phospho stat2 tyr690 antibody  (Cell Signaling Technology Inc)

Journal: Scientific Reports

Article Title: CNOT2 /c-Myc/STAT3 signaling is critically involved in glycolysis mediated apoptosis of benzyl isothiocyanate in hepatocellular carcinoma

doi: 10.1038/s41598-026-38416-8

Figure Lengend Snippet: Effect of BITC on CNOT2 and pSTAT3 and their binding in SK-Hep1 and Huh7 cells. ( A ) Overexpression of CNOT2 in liver cancer patients implies poor survival rate along with original regression ratio with STAT3. ( B ) Effect of BITC on CNOT2 and pSTAT3 in SK-Hep1 and Huh7 cells. Band intensities were quantified and normalized to β-actin. ( C ) Effect of BITC on the binding between CNOT2 and STAT3 or c-Myc in SK-Hep1 cells. All experiments were performed using biological triplicates and independently repeated three times .

Article Snippet: Blots were probed with primary antibodies against CNOT2 (#34,214, 1:1000; Cell Signaling Technology, MA, USA), c-Myc (ab32072, 1:1000, Abcam, Cambridge, UK), JAK1 (#3332, 1:1000; Cell Signaling Technology, MA, USA), STAT3 (#12,640, 1:1000; Cell Signaling Technology, MA, USA), phospho-JAK1 (#3331, 1:1000; Cell Signaling Technology, MA, USA), phospho-STAT3 (#4441, 1:1000; Cell Signaling Technology, MA, USA), pro-PARP (#9542, 1:1000; Cell Signaling Technology, MA, USA), pro-caspase-3 (#9662, 1:1000; Cell Signaling Technology, MA, USA), HK2 (#2106, 1:1000; Cell Signaling Technology, MA, USA) , PKM2 (#4053, 1:1000; Cell Signaling Technology, MA, USA), LDH (SC-133123, 1:1000; Santa Cruz, CA, USA) and β-actin (A1978, 1:10,000; Sigma-Aldrich, MO, USA).

Techniques: Binding Assay, Over Expression

Journal: Scientific Reports

Article Title: CNOT2 /c-Myc/STAT3 signaling is critically involved in glycolysis mediated apoptosis of benzyl isothiocyanate in hepatocellular carcinoma

doi: 10.1038/s41598-026-38416-8

Figure Lengend Snippet: Depletion of STAT3 or CNOT2 enhances apoptotic effect of BITC in SK-Hep1 cells. ( A ) Effect of STAT3 or CNOT2 depletion in BITC treated SK-Hep1 cells. ( B ) Effect of STAT3 depletion on pro-PARP and pro-caspase3 in BITC treated SK-Hep1 cells. ( C ) Effect of CNOT2 depletion on pro-PARP and pro-caspase3 in BITC treated SK-Hep1 cells. Band intensities were quantified and normalized to β-actin. All experiments were performed using biological triplicates and independently repeated three times .

Article Snippet: Blots were probed with primary antibodies against CNOT2 (#34,214, 1:1000; Cell Signaling Technology, MA, USA), c-Myc (ab32072, 1:1000, Abcam, Cambridge, UK), JAK1 (#3332, 1:1000; Cell Signaling Technology, MA, USA), STAT3 (#12,640, 1:1000; Cell Signaling Technology, MA, USA), phospho-JAK1 (#3331, 1:1000; Cell Signaling Technology, MA, USA), phospho-STAT3 (#4441, 1:1000; Cell Signaling Technology, MA, USA), pro-PARP (#9542, 1:1000; Cell Signaling Technology, MA, USA), pro-caspase-3 (#9662, 1:1000; Cell Signaling Technology, MA, USA), HK2 (#2106, 1:1000; Cell Signaling Technology, MA, USA) , PKM2 (#4053, 1:1000; Cell Signaling Technology, MA, USA), LDH (SC-133123, 1:1000; Santa Cruz, CA, USA) and β-actin (A1978, 1:10,000; Sigma-Aldrich, MO, USA).

