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phosphorylated p stat1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phosphorylated p stat1
    Figure 1. IFNα/β down-regulation and <t>STAT1</t> up-regulation in response to CSFV. (A,B) The mRNA levels and protein expression of IFNα and IFNβ were analyzed in PK-15 cells infected with CSFV (MOI = 1) or transfected with 50 µg poly(I:C) by RT-qPCR (A) and WB (B) analysis. (D) The mRNA levels of STAT1 were analyzed in PK-15 cells infected with the CSFV (MOI = 1) at 0, 3, 6, 9, 12, 24, 36, and 48 hpi by RT-qPCR analysis. (E) The protein levels of E1/E2, STAT1, and <t>P-STAT1</t> (Tyr 701) were analyzed by WB analysis. (C,F) The relative expression values of IFNα, IFNβ, STAT1, and P-STAT1 proteins were quantified using Image J software (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.
    Phosphorylated P Stat1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1536 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Classical Swine Fever Virus Envelope Glycoproteins E rns , E1, and E2 Activate IL-10-STAT1-MX1/OAS1 Antiviral Pathway via Replacing Classical IFNα/β."

    Article Title: Classical Swine Fever Virus Envelope Glycoproteins E rns , E1, and E2 Activate IL-10-STAT1-MX1/OAS1 Antiviral Pathway via Replacing Classical IFNα/β.

    Journal: Biomolecules

    doi: 10.3390/biom15020200

    Figure 1. IFNα/β down-regulation and STAT1 up-regulation in response to CSFV. (A,B) The mRNA levels and protein expression of IFNα and IFNβ were analyzed in PK-15 cells infected with CSFV (MOI = 1) or transfected with 50 µg poly(I:C) by RT-qPCR (A) and WB (B) analysis. (D) The mRNA levels of STAT1 were analyzed in PK-15 cells infected with the CSFV (MOI = 1) at 0, 3, 6, 9, 12, 24, 36, and 48 hpi by RT-qPCR analysis. (E) The protein levels of E1/E2, STAT1, and P-STAT1 (Tyr 701) were analyzed by WB analysis. (C,F) The relative expression values of IFNα, IFNβ, STAT1, and P-STAT1 proteins were quantified using Image J software (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.
    Figure Legend Snippet: Figure 1. IFNα/β down-regulation and STAT1 up-regulation in response to CSFV. (A,B) The mRNA levels and protein expression of IFNα and IFNβ were analyzed in PK-15 cells infected with CSFV (MOI = 1) or transfected with 50 µg poly(I:C) by RT-qPCR (A) and WB (B) analysis. (D) The mRNA levels of STAT1 were analyzed in PK-15 cells infected with the CSFV (MOI = 1) at 0, 3, 6, 9, 12, 24, 36, and 48 hpi by RT-qPCR analysis. (E) The protein levels of E1/E2, STAT1, and P-STAT1 (Tyr 701) were analyzed by WB analysis. (C,F) The relative expression values of IFNα, IFNβ, STAT1, and P-STAT1 proteins were quantified using Image J software (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.

    Techniques Used: Expressing, Infection, Transfection, Quantitative RT-PCR, Software, Western Blot

    Figure 2. STAT1 expression was up-regulated by UV-treated CSFV in vitro. (A) PK-15 cells were inoculated with CSFV and UV-treated CSFV at 101, 102, 103, and 104 TCID50. CSFV replication was assessed using an anti-CSFV E2 Ab at 48 hpi by IF analysis. CSFV gRNA (B) and STAT1 mRNA (C) levels in PK-15 cells inoculated with CSFV and UV-treated CSFV at 12, 24, 48, and 72 hpi were determined by RT-qPCR analysis. (D) CSFV E2 and STAT1 protein levels at 12, 24, 48, and 72 hpi were measured by WB analysis. (E) The relative expression values of STAT1 protein were quantified using Image J software (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.
    Figure Legend Snippet: Figure 2. STAT1 expression was up-regulated by UV-treated CSFV in vitro. (A) PK-15 cells were inoculated with CSFV and UV-treated CSFV at 101, 102, 103, and 104 TCID50. CSFV replication was assessed using an anti-CSFV E2 Ab at 48 hpi by IF analysis. CSFV gRNA (B) and STAT1 mRNA (C) levels in PK-15 cells inoculated with CSFV and UV-treated CSFV at 12, 24, 48, and 72 hpi were determined by RT-qPCR analysis. (D) CSFV E2 and STAT1 protein levels at 12, 24, 48, and 72 hpi were measured by WB analysis. (E) The relative expression values of STAT1 protein were quantified using Image J software (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.

    Techniques Used: Expressing, In Vitro, Quantitative RT-PCR, Software, Western Blot

    Figure 3. CSFV up-regulated STAT1 expression via Erns, E1, and E2, but not the Core protein. PK- 15 cells were infected with CSFV (MOI = 1) and separately transfected with 1.0 µg of the recombinant eukaryotic expression vectors pCMV-Core-HA (A–D), pCMV-Erns-His (E–H), pCMV-E1-Myc (I–L), and pCMV-E2-Flag (M–P). PK-15 cells incubated with 100 IU/mL of IFNα served as the positive control, while PK-15 cells transfected with an empty pCMV vector were utilized as the negative control. Samples were collected 72 h later for subsequent analysis. CSFV Core (A), Erns (E), E1 (I), E2 (M), and STAT1 (B,F,J,N) mRNA levels were measured by RT-qPCR analysis at 12, 24, 48, and 72 h. (C,G,K,O) CSFV E2, Core-HA, Erns-His, E1-Myc, E2-Flag, and STAT1 protein levels at 12, 24, 48, and 72 h were measured by WB analysis. (D,H,L,P) The relative expression values of STAT1 protein were quantified using Image J software (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.
    Figure Legend Snippet: Figure 3. CSFV up-regulated STAT1 expression via Erns, E1, and E2, but not the Core protein. PK- 15 cells were infected with CSFV (MOI = 1) and separately transfected with 1.0 µg of the recombinant eukaryotic expression vectors pCMV-Core-HA (A–D), pCMV-Erns-His (E–H), pCMV-E1-Myc (I–L), and pCMV-E2-Flag (M–P). PK-15 cells incubated with 100 IU/mL of IFNα served as the positive control, while PK-15 cells transfected with an empty pCMV vector were utilized as the negative control. Samples were collected 72 h later for subsequent analysis. CSFV Core (A), Erns (E), E1 (I), E2 (M), and STAT1 (B,F,J,N) mRNA levels were measured by RT-qPCR analysis at 12, 24, 48, and 72 h. (C,G,K,O) CSFV E2, Core-HA, Erns-His, E1-Myc, E2-Flag, and STAT1 protein levels at 12, 24, 48, and 72 h were measured by WB analysis. (D,H,L,P) The relative expression values of STAT1 protein were quantified using Image J software (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.

