primary antibody against pstat3 ser727 cellsignaling 9134s  (Cell Signaling Technology Inc)

Journal: Viruses

Article Title: Transcriptome Profiling Reveals That the African Swine Fever Virus C315R Exploits the IL-6 STAT3 Signaling Axis to Facilitate Virus Replication.

doi: 10.3390/v17030309

Figure Lengend Snippet: Figure 6. ASFV promotes IL-6 expression to activate STAT3 phosphorylation by C315R. (A) RT-PCR analysis of IL-6 mRNA expression in 3D4/21 cells infected with ASFV of different MOIs at different time points post infection, calculated using the 2−∆∆CT method. The data were analyzed using GraphPad Prism 8.0.2 software. The data were shown as mean ± SD based on three independent experiments. Statistical significance is denoted by asterisks (* p < 0.05; ** p < 0.01; **** p < 0.0001 determined by two-tailed Student’s t-test. ns, no significance). (B) Immunoblotting of whole cell lysates from 3D4/21 cells infected with ASFV at different time points and MOIs, probing for pSTAT3 (Ser727), STAT3, IL-6, p30, p72, and the cytosolic markers α-β-tubulin. (C) Confocal microscopy of 3D4/21 cells infected with ASFV at 24 hpi, showing colocalization of p30 (red) and pSTAT3 (Ser727) (green). Nuclei were counterstained with DAPI (blue). Scale bar: 25 µm. (D) Fluorescence intensity analysis of individual cells in C. (E) RT-PCR analysis of IL-6 expression in 3D4/21 cells transfected with either the empty vector or pcDNA3.1-HA-C315R, calculated by the 2−∆∆CT method. (F) Immunoblotting of lysates from 3D4/21 cells transfected with pcDNA3.1-HA-C315R, probing for pSTAT3 (Ser727), STAT3, IL-6, HA, and α-β-tubulin. (G) Confocal microscopy showing colocalization

Article Snippet: The following antibodies were procured from the corresponding companies: anti-β-tubulin rabbit antibody (Proteintech, 10068-1-AP, Rosemont, IL, USA), anti-GAPDH mouse antibody (Proteintech, 60004-1-Ig, Rosemont, IL, USA), anti-DYKDDDK-tag rabbit antibody (Abmart, R20008M, Shanghai, China), anti-HA-tag rabbit antibody (Abmart, P60025M, Shanghai, China), anti-STAT3 rabbit antibody (CST, 4904S, Danvers, MA, USA), anti-pSTAT3 (ser727) rabbit antibody (CST, 9134S, Danvers, MA, USA), anti-IL-6 rabbit antibody (BBI, D620828-0100, Shanghai, China), and anti-H2AC6-rabbit antibody (Solarbio, K110514P, Beijing, China).

Techniques: Expressing, Phospho-proteomics, Reverse Transcription Polymerase Chain Reaction, Infection, Software, Two Tailed Test, Western Blot, Confocal Microscopy, Fluorescence, Transfection, Plasmid Preparation

Journal: Viruses

Article Title: Transcriptome Profiling Reveals That the African Swine Fever Virus C315R Exploits the IL-6 STAT3 Signaling Axis to Facilitate Virus Replication.

doi: 10.3390/v17030309

Figure Lengend Snippet: Figure 7. Inhibition of STAT3 phosphorylation reduces ASFV replication and DNA synthesis. (A) Cy- totoxicity of Stattic was assessed using the CCK8 assay, which was represented as the average of three independent experiments. The data were analyzed using GraphPad Prism 8.0.2 software. (B) 3D4/21 cells were pretreated with Stattic or DMSO (control) for 4 h, followed by ASFV infection. IL-6 and p30 mRNA levels were analyzed by RT-PCR and calculated using the 2−∆∆CT method. Data were shown as mean ± SD based on three independent experiments. Statistical significance is denoted by asterisks (* p < 0.05; ** p < 0.01 determined by two-tailed Student’s t-test. ns, no significance). (C) Immunoblotting of 3D4/21 cell lysates treated with Stattic or DMSO for 4 h, probing for pSTAT3 (Ser727), STAT3, p30, and the cytosolic marker β-Tubulin. (D) PAMs pretreated with Stattic and infected with ASFV showed a significant reduction in viral yields from 5.37 to 2.00 HAD50/mL. The data were analyzed using GraphPad Prism 8.0.2 software. (E) 3D4/21 cells pretreated with Stattic or DMSO were infected with GFP-ASFV (green) for 24 h. Fluorescence images were captured using a fluorescence microscope (scale bar: 300 µm). (F) 3D4/21 cells, mock-infected, ASFV-infected, or ASFV-infected with Stattic for 12 h, were pulsed with 10 µM EdU for 2 h. DNA synthesis was measured by flow cytometry using a 488 nm laser for EdU detection. (G) The data from (F) were analyzed using GraphPad Prism 8.0.2 software.

Article Snippet: The following antibodies were procured from the corresponding companies: anti-β-tubulin rabbit antibody (Proteintech, 10068-1-AP, Rosemont, IL, USA), anti-GAPDH mouse antibody (Proteintech, 60004-1-Ig, Rosemont, IL, USA), anti-DYKDDDK-tag rabbit antibody (Abmart, R20008M, Shanghai, China), anti-HA-tag rabbit antibody (Abmart, P60025M, Shanghai, China), anti-STAT3 rabbit antibody (CST, 4904S, Danvers, MA, USA), anti-pSTAT3 (ser727) rabbit antibody (CST, 9134S, Danvers, MA, USA), anti-IL-6 rabbit antibody (BBI, D620828-0100, Shanghai, China), and anti-H2AC6-rabbit antibody (Solarbio, K110514P, Beijing, China).

Techniques: Inhibition, Phospho-proteomics, DNA Synthesis, CCK-8 Assay, Software, Control, Infection, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test, Western Blot, Marker, Fluorescence, Microscopy, Flow Cytometry

Journal: Viruses

Article Title: Transcriptome Profiling Reveals That the African Swine Fever Virus C315R Exploits the IL-6 STAT3 Signaling Axis to Facilitate Virus Replication.

doi: 10.3390/v17030309

Figure Lengend Snippet: Figure 8. Model of C315R-mediated activation of the IL-6 STAT3 signaling axis facilitating ASFV infection. C315R promotes the transcription of the pro-inflammatory cytokine IL-6, which in turn activates STAT3 phosphorylation (pSTAT3). Phosphorylated STAT3 translocate to the nucleus, where it enhances the transcription of viral genes, thereby supporting ASFV replication.

Article Snippet: The following antibodies were procured from the corresponding companies: anti-β-tubulin rabbit antibody (Proteintech, 10068-1-AP, Rosemont, IL, USA), anti-GAPDH mouse antibody (Proteintech, 60004-1-Ig, Rosemont, IL, USA), anti-DYKDDDK-tag rabbit antibody (Abmart, R20008M, Shanghai, China), anti-HA-tag rabbit antibody (Abmart, P60025M, Shanghai, China), anti-STAT3 rabbit antibody (CST, 4904S, Danvers, MA, USA), anti-pSTAT3 (ser727) rabbit antibody (CST, 9134S, Danvers, MA, USA), anti-IL-6 rabbit antibody (BBI, D620828-0100, Shanghai, China), and anti-H2AC6-rabbit antibody (Solarbio, K110514P, Beijing, China).

Techniques: Activation Assay, Infection, Phospho-proteomics