pstat1  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Selective JAK Inhibition Reveals Paradoxical and Hierarchical Control of interferon-γ-driven Autoimmunity in AIRE Deficiency

doi: 10.64898/2026.03.05.709894

Figure Lengend Snippet: Untreated Air e −/− mice and Air e −/− mice treated for four weeks with selective JAK1i, JAK2i, or JAK3i were analyzed. Lungs were processed for intracellular cytokine staining, qPCR, ELISA, and immunoblot analyses. ( A-B ) Representative flow cytometry plots showing IFN-γ production by CD4 + and CD8 + T cells. ( C-D ) Frequency (of total CD4 + and CD8 + T cells) and absolute numbers of IFN-γ + CD4 + and IFN-γ + CD8 + T cells in the lung. ( E-F ) Relative Ifng mRNA expression and IFN-γ protein concentrations in lung homogenates. ( G ) Relative Stat1 mRNA expression. ( H ) Representative immunoblots of phospho-STAT1 (pSTAT1), total STAT1, and GAPDH. ( I-J ) Quantification of total STAT1 and phospho-STAT1 normalized to GAPDH. For IFN- γ + CD4 + and IFN-γ + CD8 + T cells: n = 9-14 mice per group from four independent experiments. For Ifng and Cxcl9 mRNA: n = 15-22 mice per group from four independent experiments. For Stat1 _mRNA: n = 10-17 mice per group from three independent experiments. For CXCL9 protein: n = 5-10 mice per group from two independent experiments. For STAT1 and pSTAT1 immunoblots: n = 15-22 mice per group from four independent experiments). Statistical analyses were performed using one-way ANOVA or Kruskal-Wallis test with Dunn’s multiple comparisons to the untreated Air e −/− mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Primary antibodies against GAPDH (catalog no. 5174S), STAT1 (catalog no. 14995S), and pSTAT1 (catalog no. 9167S) (Cell Signaling Technologies, USA) were used at 1:1,000 dilution, with HRP-conjugated anti-rabbit IgG conjugated with HRP (catalog no.7074S; Cell Signaling Technology) as the secondary antibody.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Flow Cytometry, Expressing

Journal: PLOS Pathogens

Article Title: HCMV encoded UL84 hijacks FHL2 to suppress type I interferon production and enhance viral replication

doi: 10.1371/journal.ppat.1013895

Figure Lengend Snippet: (A) HFF cells were transfected with pGL3-luci-IFN-beta and pRL-TK reporter plasmids, along with increasing amount of the pcDNA3.1-FHL2 expression vector or the pcDNA3.1 empty vector as a control for 24 h, the cells were infected with HCMV (MOI = 1) for 24 h before the dual luciferase reporter assay. (B) HFF cells were transfected with the plasmid of pGL3-luci-IFN-beta and pRL-TK together with pcDNA3.1-FHL2 or the empty vector for 24 h, and then infected with HCMV (MOI = 1) for the indicated times before conducting dual-luciferase reporter assay. Another portion was subjected to Western blotting. (C) FHL2-knockout cell line was transfected with the plasmid of pGL3-luci-IFN-beta and pRL-TK for 24 h, followed by a 24 h, and then infected with HCMV (MOI = 1) for the indicated times before conducting dual-luciferase reporter assay. Another portion was subjected to Western blotting. (D) HFF cells were transfected with the plasmid of pcDNA3.1-FHL2 or the empty vector for 24 h, and then infected with HCMV (MOI = 1) for the indicated times before RT-qPCR analysis of the indicated antiviral interferon-stimulated genes. (E) FHL2-knockout cell line was infected with HCMV (MOI = 1) for the indicated times before RT-qPCR analysis of the indicated antiviral interferon-stimulated genes. (F) The FHL2-overexpressing cell line was infected with HCMV (MOI = 1), followed by Western blotting analysis of pSTAT1, STAT1, cytoplasmic FHL2 (cy), and nuclear FHL2 (nu). (G) The FHL2-knockout cell line was infected with HCMV (MOI = 1), followed by Western blotting analysis of pSTAT1, STAT1, cytoplasmic FHL2 (cy), and nuclear FHL2 (nu). The displayed images were representative ones from three independent experiments. For all figures, statistical analyses were performed using two-tailed t-test. Differences were considered statistically significant when * denoted p < 0.05, ** denoted p < 0.01, *** denoted p < 0.001, and **** denoted p < 0.0001.

Article Snippet: The antibodies against human IRF3 rabbit monoclonal antibody (Cat. No. 11904; diluted 1:1,000), human pIRF3 rabbit monoclonal antibody (Cat. No. E6F7Q; diluted 1:1,000), c-Jun mouse monoclonal antibody (Cat. No. 2315; diluted 1:1,000), STAT1 rabbit monoclonal antibody (Cat. No. 9172S; diluted 1:1,000) and pSTAT1 rabbit monoclonal antibody (Cat. No. 9167S; diluted 1:1,000) were obtained from CST (Boston, USA).