    Techniques Used: Expressing, Infection, Transfection, Recombinant, Incubation, Positive Control, Plasmid Preparation, Negative Control, Quantitative RT-PCR, Software, Western Blot

    Figure 4. STAT1 translocation to the nucleus induced by CSFV Erns, E1, and E2. PK-15 cells were used to express Erns, E1, and E2 proteins by transfecting with the pCMV-Erns-His, pCMV-E1-Myc, and pCMV-E2-Flag vectors, respectively. (A,C,E) The protein levels of STAT1 in the cytoplasm and nucleus of PK-15 cells were measured by WB analysis. (B,D,F) The relative expression values of STAT1 protein were quantified using Image J software. (G) HEK 293T cells were separately transfected with the pcDNA3.0-STAT1-His, pCMV-Erns-His, pCMV-E1-Myc, and pCMV-E2-Flag vectors, and treated with IFNα (100 IU/mL). The expression of STAT1 (a–f) and P-STAT1 (g–l) proteins were evaluated at 48 h using anti-STAT1 and anti-P-STAT1 antibodies by IFA analysis (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.
    Figure Legend Snippet: Figure 4. STAT1 translocation to the nucleus induced by CSFV Erns, E1, and E2. PK-15 cells were used to express Erns, E1, and E2 proteins by transfecting with the pCMV-Erns-His, pCMV-E1-Myc, and pCMV-E2-Flag vectors, respectively. (A,C,E) The protein levels of STAT1 in the cytoplasm and nucleus of PK-15 cells were measured by WB analysis. (B,D,F) The relative expression values of STAT1 protein were quantified using Image J software. (G) HEK 293T cells were separately transfected with the pcDNA3.0-STAT1-His, pCMV-Erns-His, pCMV-E1-Myc, and pCMV-E2-Flag vectors, and treated with IFNα (100 IU/mL). The expression of STAT1 (a–f) and P-STAT1 (g–l) proteins were evaluated at 48 h using anti-STAT1 and anti-P-STAT1 antibodies by IFA analysis (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.

    Techniques Used: Translocation Assay, Expressing, Software, Transfection, Western Blot

    Figure 5. Identification of genes upstream of the STAT1 pathway during CSFV infection. (A) A heatmap of changes to the transcriptional levels of IL-8, IL-10, TGFβ1, TGFβ3 CCR2, CCR3, IFNγ, and STAT1 in PBMCs of piglets infected with CSFV. (B) PK-15 cells were infected with CSFV (MOI = 1) and the CSFV gRNA copy number was measured by RT-qPCR analysis. (C) The transcriptional levels of porcine IL-8, IL-10, TGFβ1, TGFβ3 CCR2, CCR3, and IFNγ at 12, 24, 48, and 60 hpi were measured by RT-qPCR analysis. (D, E) The relative protein expression of CSFV E2 (D) and IL-10 (E) were measured by ELISA (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).
    Figure Legend Snippet: Figure 5. Identification of genes upstream of the STAT1 pathway during CSFV infection. (A) A heatmap of changes to the transcriptional levels of IL-8, IL-10, TGFβ1, TGFβ3 CCR2, CCR3, IFNγ, and STAT1 in PBMCs of piglets infected with CSFV. (B) PK-15 cells were infected with CSFV (MOI = 1) and the CSFV gRNA copy number was measured by RT-qPCR analysis. (C) The transcriptional levels of porcine IL-8, IL-10, TGFβ1, TGFβ3 CCR2, CCR3, and IFNγ at 12, 24, 48, and 60 hpi were measured by RT-qPCR analysis. (D, E) The relative protein expression of CSFV E2 (D) and IL-10 (E) were measured by ELISA (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

    Techniques Used: Infection, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay

    Figure 7. IL-10 up-regulated STAT1 expression in vitro. (A,B) Following porcine IL-10 (100 IU/mL) and porcine IFNα (100 IU/mL) treatment, the transcription and protein levels of STAT1 at 12, 24, 36, and 48 h were measured by RT-qPCR and WB analysis. (C) The relative expression values of STAT1 protein were quantified using Image J software. (D) Subcellular localization of STAT1 in PK-15 cells separately treated with IL-10 (100 IU/mL) and IFNα (100 IU/mL). The expression of STAT1 (a–c) and P-STAT1 (d–f) proteins were evaluated at 48 h using anti-STAT1 and anti-P-STAT1 antibodies by IFA analysis (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.
    Figure Legend Snippet: Figure 7. IL-10 up-regulated STAT1 expression in vitro. (A,B) Following porcine IL-10 (100 IU/mL) and porcine IFNα (100 IU/mL) treatment, the transcription and protein levels of STAT1 at 12, 24, 36, and 48 h were measured by RT-qPCR and WB analysis. (C) The relative expression values of STAT1 protein were quantified using Image J software. (D) Subcellular localization of STAT1 in PK-15 cells separately treated with IL-10 (100 IU/mL) and IFNα (100 IU/mL). The expression of STAT1 (a–c) and P-STAT1 (d–f) proteins were evaluated at 48 h using anti-STAT1 and anti-P-STAT1 antibodies by IFA analysis (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.

    Techniques Used: Expressing, In Vitro, Quantitative RT-PCR, Software, Western Blot

    Figure 8. Knockdown of IL-10 down-regulated STAT1 expression. The transcription and protein levels of Erns, E1, E2, IL-10, and STAT1 have been detected by RT-qPCR, ELISA, and WB analysis in PK-15 cells temporarily transfected with IL-10 siRNA or control siRNA (100 nM) for 6 h, and then transfected with the pCMV-Erns-His (A–F), pCMV-E1-Myc (G–L), or pCMV-E2-Flag (M–R) vectors, respectively, underlying the transfection of the empty vector pCMV (1.0 µg) as a negative control at 12, 24, 36, and 48 hpt. The mRNA levels of Erns, E1, and E2 (A,G,M), IL-10 (B,H,N), and STAT1 (D,J,P) are displayed by RT-qPCR analysis. The protein levels of IL-10 (C,I,O) and STAT1 (E,K,Q) were detected by ELISA and WB analysis. (F,L,R) The relative expression values of the STAT1 protein were quantified using Image J software (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.
    Figure Legend Snippet: Figure 8. Knockdown of IL-10 down-regulated STAT1 expression. The transcription and protein levels of Erns, E1, E2, IL-10, and STAT1 have been detected by RT-qPCR, ELISA, and WB analysis in PK-15 cells temporarily transfected with IL-10 siRNA or control siRNA (100 nM) for 6 h, and then transfected with the pCMV-Erns-His (A–F), pCMV-E1-Myc (G–L), or pCMV-E2-Flag (M–R) vectors, respectively, underlying the transfection of the empty vector pCMV (1.0 µg) as a negative control at 12, 24, 36, and 48 hpt. The mRNA levels of Erns, E1, and E2 (A,G,M), IL-10 (B,H,N), and STAT1 (D,J,P) are displayed by RT-qPCR analysis. The protein levels of IL-10 (C,I,O) and STAT1 (E,K,Q) were detected by ELISA and WB analysis. (F,L,R) The relative expression values of the STAT1 protein were quantified using Image J software (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.

