rabbit anti irf1 polyclonal antibody  (Proteintech)

Journal: Frontiers in Microbiology

Article Title: Toosendanin suppresses African swine fever virus replication through upregulating interferon regulatory factor 1 in porcine alveolar macrophage cultures

doi: 10.3389/fmicb.2022.970501

Figure Lengend Snippet: The primers used for determining the mRNA level of 9 ISGs and IFNs by qRT-PCR.

Article Snippet: Rabbit anti-IRF1 polyclonal antibody (1:1,000 dilution) were purchased from Proteintech Group, Inc. (Rosemont, United States).

Techniques: Sequencing

Journal: Frontiers in Microbiology

Article Title: Toosendanin suppresses African swine fever virus replication through upregulating interferon regulatory factor 1 in porcine alveolar macrophage cultures

doi: 10.3389/fmicb.2022.970501

Figure Lengend Snippet: Three pairs of siRNAs designed to silence the endogenous IRF1.

Article Snippet: Rabbit anti-IRF1 polyclonal antibody (1:1,000 dilution) were purchased from Proteintech Group, Inc. (Rosemont, United States).

Techniques: Sequencing

Journal: Frontiers in Microbiology

Article Title: Toosendanin suppresses African swine fever virus replication through upregulating interferon regulatory factor 1 in porcine alveolar macrophage cultures

doi: 10.3389/fmicb.2022.970501

Figure Lengend Snippet: Effects of TSN on the mRNA expression of IFNs and 9 ISGs in PAMs. 0.014 MOI ASFV infected the PAMs for 24 h to test its effect on the expression level of IFNs (IFNα, IFNβ and IFNγ) and 9 ISGs (TRIM26, IRF1, SAT1, ISG20, GBP1, PKR, MX1, DCP1A and SHFL). Besides, 1 μM TSN was added to PAMs with or without ASFV infection (0.014 MOI) for 24 h to test its effect on the expression of IFNs (IFNα, IFNβ and IFNγ) and 9 ISGs (TRIM26, IRF1, SAT1, ISG20, GBP1, PKR, MX1, DCP1A and SHFL). The vertical axis shows the relative expression level (fold-change) of IFNs or 9 ISGs mRNA. The fold-changes were calculated by the 2 ∆∆CT method and normalized by IFNβ level (A,C,E) or TRIM26 level (B,D,F) in mock or ASFV group. (A) The mRNA level of IFNα, IFNβ and IFNγ in PAMs with ASFV infection was analyzed using RT-qPCR. (B) The mRNA level of 9 ISGs in PAMs with ASFV infection was analyzed using RT-qPCR. (C) The mRNA levels of IFNα, IFNβ and IFNγ in PAMs with or without TSN treatment were analyzed using RT-qPCR. (D) The mRNA levels of 9 ISGs in PAMs with or without TSN treatment were analyzed using RT-qPCR. (E) The mRNA levels of IFNα, IFNβ and IFNγ in ASFV-infected PAMs with or without TSN treatment were analyzed using RT-qPCR. (F) The mRNA levels of 9 ISGs in ASFV-infected PAMs with or without TSN treatment were analyzed using RT-qPCR. *** denotes p < 0.001; ** denotes p < 0.01; * denotes p < 0.05; ns denotes not significant.

Article Snippet: Rabbit anti-IRF1 polyclonal antibody (1:1,000 dilution) were purchased from Proteintech Group, Inc. (Rosemont, United States).

