Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: PLOS Pathogens

Article Title: Direct and biologically significant interactions of human herpesvirus 8 interferon regulatory factor 1 with STAT3 and Janus kinase TYK2

doi: 10.1371/journal.ppat.1011806

Figure Lengend Snippet: (A) Immunoblot assessment of levels of active, autophosphorylated pTYK2 and pJAK1, reflecting activities of the respective Janus kinases, as a function of vIRF-1 expression. Cultures of 293T cells were transfected with different amounts of the kinase vectors (0.15–0.60 μg) along with vIRF-1 expression plasmid; one dose (*, 0.5 μg) of TYK2 or JAK1 vector was transfected with RFP-expressing plasmid (negative control). Relative levels of TYK2, JAK1, and vIRF-1 expression along with β-actin (loading control) were assessed by immunoblotting. (B) Luciferase reporter (4xM67-Luc)-based analysis of functional suppression of TYK2 activity by vIRF-1. 293T cells were cotransfected with the STAT3-responsive reporter and different amounts of TYK2 expression plasmid, along with either empty vector (vector) or vIRF-1 expression plasmid. Luciferase activities in cell extracts are shown relative to levels (set at 1) for empty-vector-transfected cells lacking TYK2 overexpression (-). Average values from triplicate samples along with standard deviations are shown. The blots below the chart verify appropriate, vector-dose-dependent expression of TYK2 (in combined triplicate lysates). (C) Analysis of potential vIRF-1 effects on TYK2-STAT3 and TYK2-gp130 interactions in response to vIL-6 stimulation. Transfected-cell lysates containing TYK2-S in the absence (vec) or presence of vIRF-1 (v1) were subjected to S-protein-based AP and precipitated material was immunoblotted for detection of coprecipitated gp130 and total and phosphorylated STAT3. Tyrosine-phosphorylated (active) TYK2 (pTyk2) was also detected, along with vIRF-1. (D) Interaction of bacterially-produced and purified GST-tagged vIRF-1 (v1-GST) and Flag-tagged TYK2. AP of GST-vIRF-1 or GST (control) with glutathione beads and Flag-immunoblotting for coprecipitated TYK2 identified specific and direct interaction of vIRF-1 and TYK2. The asterisk indicates degradation products.

Article Snippet: Primary antibodies used for immunoblotting were: CBD (New England BioLabs, E8034S); Flag (Sigma, F1804); β-actin (Sigma, A5316); GAPDH (Invitrogen, TAB1001); vIRF-1 (rabbit polyclonal antiserum, provided by Dr. Gary Hayward); vIL-6 (rabbit polyclonal antiserum [ ]); STAT1, STAT2, STAT3, c-Myc and GST from Santa Cruz Biotechnologies (sc-464, sc-1668, sc482, sc-764 and sc-138, respectively); pSTAT3, TYK2 and JAK1 from Cell Signaling Technology (9131, 14193, 3344); LANA (Advanced Biotechnologies, 13-210-100), p-TYK2 (Cell Signaling Technology, 68790; BD Biosciences, 558394).

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Negative Control, Control, Luciferase, Functional Assay, Activity Assay, Over Expression, Produced, Purification

Journal: PLOS Pathogens

Article Title: Direct and biologically significant interactions of human herpesvirus 8 interferon regulatory factor 1 with STAT3 and Janus kinase TYK2

doi: 10.1371/journal.ppat.1011806

Figure Lengend Snippet: (A) HHV-8 (BAC16) infected iSLK cells were depleted of either TYK2 or JAK1 via lentiviral vector-mediated shRNA expression, using two different shRNAs for each target; non-silencing (ns) shRNA was used as a control. The cultures were left untreated (latent) or treated with Dox/NaB to induce lytic replication, and cells were harvested after six days for immunoblot analysis of pSTAT3, total-STAT3, and lytically expressed vIRF-1 and vIL-6. Relative levels of pSTAT3, normalized to total-STAT3, are shown below the pSTAT3 blots. (B) Analysis of pSTAT3 and pTYK2 levels as a function of vIRF-1 expression in iSLK cells, infected with native (WT) and vIRF-1-knockout (v1 ttg ) BAC16 viruses. Cultures were ether untreated (latent) or treated with Dox/NaB (lytic) for six days. Immunoblotting of whole-cell lysates was carried out to detect levels of pSTAT3 and pTYK2 relative to total STAT3 and TYK2 and lytic markers vIRF-1 and vIL-6. Charts show quantified data from triplicate lytic cultures, with WT values set at 1. Student’s t-test P values (two-tailed) are shown.