    Techniques Used: Knockdown, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transfection, Control, Plasmid Preparation, Negative Control, Software, Western Blot

    Figure 9. Identification of downstream targets of the STAT1 pathway. (A,B) The copy number of CSFV gRNA and the mRNA levels of porcine MX1, OAS1, and ISG20 were quantified through RT-qPCR analysis at 12, 24, 36, and 48 hpi in PK-15 cells infected with CSFV (MOI = 1). (C) The protein levels were assessed using Western blot (WB) analysis. (D) The mRNA levels of porcine STAT1, MX1, OAS1, and ISG20 were quantified using RT-qPCR analysis at 12, 24, 36, and 48 hpt in PK-15 cells transfected with the pcDNA3.0-STAT1-His vector, and (E) protein levels were assessed through WB analysis. (F) The mRNA levels of porcine STAT1, MX1, OAS1, and ISG20 were quantified using RT-qPCR analysis at 12, 24, 36, and 48 hpi in CSFV-infected PK-15STAT1-/- cells and (G) protein levels were measured by WB analysis (** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.
    Figure Legend Snippet: Figure 9. Identification of downstream targets of the STAT1 pathway. (A,B) The copy number of CSFV gRNA and the mRNA levels of porcine MX1, OAS1, and ISG20 were quantified through RT-qPCR analysis at 12, 24, 36, and 48 hpi in PK-15 cells infected with CSFV (MOI = 1). (C) The protein levels were assessed using Western blot (WB) analysis. (D) The mRNA levels of porcine STAT1, MX1, OAS1, and ISG20 were quantified using RT-qPCR analysis at 12, 24, 36, and 48 hpt in PK-15 cells transfected with the pcDNA3.0-STAT1-His vector, and (E) protein levels were assessed through WB analysis. (F) The mRNA levels of porcine STAT1, MX1, OAS1, and ISG20 were quantified using RT-qPCR analysis at 12, 24, 36, and 48 hpi in CSFV-infected PK-15STAT1-/- cells and (G) protein levels were measured by WB analysis (** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.

    Techniques Used: Quantitative RT-PCR, Infection, Western Blot, Transfection, Plasmid Preparation

    Figure 10. Substitution of the classical IFNα/β-STAT1 pathway with the IL-10-STAT1 pathway during CSFV infection. The type I IFN-mediated STAT1 pathway was replaced with the alternative IL-10-STAT1 pathway during CSFV infection via the viral envelope glycoproteins Erns, E1, and E2. The classical pathway: IFNα/β-p-STAT1/p-STAT2-IRF9-ISGs (left panel). The alternative pathway: IL-10-p-STAT1-MX1/OAS1 (right panel).
    Figure Legend Snippet: Figure 10. Substitution of the classical IFNα/β-STAT1 pathway with the IL-10-STAT1 pathway during CSFV infection. The type I IFN-mediated STAT1 pathway was replaced with the alternative IL-10-STAT1 pathway during CSFV infection via the viral envelope glycoproteins Erns, E1, and E2. The classical pathway: IFNα/β-p-STAT1/p-STAT2-IRF9-ISGs (left panel). The alternative pathway: IL-10-p-STAT1-MX1/OAS1 (right panel).

    Techniques Used: Infection



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    Figure 1. IFNα/β down-regulation and STAT1 up-regulation in response to CSFV. (A,B) The mRNA levels and protein expression of IFNα and IFNβ were analyzed in PK-15 cells infected with CSFV (MOI = 1) or transfected with 50 µg poly(I:C) by RT-qPCR (A) and WB (B) analysis. (D) The mRNA levels of STAT1 were analyzed in PK-15 cells infected with the CSFV (MOI = 1) at 0, 3, 6, 9, 12, 24, 36, and 48 hpi by RT-qPCR analysis. (E) The protein levels of E1/E2, STAT1, and P-STAT1 (Tyr 701) were analyzed by WB analysis. (C,F) The relative expression values of IFNα, IFNβ, STAT1, and P-STAT1 proteins were quantified using Image J software (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.

    Journal: Biomolecules

    Article Title: Classical Swine Fever Virus Envelope Glycoproteins E rns , E1, and E2 Activate IL-10-STAT1-MX1/OAS1 Antiviral Pathway via Replacing Classical IFNα/β.

    doi: 10.3390/biom15020200

    Figure Lengend Snippet: Figure 1. IFNα/β down-regulation and STAT1 up-regulation in response to CSFV. (A,B) The mRNA levels and protein expression of IFNα and IFNβ were analyzed in PK-15 cells infected with CSFV (MOI = 1) or transfected with 50 µg poly(I:C) by RT-qPCR (A) and WB (B) analysis. (D) The mRNA levels of STAT1 were analyzed in PK-15 cells infected with the CSFV (MOI = 1) at 0, 3, 6, 9, 12, 24, 36, and 48 hpi by RT-qPCR analysis. (E) The protein levels of E1/E2, STAT1, and P-STAT1 (Tyr 701) were analyzed by WB analysis. (C,F) The relative expression values of IFNα, IFNβ, STAT1, and P-STAT1 proteins were quantified using Image J software (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.

    Article Snippet: Following blocking, the membranes were probed at 37 ◦C for 60 min with primary antibodies (Abs) against STAT1 (10144-2-AP), Myc tag (16286-1-AP), lamin B1 (12987-1-AP), OAS1 (14955-1-AP), MX1 (13750-1-AP), His tag (10001-0-AP), and Flag tag (20543-1-AP) (all from Proteintech Co., Ltd., Wuhan, China); phosphorylated (p) STAT1 (9167S), IFNα (6B18), and IFNβ (D2J1D) (from Cell Signaling Technology, Inc., Danvers, MA, USA); CSFV E1/E2 (9011) (from JENO Biotech, Seoul, Korea); CSFV E2 (orb500382) (from Biorbyt Ltd., Cambridge, England); and β-actin (CW0096A) (from Beijing CoWin Biotech Co., Ltd., Beijing, China).

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

    Figure 2. STAT1 expression was up-regulated by UV-treated CSFV in vitro. (A) PK-15 cells were inoculated with CSFV and UV-treated CSFV at 101, 102, 103, and 104 TCID50. CSFV replication was assessed using an anti-CSFV E2 Ab at 48 hpi by IF analysis. CSFV gRNA (B) and STAT1 mRNA (C) levels in PK-15 cells inoculated with CSFV and UV-treated CSFV at 12, 24, 48, and 72 hpi were determined by RT-qPCR analysis. (D) CSFV E2 and STAT1 protein levels at 12, 24, 48, and 72 hpi were measured by WB analysis. (E) The relative expression values of STAT1 protein were quantified using Image J software (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.