Techniques: Expressing, Infection, Quantitative RT-PCR

Journal: Frontiers in Microbiology

Article Title: Toosendanin suppresses African swine fever virus replication through upregulating interferon regulatory factor 1 in porcine alveolar macrophage cultures

doi: 10.3389/fmicb.2022.970501

Figure Lengend Snippet: TSN treatment upregulates the mRNA expressions of IFNs and IRF1, and the protein expression of IRF1 in PAMs. 0.014 MOI ASFV infected the PAMs for 6, 12, and 24 h to test its effect on the expression level of IFNs (IFNα, IFNβ and IFNγ) and IRF1. Besides, 1 μM TSN was added to PAMs with or without ASFV infection (0.014 MOI) for 6, 12, and 24 h to test its effect on the expression levels of IFNs (IFNα, IFNβ and IFNγ) and IRF1. The vertical axis shows the relative expression level (fold-change) of IFNs or IRF1 mRNA. The fold-changes were calculated by the 2 ∆∆CT method and normalized by IFNβ or IRF1 levels at 24 h in mock or ASFV group. (A) The mRNA levels of IFNα, IFNβ and IFNγ in PAMs with ASFV infection at 6, 12, and 24 h was analyzed using RT-qPCR. (B) The mRNA level of IRF1 in PAMs with ASFV infection at 6, 12, and 24 h was analyzed using RT-qPCR. (C) The protein level of IRF1 in PAMs with ASFV infection at 6, 12, and 24 h was analyzed using Western Blot. (D) The mRNA levels of IFNα, IFNβ and IFNγ in PAMs with or without TSN treatment at 6, 12, and 24 h was analyzed using RT-qPCR. (E) The mRNA level of IRF1 in PAMs with or without TSN treatment at 6, 12, and 24 h was analyzed using RT-qPCR. (F) The protein level of IRF1 in PAMs with or without TSN treatment at 6, 12, and 24 h was analyzed using Western Blot. (G) The mRNA levels of IFNα, IFNβ and IFNγ in ASFV-infected PAMs with or without TSN treatment at 6, 12, and 24 h was analyzed using RT-qPCR. (H) The mRNA level of IRF1 in ASFV-infected PAMs with or without TSN treatment at 6, 12, and 24 h was analyzed using RT-qPCR. (I) The protein level of IRF1 in ASFV-infected PAMs with or without TSN treatment at 6, 12, and 24 h was analyzed using Western Blot. *** denotes p < 0.001; ** denotes p < 0.01; * denotes p < 0.05; ns denotes not significant.

Article Snippet: Rabbit anti-IRF1 polyclonal antibody (1:1,000 dilution) were purchased from Proteintech Group, Inc. (Rosemont, United States).

Techniques: Expressing, Infection, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Microbiology

Article Title: Toosendanin suppresses African swine fever virus replication through upregulating interferon regulatory factor 1 in porcine alveolar macrophage cultures

doi: 10.3389/fmicb.2022.970501

Figure Lengend Snippet: TSN suppresses ASFV replication via upregulating IRF1 expression. (A) PK-15 cells were challenged with 0.014 MOI ASFV at 37°C for 2 h, then PRK5-FLAG-IRF1 was transfected into PK15 cells at 37°C for 6 h. After that, the supernatant was discarded and the cells were washed with PBS. Then fresh maintenance medium was added in PK-15 cells and incubated at 37°C for 16 h. The samples were harvested at 16 h post transfection for Western Blot. (B) PAMs were transfected with 2 μg/ml of three different siRNA against IRF1 and scramble siRNA (siRNA-Scra) at 37°C for 4 h. After that, the supernatant was discarded and the cells were washed with PBS, then fresh maintenance medium was added in PAMs. IRF1 was detected by RT-qPCR at 24 h post siRNA transfection. (C) PAMs were challenged with 0.014 MOI ASFV at 37°C for 2 h, then the siRNA-IRF1-301 or siRNA-Scra was transfected into PAMs at 37°C for 6 h. After that, the supernatant was discarded and the cells were washed with PBS followed by treatment with 1 μM TSN. After culture at 37°C for 16 h, the samples were collected for Western Blot. (D) The samples from panel (C) were collected for endpoint dilution method analysis. *** denotes p < 0.001; ** denotes p < 0.01; * denotes p < 0.05. ns denotes no significant difference.