Article Snippet: Primary antibodies used for immunoblotting were: CBD (New England BioLabs, E8034S); Flag (Sigma, F1804); β-actin (Sigma, A5316); GAPDH (Invitrogen, TAB1001); vIRF-1 (rabbit polyclonal antiserum, provided by Dr. Gary Hayward); vIL-6 (rabbit polyclonal antiserum [ ]); STAT1, STAT2, STAT3, c-Myc and GST from Santa Cruz Biotechnologies (sc-464, sc-1668, sc482, sc-764 and sc-138, respectively); pSTAT3, TYK2 and JAK1 from Cell Signaling Technology (9131, 14193, 3344); LANA (Advanced Biotechnologies, 13-210-100), p-TYK2 (Cell Signaling Technology, 68790; BD Biosciences, 558394).

Techniques: Infection, Plasmid Preparation, shRNA, Expressing, Control, Western Blot, Knock-Out, Two Tailed Test

Journal: PLOS Pathogens

Article Title: Direct and biologically significant interactions of human herpesvirus 8 interferon regulatory factor 1 with STAT3 and Janus kinase TYK2

doi: 10.1371/journal.ppat.1011806

Figure Lengend Snippet: (A) Verification of screening-identified vIRF-1Δ9 (deleted of residues 198–222) as defective for TYK2 interaction. This variant, along with STAT3-binding-defective Δ2 and Δ3 , were compared for TYK2 and STAT3 interactions by immunoprecipitation (IP) assay employing vIRF-1-Flag as “bait” and immunoblotting for vector-expressed S-tagged TYK2 or endogenous STAT3 in the precipitates. (B) Reciprocal precipitation for vIRF-1-TYK2 interaction, using affinity precipitation (AP) of TYK2-CBD and detection of coexpressed vIRF-1 or vIRF-1Δ9. (C) Testing of vIRF-1Δ9 for suppression of vIL-6-induced STAT3 signaling by transfection-based 4xM67-Luc reporter assay. The variant was inactive in this assay but retained inhibitory effect on the Smad-responsive 4xSBE-Luc reporter, used in parallel transfections. Immunoblotting for Flag-tagged vIRF-1, Smad3, and vIL-6 confirmed appropriate expression of the respective proteins in transfected-cell lysates (combined triplicates). (D) Diminished functional interaction of vIRF-1Δ9 with TYK2 was confirmed by analyzing cell lysates for pTYK2 and pSTAT3 levels in lentiviral vector-transduced, TYK2-overexpressing 292T cells (293T-TYK2) transfected with vIRF-1, vIRF-1Δ9, or RFP (negative control) expression plasmids. Levels of pTYK2 and pSTAT3, normalized to TYK2 and STAT3, respectively, and expressed relative to levels (set at 1) in the RFP vector-transfected culture are shown below the respective blots.

Article Snippet: Primary antibodies used for immunoblotting were: CBD (New England BioLabs, E8034S); Flag (Sigma, F1804); β-actin (Sigma, A5316); GAPDH (Invitrogen, TAB1001); vIRF-1 (rabbit polyclonal antiserum, provided by Dr. Gary Hayward); vIL-6 (rabbit polyclonal antiserum [ ]); STAT1, STAT2, STAT3, c-Myc and GST from Santa Cruz Biotechnologies (sc-464, sc-1668, sc482, sc-764 and sc-138, respectively); pSTAT3, TYK2 and JAK1 from Cell Signaling Technology (9131, 14193, 3344); LANA (Advanced Biotechnologies, 13-210-100), p-TYK2 (Cell Signaling Technology, 68790; BD Biosciences, 558394).