    Journal: Biomolecules

    Article Title: Classical Swine Fever Virus Envelope Glycoproteins E rns , E1, and E2 Activate IL-10-STAT1-MX1/OAS1 Antiviral Pathway via Replacing Classical IFNα/β.

    doi: 10.3390/biom15020200

    Figure Lengend Snippet: Figure 2. STAT1 expression was up-regulated by UV-treated CSFV in vitro. (A) PK-15 cells were inoculated with CSFV and UV-treated CSFV at 101, 102, 103, and 104 TCID50. CSFV replication was assessed using an anti-CSFV E2 Ab at 48 hpi by IF analysis. CSFV gRNA (B) and STAT1 mRNA (C) levels in PK-15 cells inoculated with CSFV and UV-treated CSFV at 12, 24, 48, and 72 hpi were determined by RT-qPCR analysis. (D) CSFV E2 and STAT1 protein levels at 12, 24, 48, and 72 hpi were measured by WB analysis. (E) The relative expression values of STAT1 protein were quantified using Image J software (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.

    Article Snippet: Following blocking, the membranes were probed at 37 ◦C for 60 min with primary antibodies (Abs) against STAT1 (10144-2-AP), Myc tag (16286-1-AP), lamin B1 (12987-1-AP), OAS1 (14955-1-AP), MX1 (13750-1-AP), His tag (10001-0-AP), and Flag tag (20543-1-AP) (all from Proteintech Co., Ltd., Wuhan, China); phosphorylated (p) STAT1 (9167S), IFNα (6B18), and IFNβ (D2J1D) (from Cell Signaling Technology, Inc., Danvers, MA, USA); CSFV E1/E2 (9011) (from JENO Biotech, Seoul, Korea); CSFV E2 (orb500382) (from Biorbyt Ltd., Cambridge, England); and β-actin (CW0096A) (from Beijing CoWin Biotech Co., Ltd., Beijing, China).

    Techniques: Expressing, In Vitro, Quantitative RT-PCR, Software, Western Blot

    Figure 3. CSFV up-regulated STAT1 expression via Erns, E1, and E2, but not the Core protein. PK- 15 cells were infected with CSFV (MOI = 1) and separately transfected with 1.0 µg of the recombinant eukaryotic expression vectors pCMV-Core-HA (A–D), pCMV-Erns-His (E–H), pCMV-E1-Myc (I–L), and pCMV-E2-Flag (M–P). PK-15 cells incubated with 100 IU/mL of IFNα served as the positive control, while PK-15 cells transfected with an empty pCMV vector were utilized as the negative control. Samples were collected 72 h later for subsequent analysis. CSFV Core (A), Erns (E), E1 (I), E2 (M), and STAT1 (B,F,J,N) mRNA levels were measured by RT-qPCR analysis at 12, 24, 48, and 72 h. (C,G,K,O) CSFV E2, Core-HA, Erns-His, E1-Myc, E2-Flag, and STAT1 protein levels at 12, 24, 48, and 72 h were measured by WB analysis. (D,H,L,P) The relative expression values of STAT1 protein were quantified using Image J software (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.

    Journal: Biomolecules

    Article Title: Classical Swine Fever Virus Envelope Glycoproteins E rns , E1, and E2 Activate IL-10-STAT1-MX1/OAS1 Antiviral Pathway via Replacing Classical IFNα/β.

    doi: 10.3390/biom15020200

    Figure Lengend Snippet: Figure 3. CSFV up-regulated STAT1 expression via Erns, E1, and E2, but not the Core protein. PK- 15 cells were infected with CSFV (MOI = 1) and separately transfected with 1.0 µg of the recombinant eukaryotic expression vectors pCMV-Core-HA (A–D), pCMV-Erns-His (E–H), pCMV-E1-Myc (I–L), and pCMV-E2-Flag (M–P). PK-15 cells incubated with 100 IU/mL of IFNα served as the positive control, while PK-15 cells transfected with an empty pCMV vector were utilized as the negative control. Samples were collected 72 h later for subsequent analysis. CSFV Core (A), Erns (E), E1 (I), E2 (M), and STAT1 (B,F,J,N) mRNA levels were measured by RT-qPCR analysis at 12, 24, 48, and 72 h. (C,G,K,O) CSFV E2, Core-HA, Erns-His, E1-Myc, E2-Flag, and STAT1 protein levels at 12, 24, 48, and 72 h were measured by WB analysis. (D,H,L,P) The relative expression values of STAT1 protein were quantified using Image J software (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.

    Article Snippet: Following blocking, the membranes were probed at 37 ◦C for 60 min with primary antibodies (Abs) against STAT1 (10144-2-AP), Myc tag (16286-1-AP), lamin B1 (12987-1-AP), OAS1 (14955-1-AP), MX1 (13750-1-AP), His tag (10001-0-AP), and Flag tag (20543-1-AP) (all from Proteintech Co., Ltd., Wuhan, China); phosphorylated (p) STAT1 (9167S), IFNα (6B18), and IFNβ (D2J1D) (from Cell Signaling Technology, Inc., Danvers, MA, USA); CSFV E1/E2 (9011) (from JENO Biotech, Seoul, Korea); CSFV E2 (orb500382) (from Biorbyt Ltd., Cambridge, England); and β-actin (CW0096A) (from Beijing CoWin Biotech Co., Ltd., Beijing, China).

    Techniques: Expressing, Infection, Transfection, Recombinant, Incubation, Positive Control, Plasmid Preparation, Negative Control, Quantitative RT-PCR, Software, Western Blot

    Figure 4. STAT1 translocation to the nucleus induced by CSFV Erns, E1, and E2. PK-15 cells were used to express Erns, E1, and E2 proteins by transfecting with the pCMV-Erns-His, pCMV-E1-Myc, and pCMV-E2-Flag vectors, respectively. (A,C,E) The protein levels of STAT1 in the cytoplasm and nucleus of PK-15 cells were measured by WB analysis. (B,D,F) The relative expression values of STAT1 protein were quantified using Image J software. (G) HEK 293T cells were separately transfected with the pcDNA3.0-STAT1-His, pCMV-Erns-His, pCMV-E1-Myc, and pCMV-E2-Flag vectors, and treated with IFNα (100 IU/mL). The expression of STAT1 (a–f) and P-STAT1 (g–l) proteins were evaluated at 48 h using anti-STAT1 and anti-P-STAT1 antibodies by IFA analysis (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.

    Journal: Biomolecules

    Article Title: Classical Swine Fever Virus Envelope Glycoproteins E rns , E1, and E2 Activate IL-10-STAT1-MX1/OAS1 Antiviral Pathway via Replacing Classical IFNα/β.

    doi: 10.3390/biom15020200

    Figure Lengend Snippet: Figure 4. STAT1 translocation to the nucleus induced by CSFV Erns, E1, and E2. PK-15 cells were used to express Erns, E1, and E2 proteins by transfecting with the pCMV-Erns-His, pCMV-E1-Myc, and pCMV-E2-Flag vectors, respectively. (A,C,E) The protein levels of STAT1 in the cytoplasm and nucleus of PK-15 cells were measured by WB analysis. (B,D,F) The relative expression values of STAT1 protein were quantified using Image J software. (G) HEK 293T cells were separately transfected with the pcDNA3.0-STAT1-His, pCMV-Erns-His, pCMV-E1-Myc, and pCMV-E2-Flag vectors, and treated with IFNα (100 IU/mL). The expression of STAT1 (a–f) and P-STAT1 (g–l) proteins were evaluated at 48 h using anti-STAT1 and anti-P-STAT1 antibodies by IFA analysis (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.