Techniques:

Journal: Scientific Reports

Article Title: CNOT2 /c-Myc/STAT3 signaling is critically involved in glycolysis mediated apoptosis of benzyl isothiocyanate in hepatocellular carcinoma

doi: 10.1038/s41598-026-38416-8

Figure Lengend Snippet: Effect of BITC on Warburg effect in SK-Hep1 and Huh7 cells. ( A ) Effect of BITC on Warburg effect proteins in SK-Hep1 and Huh7 cells. ( B ) Effect of BITC on LDH production in SK-Hep1 and Huh7 cells. STAT3 or CNOT2 depletion vs untreated control. ***p < 0.001 vs untreated control. ( C ) Effect of BITC on glucose consumption in SK-Hep1 and Huh7 cells. *p < 0.05, ***p < 0.001 vs untreated control. ( D ) Effect of pyruvate treatment or overexpression of CNOT2 or c-Myc on apoptosis and glycolysis proteins in BITC treated SK-Hep1 cells. (E) Effect of siSTAT3 or/and siCNOT2 on HK2, PKM2 and LDH in SK-Hep1 cells transfected with or without c-Myc overexpression plasmid. Cells were co-transfected with control vector, siSTAT3, siCNOT2 and siCNOT2 + c-Myc OE (overexpression) plasmids as indicated in each lane. Band intensities were quantified and normalized to β-actin. All experiments were performed using biological triplicates and independently repeated three times .

Article Snippet: Blots were probed with primary antibodies against CNOT2 (#34,214, 1:1000; Cell Signaling Technology, MA, USA), c-Myc (ab32072, 1:1000, Abcam, Cambridge, UK), JAK1 (#3332, 1:1000; Cell Signaling Technology, MA, USA), STAT3 (#12,640, 1:1000; Cell Signaling Technology, MA, USA), phospho-JAK1 (#3331, 1:1000; Cell Signaling Technology, MA, USA), phospho-STAT3 (#4441, 1:1000; Cell Signaling Technology, MA, USA), pro-PARP (#9542, 1:1000; Cell Signaling Technology, MA, USA), pro-caspase-3 (#9662, 1:1000; Cell Signaling Technology, MA, USA), HK2 (#2106, 1:1000; Cell Signaling Technology, MA, USA) , PKM2 (#4053, 1:1000; Cell Signaling Technology, MA, USA), LDH (SC-133123, 1:1000; Santa Cruz, CA, USA) and β-actin (A1978, 1:10,000; Sigma-Aldrich, MO, USA).

Techniques: Control, Over Expression, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: CNOT2 /c-Myc/STAT3 signaling is critically involved in glycolysis mediated apoptosis of benzyl isothiocyanate in hepatocellular carcinoma

doi: 10.1038/s41598-026-38416-8

Figure Lengend Snippet: Scheme on mechanism of BITC via CNOT2/c-Myc/STAT3 signaling in HCCs.

Article Snippet: Blots were probed with primary antibodies against CNOT2 (#34,214, 1:1000; Cell Signaling Technology, MA, USA), c-Myc (ab32072, 1:1000, Abcam, Cambridge, UK), JAK1 (#3332, 1:1000; Cell Signaling Technology, MA, USA), STAT3 (#12,640, 1:1000; Cell Signaling Technology, MA, USA), phospho-JAK1 (#3331, 1:1000; Cell Signaling Technology, MA, USA), phospho-STAT3 (#4441, 1:1000; Cell Signaling Technology, MA, USA), pro-PARP (#9542, 1:1000; Cell Signaling Technology, MA, USA), pro-caspase-3 (#9662, 1:1000; Cell Signaling Technology, MA, USA), HK2 (#2106, 1:1000; Cell Signaling Technology, MA, USA) , PKM2 (#4053, 1:1000; Cell Signaling Technology, MA, USA), LDH (SC-133123, 1:1000; Santa Cruz, CA, USA) and β-actin (A1978, 1:10,000; Sigma-Aldrich, MO, USA).

Techniques:

Journal: Virus Research

Article Title: Foot-and-mouth disease virus 3D polymerase antagonizes the interferon signaling pathway by blocking STAT2 nuclear translocation

doi: 10.1016/j.virusres.2025.199671

Figure Lengend Snippet: FMDV 3D protein interacts with STAT2. (A) HEK-293T cells were co-transfected with Flag-3D and various Myc-tagged innate immune molecule-expressing plasmids (JAK1, TYK2, STAT1, STAT2, or IRF9). At 36 hpt, the cell lysates were subjected to Co-IP assay analysis. Immunoprecipitated proteins and whole-cell lysates (WCL) were analyzed by western blotting using specified antibodies. ( B) HEK-293T cells were co-transfected with Myc-STAT2 and either an empty vector or Flag-3D expressing plasmids for 36 h. Cell lysates were immunoprecipitated with anti-Myc or control IgG antibodies and analyzed by western blotting. ( C) HEK-293T cells were co-transfected with Flag-3D along with Vec or Myc-STAT2. At 36 hpt, cell lysates were subjected to Co-IP assay. Immunoprecipitated proteins and WCL were analyzed by western blotting. (D) PK-15 cells were transfected with Flag-3D or empty vector plasmids. At 36 hpt, cell lysates were immunoprecipitated with anti-Flag antibodies and analyzed by western blotting. (E) PK-15 cells were mock-infected or infected with FMDV for 12 h, cell lysates were immunoprecipitated with anti-3D antibodies and analyzed by western blotting with the indicated antibodies. (F) PK-15 cells were transfected with porcine HA-STAT2 expressing plasmids for 24 h, then mock-infected or infected with FMDV (MOI = 0.1) for 10 h. Colocalization of HA-STAT2 (red) and FMDV 3D (green) was assessed by immunofluorescence assay (IFA). Nuclei were counterstained with DAPI (blue).