Techniques: Variant Assay, Binding Assay, Immunoprecipitation, Western Blot, Plasmid Preparation, Affinity Precipitation, Transfection, Reporter Assay, Expressing, Functional Assay, Negative Control

Journal: PLOS Pathogens

Article Title: Direct and biologically significant interactions of human herpesvirus 8 interferon regulatory factor 1 with STAT3 and Janus kinase TYK2

doi: 10.1371/journal.ppat.1011806

Figure Lengend Snippet: (A) Restriction profiling, using SpeI endonuclease, of wild-type (WT), vIRF-1Δ9-expressing (Δ9), and Δ9-mutation-repaired (Δ9 R ) HHV-8 BAC16 genomes. The white and grey arrowheads indicate the positions of DNA fragments containing WT/repaired and Δ9 vIRF-1 ORFs, respectively. (B) LANA-immunoblot verification of equivalent latent viral loads in iSLK cultures infected with the same infectious doses of native, vIRF-1 Δ9-mutated, and repaired (control) viruses. (C) Assessment of active STAT3 in recombinant HHV-8-infected and lytically reactivated iSLK cultures using the STAT3-responsive 4xM67-Luc reporter vector, transfected into the respective cultures. Luciferase activities from triplicate cultures are expressed as means, with standard deviations indicated; the mean value for WT virus is set at 1. Significance ( P ) of the increased luciferase activity in the vIRF-1Δ9-expressing cells was determined by student’s t-test. (D) Immunoblot analysis of pSTAT3 levels in lytically reactivated iSLK cells infected with WT, Δ9, and Δ9 R viruses. Cells were harvested at the indicated times after Dox/NaB treatment and lysates probed for total- and phospho-STAT3, in addition to LANA and lytically-induced vIRF-1 and vIL-6. Quantified levels of pSTAT3, normalized to total-STAT3 and relative to “WT” pSTAT3 levels (set at 1), are shown below the pSTAT3 blots. (E) Combined “day-2” pSTAT3/STAT3 data from panel D and two additional experiments. Average values relative to wild-type (set at 1), standard deviations, and P value (student’s t-test, two-tailed) for Δ9 against wild-type virus are shown. (F) Analysis of pSTAT3 and pTYK2 levels in lysates of lytically reactivated iSLK/BAC16 cultures, incorporating vIL-6-null (v6 ttg ) and vIL-6-null/vIRF-1Δ9 (v6 ttg +Δ9) viruses, harvested 2 days after Dox/NaB treatment. Levels of pSTAT3 and pTYK2, normalized to total-STAT3 and -TYK2 and expressed relative to levels (set at 1) in WT BAC16-infected cells, are indicated below the respective blots. (G) A similar analysis of pSTAT3 levels was undertaken using iTIME cells, harvested 2 days after lytic induction.

Article Snippet: Primary antibodies used for immunoblotting were: CBD (New England BioLabs, E8034S); Flag (Sigma, F1804); β-actin (Sigma, A5316); GAPDH (Invitrogen, TAB1001); vIRF-1 (rabbit polyclonal antiserum, provided by Dr. Gary Hayward); vIL-6 (rabbit polyclonal antiserum [ ]); STAT1, STAT2, STAT3, c-Myc and GST from Santa Cruz Biotechnologies (sc-464, sc-1668, sc482, sc-764 and sc-138, respectively); pSTAT3, TYK2 and JAK1 from Cell Signaling Technology (9131, 14193, 3344); LANA (Advanced Biotechnologies, 13-210-100), p-TYK2 (Cell Signaling Technology, 68790; BD Biosciences, 558394).