    Article Snippet: Following blocking, the membranes were probed at 37 ◦C for 60 min with primary antibodies (Abs) against STAT1 (10144-2-AP), Myc tag (16286-1-AP), lamin B1 (12987-1-AP), OAS1 (14955-1-AP), MX1 (13750-1-AP), His tag (10001-0-AP), and Flag tag (20543-1-AP) (all from Proteintech Co., Ltd., Wuhan, China); phosphorylated (p) STAT1 (9167S), IFNα (6B18), and IFNβ (D2J1D) (from Cell Signaling Technology, Inc., Danvers, MA, USA); CSFV E1/E2 (9011) (from JENO Biotech, Seoul, Korea); CSFV E2 (orb500382) (from Biorbyt Ltd., Cambridge, England); and β-actin (CW0096A) (from Beijing CoWin Biotech Co., Ltd., Beijing, China).

    Techniques: Translocation Assay, Expressing, Software, Transfection, Western Blot

    Figure 5. Identification of genes upstream of the STAT1 pathway during CSFV infection. (A) A heatmap of changes to the transcriptional levels of IL-8, IL-10, TGFβ1, TGFβ3 CCR2, CCR3, IFNγ, and STAT1 in PBMCs of piglets infected with CSFV. (B) PK-15 cells were infected with CSFV (MOI = 1) and the CSFV gRNA copy number was measured by RT-qPCR analysis. (C) The transcriptional levels of porcine IL-8, IL-10, TGFβ1, TGFβ3 CCR2, CCR3, and IFNγ at 12, 24, 48, and 60 hpi were measured by RT-qPCR analysis. (D, E) The relative protein expression of CSFV E2 (D) and IL-10 (E) were measured by ELISA (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

    Journal: Biomolecules

    Article Title: Classical Swine Fever Virus Envelope Glycoproteins E rns , E1, and E2 Activate IL-10-STAT1-MX1/OAS1 Antiviral Pathway via Replacing Classical IFNα/β.

    doi: 10.3390/biom15020200

    Figure Lengend Snippet: Figure 5. Identification of genes upstream of the STAT1 pathway during CSFV infection. (A) A heatmap of changes to the transcriptional levels of IL-8, IL-10, TGFβ1, TGFβ3 CCR2, CCR3, IFNγ, and STAT1 in PBMCs of piglets infected with CSFV. (B) PK-15 cells were infected with CSFV (MOI = 1) and the CSFV gRNA copy number was measured by RT-qPCR analysis. (C) The transcriptional levels of porcine IL-8, IL-10, TGFβ1, TGFβ3 CCR2, CCR3, and IFNγ at 12, 24, 48, and 60 hpi were measured by RT-qPCR analysis. (D, E) The relative protein expression of CSFV E2 (D) and IL-10 (E) were measured by ELISA (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

    Article Snippet: Following blocking, the membranes were probed at 37 ◦C for 60 min with primary antibodies (Abs) against STAT1 (10144-2-AP), Myc tag (16286-1-AP), lamin B1 (12987-1-AP), OAS1 (14955-1-AP), MX1 (13750-1-AP), His tag (10001-0-AP), and Flag tag (20543-1-AP) (all from Proteintech Co., Ltd., Wuhan, China); phosphorylated (p) STAT1 (9167S), IFNα (6B18), and IFNβ (D2J1D) (from Cell Signaling Technology, Inc., Danvers, MA, USA); CSFV E1/E2 (9011) (from JENO Biotech, Seoul, Korea); CSFV E2 (orb500382) (from Biorbyt Ltd., Cambridge, England); and β-actin (CW0096A) (from Beijing CoWin Biotech Co., Ltd., Beijing, China).

    Techniques: Infection, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay

    Figure 7. IL-10 up-regulated STAT1 expression in vitro. (A,B) Following porcine IL-10 (100 IU/mL) and porcine IFNα (100 IU/mL) treatment, the transcription and protein levels of STAT1 at 12, 24, 36, and 48 h were measured by RT-qPCR and WB analysis. (C) The relative expression values of STAT1 protein were quantified using Image J software. (D) Subcellular localization of STAT1 in PK-15 cells separately treated with IL-10 (100 IU/mL) and IFNα (100 IU/mL). The expression of STAT1 (a–c) and P-STAT1 (d–f) proteins were evaluated at 48 h using anti-STAT1 and anti-P-STAT1 antibodies by IFA analysis (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.

    Journal: Biomolecules

    Article Title: Classical Swine Fever Virus Envelope Glycoproteins E rns , E1, and E2 Activate IL-10-STAT1-MX1/OAS1 Antiviral Pathway via Replacing Classical IFNα/β.

    doi: 10.3390/biom15020200

    Figure Lengend Snippet: Figure 7. IL-10 up-regulated STAT1 expression in vitro. (A,B) Following porcine IL-10 (100 IU/mL) and porcine IFNα (100 IU/mL) treatment, the transcription and protein levels of STAT1 at 12, 24, 36, and 48 h were measured by RT-qPCR and WB analysis. (C) The relative expression values of STAT1 protein were quantified using Image J software. (D) Subcellular localization of STAT1 in PK-15 cells separately treated with IL-10 (100 IU/mL) and IFNα (100 IU/mL). The expression of STAT1 (a–c) and P-STAT1 (d–f) proteins were evaluated at 48 h using anti-STAT1 and anti-P-STAT1 antibodies by IFA analysis (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.

    Article Snippet: Following blocking, the membranes were probed at 37 ◦C for 60 min with primary antibodies (Abs) against STAT1 (10144-2-AP), Myc tag (16286-1-AP), lamin B1 (12987-1-AP), OAS1 (14955-1-AP), MX1 (13750-1-AP), His tag (10001-0-AP), and Flag tag (20543-1-AP) (all from Proteintech Co., Ltd., Wuhan, China); phosphorylated (p) STAT1 (9167S), IFNα (6B18), and IFNβ (D2J1D) (from Cell Signaling Technology, Inc., Danvers, MA, USA); CSFV E1/E2 (9011) (from JENO Biotech, Seoul, Korea); CSFV E2 (orb500382) (from Biorbyt Ltd., Cambridge, England); and β-actin (CW0096A) (from Beijing CoWin Biotech Co., Ltd., Beijing, China).

    Techniques: Expressing, In Vitro, Quantitative RT-PCR, Software, Western Blot

    Figure 8. Knockdown of IL-10 down-regulated STAT1 expression. The transcription and protein levels of Erns, E1, E2, IL-10, and STAT1 have been detected by RT-qPCR, ELISA, and WB analysis in PK-15 cells temporarily transfected with IL-10 siRNA or control siRNA (100 nM) for 6 h, and then transfected with the pCMV-Erns-His (A–F), pCMV-E1-Myc (G–L), or pCMV-E2-Flag (M–R) vectors, respectively, underlying the transfection of the empty vector pCMV (1.0 µg) as a negative control at 12, 24, 36, and 48 hpt. The mRNA levels of Erns, E1, and E2 (A,G,M), IL-10 (B,H,N), and STAT1 (D,J,P) are displayed by RT-qPCR analysis. The protein levels of IL-10 (C,I,O) and STAT1 (E,K,Q) were detected by ELISA and WB analysis. (F,L,R) The relative expression values of the STAT1 protein were quantified using Image J software (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.

