anti-Flag and anti-IRF3) and secondary antibodies (anti-Alexa Fluor 488 and anti-Alexa Fluor 648). Scale bar, 10 µm. Representative blots and fluorescence pictures of 3 independent experiments were shown. The percentages of IRF3 in the nucleus (out of total IRF3 signal) were quantified by ImageJ software. ***P<0.001, t -test.

Article Snippet: The following antibodies were used: rabbit anti-DYKDDDDK tag (ABclonal, Cat # AE005), rabbit anti-HA (CST, Cat # 3724S), rabbit anti-IRF3 (ABclonal, Cat # D199862-0100), rabbit anti-p-IRF3 (CST, Cat # 4947S), rabbit anti-TBK1 (4A Biotech co.Ltd, Cat# 4ab032308cs), rabbit anti-p-TBK1 (Absin, Cat # abs140019), rabbit anti-KPNA6 (ImmunoWay, Cat # YN3049), rabbit anti-STAT2 (CST, Cat # 72604S), rabbit anti-p-STAT2 (CST, Cat # 88410S), rabbit anti-p-STAT1 (CST, Cat # 9167S), rabbit anti-p-TYK2 (Absin, Cat # abs131318), rabbit anti-TYK2 (Absin, Cat # abs131318a), rabbit anti-Myc (Proteintech, Cat # I6286-I-AP), anti-rabbit IgG HRP-linked antibody (CST, Cat # 7074), mouse anti-DYKDDDDK tag (Bioworld, Cat # AP0007), mouse anti-GAPDH (Proteintech, Cat # 60004-I-Ig), mouse anti-VSV-G (Abgent, Cat # AP1016a) and HRP goat anti-mouse IgG (BioLegend, Cat # 405306).

Techniques: Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Magnetic Beads, Western Blot, Mass Spectrometry, Software, Translocation Assay, Blocking Assay, Fluorescence