Techniques: Expressing, Mutagenesis, Western Blot, Infection, Control, Recombinant, Plasmid Preparation, Transfection, Luciferase, Virus, Activity Assay, Two Tailed Test

Journal: PLOS Pathogens

Article Title: Direct and biologically significant interactions of human herpesvirus 8 interferon regulatory factor 1 with STAT3 and Janus kinase TYK2

doi: 10.1371/journal.ppat.1011806

Figure Lengend Snippet: Data presented here have revealed direct interaction of vIRF-1 with STAT3 (phosphorylated and unphosphorylated) and its kinase TYK2. Abrogation of TYK2 interaction by the Δ9 mutation in vIRF-1 led to loss of suppression by vIRF-1 of active, phosphorylated STAT3 (pSTAT3) whereas STAT3 targeting, disrupted by vIRF-1 Δ2 and Δ3 mutations, had no effect. However, the latter interaction correlated with normal expression of vIRF-1, which was reduced in infected cells by the Δ2 and Δ3 mutations (effecting greatly diminished virus replication) and enhanced by peptide 1–72 (pep.1-72) that promotes vIRF-1-STAT3 interaction. Also, STAT3 depletion in infected cells led to reduced expression of vIRF-1 during lytic replication (not indicated). While the Δ9 mutation diminished virus productive replication in recombinant virus-infected cells, pep.1-72 promoted virus production. Levels of pSTAT3 in recombinant vIRF-1Δ9-virus-infected cells supporting lytic reactivation were higher than in cells infected with wild-type virus or vIRF-1Δ2 or vIRF-1Δ3 viruses. Introduction of the hyper-active, dephosphorylation-resistant STAT3C variant into infected cells inhibited productive replication, indicating that excessive pSTAT3 is detrimental to virus replication. Overall, the presented data show that vIRF-1 can suppress levels of active STAT3 via interaction with and inhibition of TYK2, likely helping to achieve levels of pSTAT3 compatible with efficient virus replication, while vIRF-1 interaction with STAT3 can effect positive regulation of vIRF-1, a viral protein known to support productive replication, and promote replication via this and/or other mechanisms.

Article Snippet: Primary antibodies used for immunoblotting were: CBD (New England BioLabs, E8034S); Flag (Sigma, F1804); β-actin (Sigma, A5316); GAPDH (Invitrogen, TAB1001); vIRF-1 (rabbit polyclonal antiserum, provided by Dr. Gary Hayward); vIL-6 (rabbit polyclonal antiserum [ ]); STAT1, STAT2, STAT3, c-Myc and GST from Santa Cruz Biotechnologies (sc-464, sc-1668, sc482, sc-764 and sc-138, respectively); pSTAT3, TYK2 and JAK1 from Cell Signaling Technology (9131, 14193, 3344); LANA (Advanced Biotechnologies, 13-210-100), p-TYK2 (Cell Signaling Technology, 68790; BD Biosciences, 558394).

Techniques: Mutagenesis, Expressing, Infection, Virus, Recombinant, De-Phosphorylation Assay, Variant Assay, Inhibition

Journal: International journal of cancer

Article Title: Fibroblast nemosis arrests growth and induces differentiation of human leukemia cells.

doi: 10.1002/ijc.23179

Figure Lengend Snippet: FIGURE 7 – Immunoblot analysis of apoptosis-related and intracel- lular signaling proteins in leukemia cells. (a) Expression of apoptosis- related molecules in leukemia cell lines KG-1, THP-1 and U-937 with (1) or without (2) stimulation by nemotic fibroblast spheroids in co- culture for 96 hr. In nemosis-responsive cell lines KG-1 and THP-1, activation-associated cleavage of caspase-3 and -8 is evident. No cleavage products of caspase-9 and reduced expression of the cleaved form of poly(ADP-ribose)polymerase (PARP) are visible. Phenotype differences between nemosis-responders and the nemosis-unrespon- sive cell line U-937 evident in expression levels of the pro-apoptotic Bax protein. (b) Expression of phosphorylated and total levels of mito- gen-activated protein kinases (MAPK) p38, JNK, ERK1/2 and the Akt kinase in comparison to expression of the differentiation-associated Janus-kinase family JAK1, JAK2, JAK3 and TYK2 in leukemia cell lines KG-1, THP-1 and U-937, stimulated (1) or unstimulated (2) with nemotic fibroblast spheroids. Increased dephosphorylation of p38 and ERK1/2 is evident in the nemosis-responsive cell lines KG-1 and THP-1 together with increased expression of JAK1 and JAK3. The nemosis-unresponsive cell line U-937 showed no expressional differ- ences for these proteins.