    Journal: Biomolecules

    Article Title: Classical Swine Fever Virus Envelope Glycoproteins E rns , E1, and E2 Activate IL-10-STAT1-MX1/OAS1 Antiviral Pathway via Replacing Classical IFNα/β.

    doi: 10.3390/biom15020200

    Figure Lengend Snippet: Figure 8. Knockdown of IL-10 down-regulated STAT1 expression. The transcription and protein levels of Erns, E1, E2, IL-10, and STAT1 have been detected by RT-qPCR, ELISA, and WB analysis in PK-15 cells temporarily transfected with IL-10 siRNA or control siRNA (100 nM) for 6 h, and then transfected with the pCMV-Erns-His (A–F), pCMV-E1-Myc (G–L), or pCMV-E2-Flag (M–R) vectors, respectively, underlying the transfection of the empty vector pCMV (1.0 µg) as a negative control at 12, 24, 36, and 48 hpt. The mRNA levels of Erns, E1, and E2 (A,G,M), IL-10 (B,H,N), and STAT1 (D,J,P) are displayed by RT-qPCR analysis. The protein levels of IL-10 (C,I,O) and STAT1 (E,K,Q) were detected by ELISA and WB analysis. (F,L,R) The relative expression values of the STAT1 protein were quantified using Image J software (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.

    Article Snippet: Following blocking, the membranes were probed at 37 ◦C for 60 min with primary antibodies (Abs) against STAT1 (10144-2-AP), Myc tag (16286-1-AP), lamin B1 (12987-1-AP), OAS1 (14955-1-AP), MX1 (13750-1-AP), His tag (10001-0-AP), and Flag tag (20543-1-AP) (all from Proteintech Co., Ltd., Wuhan, China); phosphorylated (p) STAT1 (9167S), IFNα (6B18), and IFNβ (D2J1D) (from Cell Signaling Technology, Inc., Danvers, MA, USA); CSFV E1/E2 (9011) (from JENO Biotech, Seoul, Korea); CSFV E2 (orb500382) (from Biorbyt Ltd., Cambridge, England); and β-actin (CW0096A) (from Beijing CoWin Biotech Co., Ltd., Beijing, China).

    Techniques: Knockdown, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transfection, Control, Plasmid Preparation, Negative Control, Software, Western Blot

    Figure 9. Identification of downstream targets of the STAT1 pathway. (A,B) The copy number of CSFV gRNA and the mRNA levels of porcine MX1, OAS1, and ISG20 were quantified through RT-qPCR analysis at 12, 24, 36, and 48 hpi in PK-15 cells infected with CSFV (MOI = 1). (C) The protein levels were assessed using Western blot (WB) analysis. (D) The mRNA levels of porcine STAT1, MX1, OAS1, and ISG20 were quantified using RT-qPCR analysis at 12, 24, 36, and 48 hpt in PK-15 cells transfected with the pcDNA3.0-STAT1-His vector, and (E) protein levels were assessed through WB analysis. (F) The mRNA levels of porcine STAT1, MX1, OAS1, and ISG20 were quantified using RT-qPCR analysis at 12, 24, 36, and 48 hpi in CSFV-infected PK-15STAT1-/- cells and (G) protein levels were measured by WB analysis (** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.

    Journal: Biomolecules

    Article Title: Classical Swine Fever Virus Envelope Glycoproteins E rns , E1, and E2 Activate IL-10-STAT1-MX1/OAS1 Antiviral Pathway via Replacing Classical IFNα/β.

    doi: 10.3390/biom15020200

    Figure Lengend Snippet: Figure 9. Identification of downstream targets of the STAT1 pathway. (A,B) The copy number of CSFV gRNA and the mRNA levels of porcine MX1, OAS1, and ISG20 were quantified through RT-qPCR analysis at 12, 24, 36, and 48 hpi in PK-15 cells infected with CSFV (MOI = 1). (C) The protein levels were assessed using Western blot (WB) analysis. (D) The mRNA levels of porcine STAT1, MX1, OAS1, and ISG20 were quantified using RT-qPCR analysis at 12, 24, 36, and 48 hpt in PK-15 cells transfected with the pcDNA3.0-STAT1-His vector, and (E) protein levels were assessed through WB analysis. (F) The mRNA levels of porcine STAT1, MX1, OAS1, and ISG20 were quantified using RT-qPCR analysis at 12, 24, 36, and 48 hpi in CSFV-infected PK-15STAT1-/- cells and (G) protein levels were measured by WB analysis (** p < 0.01, *** p < 0.001, **** p < 0.0001). Original images of the western blots can be found at Supplementary Materials.

    Article Snippet: Following blocking, the membranes were probed at 37 ◦C for 60 min with primary antibodies (Abs) against STAT1 (10144-2-AP), Myc tag (16286-1-AP), lamin B1 (12987-1-AP), OAS1 (14955-1-AP), MX1 (13750-1-AP), His tag (10001-0-AP), and Flag tag (20543-1-AP) (all from Proteintech Co., Ltd., Wuhan, China); phosphorylated (p) STAT1 (9167S), IFNα (6B18), and IFNβ (D2J1D) (from Cell Signaling Technology, Inc., Danvers, MA, USA); CSFV E1/E2 (9011) (from JENO Biotech, Seoul, Korea); CSFV E2 (orb500382) (from Biorbyt Ltd., Cambridge, England); and β-actin (CW0096A) (from Beijing CoWin Biotech Co., Ltd., Beijing, China).

    Techniques: Quantitative RT-PCR, Infection, Western Blot, Transfection, Plasmid Preparation

    Figure 10. Substitution of the classical IFNα/β-STAT1 pathway with the IL-10-STAT1 pathway during CSFV infection. The type I IFN-mediated STAT1 pathway was replaced with the alternative IL-10-STAT1 pathway during CSFV infection via the viral envelope glycoproteins Erns, E1, and E2. The classical pathway: IFNα/β-p-STAT1/p-STAT2-IRF9-ISGs (left panel). The alternative pathway: IL-10-p-STAT1-MX1/OAS1 (right panel).

    Journal: Biomolecules

    Article Title: Classical Swine Fever Virus Envelope Glycoproteins E rns , E1, and E2 Activate IL-10-STAT1-MX1/OAS1 Antiviral Pathway via Replacing Classical IFNα/β.

    doi: 10.3390/biom15020200

    Figure Lengend Snippet: Figure 10. Substitution of the classical IFNα/β-STAT1 pathway with the IL-10-STAT1 pathway during CSFV infection. The type I IFN-mediated STAT1 pathway was replaced with the alternative IL-10-STAT1 pathway during CSFV infection via the viral envelope glycoproteins Erns, E1, and E2. The classical pathway: IFNα/β-p-STAT1/p-STAT2-IRF9-ISGs (left panel). The alternative pathway: IL-10-p-STAT1-MX1/OAS1 (right panel).