Article Snippet: Rabbit anti-IRF1 polyclonal antibody (1:1,000 dilution) were purchased from Proteintech Group, Inc. (Rosemont, United States).

Techniques: Expressing, Transfection, Incubation, Western Blot, Quantitative RT-PCR

Journal: Experimental & Molecular Medicine

Article Title: IRF1 is critical for the TNF-driven interferon response in rheumatoid fibroblast-like synoviocytes

doi: 10.1038/s12276-019-0267-6

Figure Lengend Snippet: a Representative immunostaining for IRF1 (brown staining) in rheumatoid arthritis (RA) and osteoarthritis (OA) synovial tissue samples (upper panel). Staining with an isotype-matched control antibody (CTRL) is presented in the lower panel. b Synovial tissue samples from 12 RA and 8 OA patients evaluated for IRF1 expression using a semiquantitative score (0 = no staining, 3 = high staining). Lining: Mann–Whitney U test, ** p = 0.0016; Sublining: Student’s t -test, **** p < 0.0001. c Quantitative RT-PCR analysis of the IRF1 mRNA levels in hind paws obtained from wild-type (WT) and hTNFtg mice. Mann-Whitney U test, *** p = 0.0008. d Immunohistochemical detection of IRF1 (brown staining) in hind paw tissue from WT and hTNFtg mice. e Western blot analysis of TNF-stimulated (10 ng/ml) RA-FLSs. Blots representative of at least five independent experiments with FLSs from different donors are shown. f RA-FLSs cultured in micromass organ cultures for 7 days in the presence or absence of TNF (10 ng/ml). Micromasses were fixed, sectioned, and stained with hematoxylin and a specific antibody against IRF1 (brown staining). Representative images from three independent experiments performed with FLSs from three RA patients are shown (upper panel). Staining with an isotype-matched control antibody (CTRL) is presented in the lower panel

Article Snippet: Synovial IRF1 expression was detected with a polyclonal rabbit anti-IRF1 antibody (Cell Signaling Technology).

Techniques: Immunostaining, Staining, Control, Expressing, MANN-WHITNEY, Quantitative RT-PCR, Immunohistochemical staining, Western Blot, Cell Culture

Journal: Experimental & Molecular Medicine

Article Title: IRF1 is critical for the TNF-driven interferon response in rheumatoid fibroblast-like synoviocytes

doi: 10.1038/s12276-019-0267-6

Figure Lengend Snippet: a Workflow outlining the RNA sequencing (RNA-seq) experiment. RA-FLSs from three different RA patients were transfected with nontargeting (siCTRL) or IRF1-targeting siRNA pools and stimulated with TNF (10 ng/ml) for 3 h. RNA was isolated and processed for transcriptomic profiling. b Representative immunoblots of IRF1 expression in RA-FLSs after siRNA transfection and stimulation with TNF (10 ng/ml) for 3 h. c Scatter-plot of the RNA-seq data (panel A) showing the impact of IRF1 knockdown on TNF-regulated genes. Dots in the lower right quadrant (red box) represent genes with expression upregulated by TNF, but impaired by the siRNA-mediated knockdown of IRF1 expression. Dashed blue lines indicate a twofold change in gene expression. d Network analysis of GO term (biological process, BP) enrichment among significantly regulated TNF-IRF1-dependent genes (as depicted in panel C, red box). The resulting network was calculated and visualized using EnrichmentMap. Groups of similar GO terms were manually circled. Line thickness is proportional to the similarity coefficient between the connected nodes. Node color is proportional to the FDR-adjusted P -value of the enrichment. e Enriched GO terms (BP, top 8) of the genes within the bottom right quadrant (red box) in the scatter-plot (Fig. 2c). Circle size shows the relative amount of significant genes associated with the GO term

Article Snippet: Synovial IRF1 expression was detected with a polyclonal rabbit anti-IRF1 antibody (Cell Signaling Technology).