Article Snippet: The commercial antibodies used in this study include: anti-Flag mouse Ab (Sigma, F1804), anti-Myc mouse Ab (Sigma, M5546), anti-HA mouse Ab (Sigma, H9658), and anti-GAPDH mouse Ab (Abclonal, AC002), anti-JAK1 rabbit Ab (Cell Signaling Technology, 3332), anti-TYK2 rabbit Ab (Cell Signaling Technology, 9312), anti-STAT1 rabbit Ab (Cell Signaling Technology, 9172), anti-STAT2 rabbit Ab (Cell Signaling Technology, 4594), anti-p-STAT2 rabbit Ab (Cell Signaling Technology, 4441), anti-IRF9 rabbit Ab (Cell Signaling Technology, 76,684).

Techniques: Transfection, Expressing, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Plasmid Preparation, Control, Infection, Immunofluorescence

Journal: Virus Research

Article Title: Foot-and-mouth disease virus 3D polymerase antagonizes the interferon signaling pathway by blocking STAT2 nuclear translocation

doi: 10.1016/j.virusres.2025.199671

Figure Lengend Snippet: FMDV 3D protein does not alter the protein level of STAT2. (A) PK-15 cells were transfected with empty vector or Flag-3D expressing plasmids. At 24 hpt, cell lysates were analyzed by western blotting with specified antibodies. (B) PK-15 cells were transfected with 0, 0.25, 0.5 or 1 μg of Flag-3D expressing plasmids for 24 h. Endogenous STAT2 protein expression levels were detected by western blotting. (C) PK-15 cells were transfected with an increasing amount of Flag-3D expressing plasmids (0, 0.25, 0.5 or 1 μg) for 24 h. Total RNA was extracted, and STAT2 mRNA levels were quantified by qPCR. (D) PK-15 cells mock-infected or infected with FMDV (MOI=0.1) for 6 and 12 h, followed by IFN-β (1000 U/mL) or mock treatment for 30 min. The expression and phosphorylation levels of STAT2 were detected by western blotting.

Article Snippet: The commercial antibodies used in this study include: anti-Flag mouse Ab (Sigma, F1804), anti-Myc mouse Ab (Sigma, M5546), anti-HA mouse Ab (Sigma, H9658), and anti-GAPDH mouse Ab (Abclonal, AC002), anti-JAK1 rabbit Ab (Cell Signaling Technology, 3332), anti-TYK2 rabbit Ab (Cell Signaling Technology, 9312), anti-STAT1 rabbit Ab (Cell Signaling Technology, 9172), anti-STAT2 rabbit Ab (Cell Signaling Technology, 4594), anti-p-STAT2 rabbit Ab (Cell Signaling Technology, 4441), anti-IRF9 rabbit Ab (Cell Signaling Technology, 76,684).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Infection, Phospho-proteomics

Journal: Virus Research

Article Title: Foot-and-mouth disease virus 3D polymerase antagonizes the interferon signaling pathway by blocking STAT2 nuclear translocation

doi: 10.1016/j.virusres.2025.199671

Figure Lengend Snippet: FMDV 3D protein suppresses phosphorylation and nuclear translocation of STAT2. (A) PK-15 cells were transfected with either an empty vector or Flag-3D expressing plasmids. At 24 hpt, cells were treated with IFN-β (1000 U/mL) or solvent control for 30 min. Cell lysates were analyzed by western blotting with specified antibodies. (B) PK-15 cells were transfected with increasing amounts of Flag-3D expressing plasmids (0, 0.25, 0.5, or 1 μg). At 24 hpt, cells were treated with IFN-β (1000 U/mL) for 30 min. Cell lysates were analyzed by western blotting with indicated antibodies. (C) HEK-293T cells were co-transfected with Myc-STAT2 and HA-STAT1 or HA-vector, along with increasing amounts Flag-3D expressing plasmids. At 36 hpt, the cells were treated with IFN-β (1000 U/mL) for 30 min. The cell lysates were immunoprecipitated with anti-Myc antibodies. The immunoprecipitated proteins and WCL were analyzed by western blotting using the specified antibodies. (D) PK-15 cells mock-infected or infected with FMDV (MOI=0.1) for 10 h, followed by IFN-β (1000 U/mL) or mock treatment for 30 min. The subcellular localization of STAT2 and FMDV 3D was assessed by IFA. Nuclei were stained with DAPI (blue), and fluorescence was visualized for 3D (green) and STAT2 (red). (E) PK-15 cells were transfected with an increasing amounts Flag-3D expressing plasmids for 24 h, the cell lysates were analyzed by western blotting using the specified antibodies. (F) PK-15 cells were transfected with Flag-3D expressing plasmids for 24 h, then mock-infected or infected with FMDV (MOI=0.1) for 12 h. Cell lysates were analyzed by western blotting with indicated antibodies.