    Article Snippet: Following blocking, the membranes were probed at 37 ◦C for 60 min with primary antibodies (Abs) against STAT1 (10144-2-AP), Myc tag (16286-1-AP), lamin B1 (12987-1-AP), OAS1 (14955-1-AP), MX1 (13750-1-AP), His tag (10001-0-AP), and Flag tag (20543-1-AP) (all from Proteintech Co., Ltd., Wuhan, China); phosphorylated (p) STAT1 (9167S), IFNα (6B18), and IFNβ (D2J1D) (from Cell Signaling Technology, Inc., Danvers, MA, USA); CSFV E1/E2 (9011) (from JENO Biotech, Seoul, Korea); CSFV E2 (orb500382) (from Biorbyt Ltd., Cambridge, England); and β-actin (CW0096A) (from Beijing CoWin Biotech Co., Ltd., Beijing, China).

    Techniques: Infection

    ( A ) Principal component analysis (PCA) plot for the sequenced samples of WT ( n = 6), KO ( n = 5), WT+NPCs ( n = 5) and KO+NPCs ( n = 6) cerebella. Percentage of variance is reported for both PC1 (first component) and PC2 (second component). ( B ) The number of deregulated genes (DEGs) for the different comparison is reported, considering a p.adj < 0.05. The number of DEGs with a LogFoldChange lower than −1 (down-regulated genes) or LogFoldChange greater than 1 (upregulated genes) is also indicated. ( C ) Dot plot of Gene Ontology (GO) enriched pathway analysis in the cerebellum, indicating the top 30 most enriched pathways of the comparison between KO+NPCs versus KO samples. For a description of the statistical method implemented in DESeq2 or ORA see Boyle et al and Yu et al , respectively. ( D ) The graph shows the LogFoldChange (LFC) of genes belonging to the GO pathway “Interferon-γ response” in the comparisons KO versus WT, WT + NPC versus WT, and KO+NPCs versus KO. ( E ) Gene set enrichment analysis (GSEA) of IFNγ response in cerebella, indicating a significant enrichment of the gene set in KO+NPCs vs KO comparison, as well as in KO+NPCs vs WT comparison. ( F ) The histogram reports the transcriptional levels of genes associated to IFNγ pathway. Data, expressed as percentage of WT, are shown as mean ± SEM. Parp9: * p = 0.0292 WT vs KO, § p = 0.0508 KO vs KO+NPCs; Iftm3: *** p = 0.0007 WT vs KO, *** p = 0.0008 KO vs KO+NPCs; Irf8: *** p = 0.0002 KO vs KO+NPCs; Bst2: * p = 0.0274 WT vs KO, *** p = 0.0010 KO vs KO+NPCs by two-way ANOVA followed by Sidak’s post-hoc test. n = 5 WT, n = 6 WT+NPCs, n = 5 KO, n = 6 KO+NPCs. ( G ) Western blot analysis of phosphorylated STAT1 (P-STAT1) in WT or KO neurons cultured with NPCs (for 14 days) ( n = 7 mouse embryos/genotype). Data are represented as mean ± SEM and expressed as percentage of WT neurons cultured alone. * p = 0.0242 by two-way ANOVA followed by Sidak’s post hoc test. Representative bands of P-STAT1 and the corresponding lanes of TGX-stain free gel are reported. .

    Journal: EMBO Molecular Medicine

    Article Title: Neural precursor cells rescue symptoms of Rett syndrome by activation of the Interferon γ pathway

    doi: 10.1038/s44321-024-00144-9

    Figure Lengend Snippet: ( A ) Principal component analysis (PCA) plot for the sequenced samples of WT ( n = 6), KO ( n = 5), WT+NPCs ( n = 5) and KO+NPCs ( n = 6) cerebella. Percentage of variance is reported for both PC1 (first component) and PC2 (second component). ( B ) The number of deregulated genes (DEGs) for the different comparison is reported, considering a p.adj < 0.05. The number of DEGs with a LogFoldChange lower than −1 (down-regulated genes) or LogFoldChange greater than 1 (upregulated genes) is also indicated. ( C ) Dot plot of Gene Ontology (GO) enriched pathway analysis in the cerebellum, indicating the top 30 most enriched pathways of the comparison between KO+NPCs versus KO samples. For a description of the statistical method implemented in DESeq2 or ORA see Boyle et al and Yu et al , respectively. ( D ) The graph shows the LogFoldChange (LFC) of genes belonging to the GO pathway “Interferon-γ response” in the comparisons KO versus WT, WT + NPC versus WT, and KO+NPCs versus KO. ( E ) Gene set enrichment analysis (GSEA) of IFNγ response in cerebella, indicating a significant enrichment of the gene set in KO+NPCs vs KO comparison, as well as in KO+NPCs vs WT comparison. ( F ) The histogram reports the transcriptional levels of genes associated to IFNγ pathway. Data, expressed as percentage of WT, are shown as mean ± SEM. Parp9: * p = 0.0292 WT vs KO, § p = 0.0508 KO vs KO+NPCs; Iftm3: *** p = 0.0007 WT vs KO, *** p = 0.0008 KO vs KO+NPCs; Irf8: *** p = 0.0002 KO vs KO+NPCs; Bst2: * p = 0.0274 WT vs KO, *** p = 0.0010 KO vs KO+NPCs by two-way ANOVA followed by Sidak’s post-hoc test. n = 5 WT, n = 6 WT+NPCs, n = 5 KO, n = 6 KO+NPCs. ( G ) Western blot analysis of phosphorylated STAT1 (P-STAT1) in WT or KO neurons cultured with NPCs (for 14 days) ( n = 7 mouse embryos/genotype). Data are represented as mean ± SEM and expressed as percentage of WT neurons cultured alone. * p = 0.0242 by two-way ANOVA followed by Sidak’s post hoc test. Representative bands of P-STAT1 and the corresponding lanes of TGX-stain free gel are reported. .

    Article Snippet: Membranes were incubated for 1 h in blocking solution [5% BSA in 0.1% Tween-20 in Tris-buffered saline containing (TBST)], and incubated overnight at 4 °C with the following primary antibodies: rabbit anti-phosphorylated Tyr701 Stat1 (clone 58D6, 1:1000; #9167, Cell Signalling; RRID:AB_561284) or rabbit anti-Bdnf (1:1000; ab108319, Abcam; RRID:AB_10862052).

    Techniques: Comparison, Western Blot, Cell Culture, Staining

    ( A ) The histogram represents the cell survival (% untreated WT neurons) ± SEM, assessed by MTT assay. IFNγ was added for 24 h in DIV13 primary neurons and three doses were tested: 25, 75, and 100 ng/ml. ( B ) The histogram reports the mean ± SEM of the levels of phosphorylated STAT1 after IFNγ treatment. Data are normalized to total protein content, visualized by a TGX stain-free technology. Representative bands of phosphorylated STAT1, and the corresponding lanes of TGX-stain-free gel, in WT and KO neurons treated or not with IFNγ are depicted. § p = 0.0003 WT-UT vs WT 100 ng/ml and § p < 0.0001 for the other comparisons, by two-way ANOVA followed by Tukey’s post-hoc test. § denotes a significant difference respect to the corresponding untreated control of the same genotype. WT and KO neurons derived from 3 different mice/genotype.