Techniques: RNA Sequencing, Transfection, Isolation, Western Blot, Expressing, Knockdown, Gene Expression

Journal: Experimental & Molecular Medicine

Article Title: IRF1 is critical for the TNF-driven interferon response in rheumatoid fibroblast-like synoviocytes

doi: 10.1038/s12276-019-0267-6

Figure Lengend Snippet: a – c FLSs were transfected with nontargeting (siCTRL) or IRF1-targeting siRNA pools. a Transfected FLSs from seven donors with RA were treated with TNF (10 ng/ml) for 6 h. Gene expression was determined by qPCR. Expression in the treated cells is presented relative to that in the unstimulated cells. Values are shown as the mean ± SEM. ** p < 0.01, Wilcoxon matched-pairs test. b RA-FLSs were stimulated with TNF for 24 h. Representative western blots of at least four experiments with different RA-FLS cell lines are shown. c . IRF1 expression knockdown FLSs ( n = 4) were treated with TNF (10 ng/ml) for 24 h. Supernatants were analyzed for CXCL10 expression by ELISA. Values are shown as the mean ± SEM. d qPCR was used to analyze gene expression in human RA-FLSs treated with TNF in the absence or presence of cycloheximide (CHX, 20 μg/ml). Each experiment was performed in technical triplicates (error bars, SEM of triplicates). Expression in the treated cells is presented relative to that in the unstimulated cells

Article Snippet: Synovial IRF1 expression was detected with a polyclonal rabbit anti-IRF1 antibody (Cell Signaling Technology).

Techniques: Transfection, Gene Expression, Expressing, Western Blot, Knockdown, Enzyme-linked Immunosorbent Assay

Journal: Experimental & Molecular Medicine

Article Title: IRF1 is critical for the TNF-driven interferon response in rheumatoid fibroblast-like synoviocytes

doi: 10.1038/s12276-019-0267-6

Figure Lengend Snippet: a Western blot analysis of IRF1 expression and STAT1 phosphorylation in TNF-stimulated (10 ng/ml) FLSs. Blots representative of three independent experiments with different RA-FLS cell lines are shown. b Western blots showing the IRF1 expression and STAT1 phosphorylation in TNF-treated (10 ng/ml; 3 h) RA-FLSs that were transfected with either nontargeting or IRF1-targeting siRNA pools. Blots representative of three independent experiments with FLSs from different RA donors are shown. c – e FLSs were transfected with nontargeting (siCTRL) or IFNβ-targeting siRNA pools and then stimulated with TNF (10 ng/ml). c Transfected FLSs stimulated with TNF for 24 h. Western blots representative of at least four experiments performed with different RA-FLS cell lines are shown. d Transfected FLSs from seven donors with RA treated with TNF (10 ng/ml) for 6 h. Gene expression was determined by qPCR. Expression in the treated cells is presented relative to that in the unstimulated cells. Values are shown as the mean ± SEM. * p < 0.05, ** p < 0.01; Wilcoxon matched-pairs test. e Transfected RA-FLSs from seven different donors suffering from RA treated with TNF (10 ng/ml) for 24 h. Supernatants were analyzed for CXCL10 expression by ELISA. Values are shown as the mean ± SEM. ** p < 0.01, Wilcoxon matched-pairs test. f FLSs ( n = 6) transfected with nontargeting (siCTRL) or IFNAR1-targeting siRNA pools and then stimulated with TNF for 6 h. Gene expression was determined by qPCR. Expression in the treated cells is presented relative to that in the unstimulated cells. Values are shown as the mean ± SEM. * p < 0.05, Wilcoxon matched-pairs test. Western blots representative of at least four experiments with different RA-FLS cell lines are shown

Article Snippet: Synovial IRF1 expression was detected with a polyclonal rabbit anti-IRF1 antibody (Cell Signaling Technology).

Techniques: Western Blot, Expressing, Phospho-proteomics, Transfection, Gene Expression, Enzyme-linked Immunosorbent Assay