Article Snippet: The commercial antibodies used in this study include: anti-Flag mouse Ab (Sigma, F1804), anti-Myc mouse Ab (Sigma, M5546), anti-HA mouse Ab (Sigma, H9658), and anti-GAPDH mouse Ab (Abclonal, AC002), anti-JAK1 rabbit Ab (Cell Signaling Technology, 3332), anti-TYK2 rabbit Ab (Cell Signaling Technology, 9312), anti-STAT1 rabbit Ab (Cell Signaling Technology, 9172), anti-STAT2 rabbit Ab (Cell Signaling Technology, 4594), anti-p-STAT2 rabbit Ab (Cell Signaling Technology, 4441), anti-IRF9 rabbit Ab (Cell Signaling Technology, 76,684).

Techniques: Phospho-proteomics, Translocation Assay, Transfection, Plasmid Preparation, Expressing, Solvent, Control, Western Blot, Immunoprecipitation, Infection, Staining, Fluorescence

Journal: Virus Research

Article Title: Foot-and-mouth disease virus 3D polymerase antagonizes the interferon signaling pathway by blocking STAT2 nuclear translocation

doi: 10.1016/j.virusres.2025.199671

Figure Lengend Snippet: Prediction the key interaction residues between STAT2 and FMDV-3D The interaction model between STAT2 and FMDV-3D was constructed using the ZDOCK online service. Predicted salt bridge interactions, Pi-Alkyl interaction of STAT2 phosphorylation residue Y690 with 3D P23, and key residues STAT2 R687, E686, N683, H693 and 3D E33, R174, R174, G28 were highlighted using PyMOL software.

Article Snippet: The commercial antibodies used in this study include: anti-Flag mouse Ab (Sigma, F1804), anti-Myc mouse Ab (Sigma, M5546), anti-HA mouse Ab (Sigma, H9658), and anti-GAPDH mouse Ab (Abclonal, AC002), anti-JAK1 rabbit Ab (Cell Signaling Technology, 3332), anti-TYK2 rabbit Ab (Cell Signaling Technology, 9312), anti-STAT1 rabbit Ab (Cell Signaling Technology, 9172), anti-STAT2 rabbit Ab (Cell Signaling Technology, 4594), anti-p-STAT2 rabbit Ab (Cell Signaling Technology, 4441), anti-IRF9 rabbit Ab (Cell Signaling Technology, 76,684).

Techniques: Construct, Phospho-proteomics, Residue, Software

Journal: Virus Research

Article Title: Foot-and-mouth disease virus 3D polymerase antagonizes the interferon signaling pathway by blocking STAT2 nuclear translocation

doi: 10.1016/j.virusres.2025.199671

Figure Lengend Snippet: Mechanisms by which FMDV 3D protein targets STAT2 to impede the activation of the JAK-STAT signaling pathway. Upon FMDV infection of host cells, the viral 3D protein interacts with STAT2, hinders the phosphorylation and inhibits the nuclear translocation of STAT2, thereby blocks the activation of the JAK-STAT signaling pathway and suppresses host antiviral response.

Article Snippet: The commercial antibodies used in this study include: anti-Flag mouse Ab (Sigma, F1804), anti-Myc mouse Ab (Sigma, M5546), anti-HA mouse Ab (Sigma, H9658), and anti-GAPDH mouse Ab (Abclonal, AC002), anti-JAK1 rabbit Ab (Cell Signaling Technology, 3332), anti-TYK2 rabbit Ab (Cell Signaling Technology, 9312), anti-STAT1 rabbit Ab (Cell Signaling Technology, 9172), anti-STAT2 rabbit Ab (Cell Signaling Technology, 4594), anti-p-STAT2 rabbit Ab (Cell Signaling Technology, 4441), anti-IRF9 rabbit Ab (Cell Signaling Technology, 76,684).

Techniques: Activation Assay, Infection, Phospho-proteomics, Translocation Assay