Article Snippet: Antibodies for immunoblotting were rabbit anti-p38 antibody (Ab) (sc-535, Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-p-p38 Tyr182 monoclonal antibody (MAb) (sc-7973), rabbit anti-JNK Ab (CST-0252, Cell Signaling Technology, Danvers, MA), mouse anti-p-JNK Thr183/Tyr185 MAb (sc-6254), rabbit anti-ERK1/2 Ab (sc-94), mouse anti-p-ERK1/2 Tyr204 MAb (sc7383), rabbit anti-Akt Ab (CST-9272), rabbit anti-p-Akt Ser473 Ab (CST-9271), rabbit anti-JAK1 Ab (sc-7228), rabbit anti-JAK2 Ab (sc-294), rabbit anti-JAK3 Ab (sc-513), rabbit anti-TYK2 Ab (sc-169), rabbit anti-cleaved caspase-3 Asp175 Ab (CST-9661), This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/0020-7136/suppmat.

Techniques: Western Blot, Expressing, Co-Culture Assay, Activation Assay, Comparison, De-Phosphorylation Assay

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: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Increased IL-10 mRNA expression in tumor-associated macrophage correlated with late stage of lung cancer

doi: 10.1186/1756-9966-30-62

Figure Lengend Snippet: The mRNA expression levels of IL-10, cathepsin B and cathepsin S in normal macrophages . Results are given as fold increase in mRNA expression with respect to expression in D0 monocytes. Data were normalized to expression of the β-actin gene. A: Monocytes(D0) was used as a calibrator. B, monocytes culture without rhM-CSF was used as a calibrator (Ctrl). Error bar is SD, Independent experiments were repeated three times, all #p > 0.05(by student t-test).

Article Snippet: Slides were incubated with the primary antibodies directed against monoclonal anti-human CD68 antibody (1:200 dilution, sc-20060, Santa Cruz, CA, USA), monoclonal anti-human IL-10 antibody (1:100 dilution, BA1201,Boster, WuHan, China) or polyclonal anti-human cathepsin B antibody (1:100 dilution, ab49232, Abcam, MA, USA).

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Increased IL-10 mRNA expression in tumor-associated macrophage correlated with late stage of lung cancer

doi: 10.1186/1756-9966-30-62

Figure Lengend Snippet: The mRNA expression levels of IL-10, cathepsin B and cathepsin S in TAM changes in primary culture . Results are given as fold increase in mRNA expression with respect to expression in ctrl (normal macrophages). Data were normalized to expression of the β-actin gene. Normal macrophages were used as a calibrator. Error bar is SD; Independent experiments were repeated three times.

Article Snippet: Slides were incubated with the primary antibodies directed against monoclonal anti-human CD68 antibody (1:200 dilution, sc-20060, Santa Cruz, CA, USA), monoclonal anti-human IL-10 antibody (1:100 dilution, BA1201,Boster, WuHan, China) or polyclonal anti-human cathepsin B antibody (1:100 dilution, ab49232, Abcam, MA, USA).

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Increased IL-10 mRNA expression in tumor-associated macrophage correlated with late stage of lung cancer

doi: 10.1186/1756-9966-30-62

Figure Lengend Snippet: Immunohistochemical expression of IL-10 , cathepsin B and CD68 in macrophage . A-B, High IL-10 expression in macrophage, A, IL-10 staining in macrophage (strong positivity); B, CD68 staining. C-D, Cathepsin B expression in macrophage; C, cathepsin B staining in macrophage (most cells were moderate positivity, only a few cells were strong staining); D, CD68 staining. Scale bar indicates 50 μm. Original magnification, × 400.

Article Snippet: Slides were incubated with the primary antibodies directed against monoclonal anti-human CD68 antibody (1:200 dilution, sc-20060, Santa Cruz, CA, USA), monoclonal anti-human IL-10 antibody (1:100 dilution, BA1201,Boster, WuHan, China) or polyclonal anti-human cathepsin B antibody (1:100 dilution, ab49232, Abcam, MA, USA).

Techniques: Immunohistochemical staining, Expressing, Staining