    Journal: EMBO Molecular Medicine

    Article Title: Neural precursor cells rescue symptoms of Rett syndrome by activation of the Interferon γ pathway

    doi: 10.1038/s44321-024-00144-9

    Figure Lengend Snippet: ( A ) The histogram represents the cell survival (% untreated WT neurons) ± SEM, assessed by MTT assay. IFNγ was added for 24 h in DIV13 primary neurons and three doses were tested: 25, 75, and 100 ng/ml. ( B ) The histogram reports the mean ± SEM of the levels of phosphorylated STAT1 after IFNγ treatment. Data are normalized to total protein content, visualized by a TGX stain-free technology. Representative bands of phosphorylated STAT1, and the corresponding lanes of TGX-stain-free gel, in WT and KO neurons treated or not with IFNγ are depicted. § p = 0.0003 WT-UT vs WT 100 ng/ml and § p < 0.0001 for the other comparisons, by two-way ANOVA followed by Tukey’s post-hoc test. § denotes a significant difference respect to the corresponding untreated control of the same genotype. WT and KO neurons derived from 3 different mice/genotype.

    Article Snippet: Membranes were incubated for 1 h in blocking solution [5% BSA in 0.1% Tween-20 in Tris-buffered saline containing (TBST)], and incubated overnight at 4 °C with the following primary antibodies: rabbit anti-phosphorylated Tyr701 Stat1 (clone 58D6, 1:1000; #9167, Cell Signalling; RRID:AB_561284) or rabbit anti-Bdnf (1:1000; ab108319, Abcam; RRID:AB_10862052).

    Techniques: MTT Assay, Staining, Control, Derivative Assay

    Reagents and tools table

    Journal: EMBO Molecular Medicine

    Article Title: Neural precursor cells rescue symptoms of Rett syndrome by activation of the Interferon γ pathway

    doi: 10.1038/s44321-024-00144-9

    Figure Lengend Snippet: Reagents and tools table

    Article Snippet: Membranes were incubated for 1 h in blocking solution [5% BSA in 0.1% Tween-20 in Tris-buffered saline containing (TBST)], and incubated overnight at 4 °C with the following primary antibodies: rabbit anti-phosphorylated Tyr701 Stat1 (clone 58D6, 1:1000; #9167, Cell Signalling; RRID:AB_561284) or rabbit anti-Bdnf (1:1000; ab108319, Abcam; RRID:AB_10862052).

    Techniques: Modification, Isolation, Amplification, SYBR Green Assay, Recombinant, Plasmid Preparation, Purification, Sequencing, Software, Cell Culture, Membrane, Staining

    Diet-induced obesity prevents beige fat biogenesis and promotes Pdgfrb expression. A. Experimental design: C57Bl6/J-129SV mice were fed a chow or HFD diet for 4 or 8 weeks. At denoted times mice were subsequently cold exposed for seven days and phenotypically evaluated. B, C. Rectal temperature of male (B) and female (C) chow or HFD fed mice throughout CE (n = 3-5 biologically independent mice). D, E. iWAT (D) and gWAT (E) weight from chow and HFD male mice (n = 3-5 biologically independent mice). F, G. iWAT (F) and gWAT (G) weight from chow and HFD female mice (n = 3-5 biologically independent mice). H, I. Representative images of H&E staining of iWAT sections from male (H) or female (I) chow and HFD fed mice at 4 and 8 weeks after CE. J, K. Representative images of Ucp1 immunostaining of iWAT sections from male (J) or female (K) chow and HFD fed mice at 4 and 8 weeks after CE. L. Relative mRNA levels of Ucp1 expression within iWAT depots from male mice fed chow or HFD for 8 weeks after CE (n = 3-4 biologically independent mice/group). M. Relative mRNA levels of Pdgfrb expression within iWAT depots from male mice fed chow or HFD for 8 weeks maintained at room temperature (RT) (n = 4 biologically independent mice). N. Representative flow cytometric histograms of PDGFRb expression within SMA-positive cells from male mice fed chow or HFD for 8 weeks maintained at RT. O. Representative flow cytometric histograms of phosphorylated STAT1 within SMA-positive cells from male mice fed chow or HFD for 8 weeks maintained at RT. Data are means with individual data points ±S.E.M. Statistical significance was determined using unpaired two-tailed Student’s t test (B-G, L, and M). Scale bar = 100 µm.

    Journal: Molecular Metabolism

    Article Title: Reversing Pdgfrβ signaling restores metabolically active beige adipocytes by alleviating ILC2 suppression in aged and obese mice

    doi: 10.1016/j.molmet.2024.102028

    Figure Lengend Snippet: Diet-induced obesity prevents beige fat biogenesis and promotes Pdgfrb expression. A. Experimental design: C57Bl6/J-129SV mice were fed a chow or HFD diet for 4 or 8 weeks. At denoted times mice were subsequently cold exposed for seven days and phenotypically evaluated. B, C. Rectal temperature of male (B) and female (C) chow or HFD fed mice throughout CE (n = 3-5 biologically independent mice). D, E. iWAT (D) and gWAT (E) weight from chow and HFD male mice (n = 3-5 biologically independent mice). F, G. iWAT (F) and gWAT (G) weight from chow and HFD female mice (n = 3-5 biologically independent mice). H, I. Representative images of H&E staining of iWAT sections from male (H) or female (I) chow and HFD fed mice at 4 and 8 weeks after CE. J, K. Representative images of Ucp1 immunostaining of iWAT sections from male (J) or female (K) chow and HFD fed mice at 4 and 8 weeks after CE. L. Relative mRNA levels of Ucp1 expression within iWAT depots from male mice fed chow or HFD for 8 weeks after CE (n = 3-4 biologically independent mice/group). M. Relative mRNA levels of Pdgfrb expression within iWAT depots from male mice fed chow or HFD for 8 weeks maintained at room temperature (RT) (n = 4 biologically independent mice). N. Representative flow cytometric histograms of PDGFRb expression within SMA-positive cells from male mice fed chow or HFD for 8 weeks maintained at RT. O. Representative flow cytometric histograms of phosphorylated STAT1 within SMA-positive cells from male mice fed chow or HFD for 8 weeks maintained at RT. Data are means with individual data points ±S.E.M. Statistical significance was determined using unpaired two-tailed Student’s t test (B-G, L, and M). Scale bar = 100 µm.

    Article Snippet: Cells were fixed for 30 min in 4% paraformaldehyde on ice, permeabilized, stained and analyzed for phosphorylated Stat1 (1:200; 9167 S Cell Signaling) or total Pdgfrβ (1:200; 4564 S Cell Signaling) conjugated to Cy5 donkey anti-rabbit secondary antibody (1:200; A10523 Invitrogen).

    Techniques: Expressing, Staining, Immunostaining, Two Tailed Test