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Infection, Luciferase, Reporter Assay, Western Blot, Knock-Out, Quantitative RT-PCR, Two Tailed Test

Journal: PLOS Pathogens

Article Title: HCMV encoded UL84 hijacks FHL2 to suppress type I interferon production and enhance viral replication

doi: 10.1371/journal.ppat.1013895

Figure Lengend Snippet: (A) UL84 interacts with FHL2 to repress IFN-β transcription. HFFs were transiently transfected with expression plasmids for pGL3-luci-IFN-beta, FHL2, UL84, UL84 (Δ400-460) for 24 h, HCMV (MOI = 1) reinfection of cells for 6h before luciferase assay. (B) The UL84-overexpressing cell line was infected with HCMV (MOI = 1), followed by Western blotting analysis of pSTAT1, STAT1, IRF3, pIRF3, cytoplasmic FHL2 (cy), and nuclear FHL2 (nu). (C) HFF cells were transfected with the Con-RNAi, UL84-RNAi-#1, and then infected with HCMV (MOI = 1), followed by Western blotting analysis of pSTAT1, STAT1, IRF3, pIRF3, cytoplasmic FHL2 (cy), and nuclear FHL2 (nu). (D) HFF cells were transfected with the Con-RNAi, UL84-RNAi-#2, and then infected with HCMV (MOI = 1), followed by Western blotting analysis of pSTAT1, STAT1, IRF3, pIRF3, cytoplasmic FHL2 (cy), and nuclear FHL2 (nu). (E) Co-IP analysis of UL84 interaction with FHL2, IRF3, c-Jun, or p300. Expression plasmids of UL84, FHL2, TBP, IRF3, p300 and c-Jun were transiently transfected into HEK293T cells for 48 h, and then the total cell lysates were prepared and immunoprecipitated with anti-UL84, anti-FHL2, anti-IRF3, anti-c-Jun or anti-p300 antibodies, followed by immunoblot analysis. (F) Co-IP analysis of UL84 (Δ400-460) interaction with FHL2, IRF3, c-Jun, or p300. Expression plasmids of UL84(Δ400-460), FHL2, TBP, IRF3, p300 and c-Jun were transiently transfected into HEK293T cells for 48 h, and then the total cell lysates were prepared and immunoprecipitated with anti-Myc, anti-FHL2, anti-IRF3, anti-c-Jun or anti-p300 antibodies, followed by immunoblot analysis. (G) HEK293T cells were transfected with pCDNA3.1-FHL2, pCDNA3.1-IRF3, pCDNA3.1-p300, and pCDNA3.1-c-Jun plasmids, along with increasing amounts of the pCDNA3.1-UL84 expression vector for 48 h. Co-IP analysis of UL84 interaction with FHL2, IRF3, c-Jun, or p300. (H) Effect of different mutations on the interaction of FHL2 with UL84. HEK293T cells were transiently transfected with Flag-tagged FHL2 (four single point mutants) and pcDNA3.1-UL84 for 48 h before Co-IP analysis. (I) Co-IP analysis of UL84 interactions with TBP. HEK293T cells were transiently transfected with the indicated plasmids for 48 h, using as the indicated anti-UL84, anti-Flag or anti-TBP antibody before Co-IP analysis was performed. (J) Molecular docking simulation of the complex of UL84-FHL2-TBP. The structure of the IRF3, FHL2 and TBP was downloaded from the Protein Data Bank, green refers to FHL2, grey refers to TBP, and blue refers to UL84. The displayed images were representative ones from three independent experiments. For all figures, statistical analyses were performed using two-tailed t-test. Differences were considered statistically significant when * denoted p < 0.05, ** denoted p < 0.01, *** denoted p < 0.001, and **** denoted p < 0.0001.

Article Snippet: The antibodies against human IRF3 rabbit monoclonal antibody (Cat. No. 11904; diluted 1:1,000), human pIRF3 rabbit monoclonal antibody (Cat. No. E6F7Q; diluted 1:1,000), c-Jun mouse monoclonal antibody (Cat. No. 2315; diluted 1:1,000), STAT1 rabbit monoclonal antibody (Cat. No. 9172S; diluted 1:1,000) and pSTAT1 rabbit monoclonal antibody (Cat. No. 9167S; diluted 1:1,000) were obtained from CST (Boston, USA).

Techniques: Transfection, Expressing, Luciferase, Infection, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation, Plasmid Preparation, Two Tailed Test