Journal: Cell reports

Article Title: BATF2 is a regulator of interferon-γ signaling in astrocytes during neuroinflammation

doi: 10.1016/j.celrep.2025.115393

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: IRF-1 (D5E4) XP ® Rabbit mAb , Cell Signaling , AB_10949108.

Techniques: Recombinant, Control, Protease Inhibitor, Western Blot, Electron Microscopy, Amplification, Reverse Transcription, SYBR Green Assay, Bicinchoninic Acid Protein Assay, RNA Sequencing, Software, Real-time Polymerase Chain Reaction

Journal: Virology

Article Title: Lipopolysaccharide restricts murine norovirus infection in macrophages mainly through NF-kB and JAK-STAT signaling pathway.

doi: 10.1016/j.virol.2020.04.010

Figure Lengend Snippet: Fig. 5. NF-ĸB signaling is important for LPS- induced IFN-β and ISG transcription. Cells were untreated or treated with LPS (100 ng/ ml), Bay 11–7085 (5 μM) or combination for 6 h qRT-PCR analysis of IFN-β (A) and ISG (B) mRNA levels in RAW264.7 cells (n = 6). qRT- PCR analysis of IFN-β (C) and ISG (D) mRNA levels in J774A.1 cells (n = 6). Western blot- ting analysis of STAT1 expression and phos- phorylation, and expression of IRF1 and GBP2 in RAW264.7 cells (E) and J774A.1 cells (F) untreated or treated with LPS (100 ng/ml), IFN-γ (100 U/ml), Bay 11–7085 (5 μM) or combination for 6 h qRT-PCR analysis of mRNA levels of IFN-β (G) (n = 6) and ISGs (H) (n = 6) in WT and NF-ĸB−/−MEFs untreated or treated with LPS (100 ng/ml) for 6 h. Data in (A, B, C, D and G) were normalized to the untreated control (CTR, set as 1). Data in (H) were normalized to untreated WT and NF- ĸB−/−MEFs, respectively (both set as 1). *P < 0.05; **P < 0.01. β-actin was used as a loading control.

Article Snippet: Antibodies against phosphoSTAT1 (Ser727) (#9177), STAT1 (#9172), MDA5 (D74E4, #5321) and IRF1 (D5E4, #8478), were purchased from Cell Signaling Technology.

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

Journal: Journal of Virology

Article Title: Differential Responses by Human Respiratory Epithelial Cell Lines to Respiratory Syncytial Virus Reflect Distinct Patterns of Infection Control

doi: 10.1128/JVI.02202-17

Figure Lengend Snippet: Summary of differential gene expression kinetics between A549 and BEAS-2B cells in response to low doses of RSV

Article Snippet: Levels of IRF1, IRF9, and RIG-I were visualized by chemiluminescence using rabbit anti-IRF1 monoclonal antibody (MAb) (clone D5E4; Cell Signaling Technology), rabbit anti-ISGF-3γ polyclonal antibody (PAb) (Santa Cruz, Dallas, TX), mouse anti-RIG-I MAb (clone 1c3; Millipore), and mouse anti-β-actin MAb (clone C4; Santa Cruz) as a control for protein loading.

Techniques: Expressing

Journal: Journal of Virology

Article Title: Differential Responses by Human Respiratory Epithelial Cell Lines to Respiratory Syncytial Virus Reflect Distinct Patterns of Infection Control

doi: 10.1128/JVI.02202-17

Figure Lengend Snippet: RSV-infected BEAS-2B cells express more IRF-1 but activate less IRF-3 and NF-κB than A549 cells. A549 or BEAS-2B cells were mock infected (medium only) or infected with rgRSV at an MOI of 0.1 or 0.3 for the indicated times. (A) Transcription factor gene expression was measured by RT-qPCR. The data shown are means ± SD from the results of four experiments performed in duplicate. Statistical differences between cell lines at each time point were calculated using a one-way ANOVA model without adjustment for mock infection on log10-transformed values as described in Materials and Methods. *, P < 0.05 for A549 compared to BEAS-2B mock infection at 0 h; ++, P < 0.05 for A549 compared to BEAS-2B at MOIs of 0.1 and 0.3; +, P < 0.05 at an MOI of either 0.1 or 0.3. (B and D) IRF3 dimerization (B) and IRF1 protein expression (D) were measured by Western blotting. The data represent the results of experiments on two separate cultures. (C) EMSA was performed to determine NF-κB activation.

Article Snippet: Levels of IRF1, IRF9, and RIG-I were visualized by chemiluminescence using rabbit anti-IRF1 monoclonal antibody (MAb) (clone D5E4; Cell Signaling Technology), rabbit anti-ISGF-3γ polyclonal antibody (PAb) (Santa Cruz, Dallas, TX), mouse anti-RIG-I MAb (clone 1c3; Millipore), and mouse anti-β-actin MAb (clone C4; Santa Cruz) as a control for protein loading.

Techniques: Infection, Expressing, Quantitative RT-PCR, Transformation Assay, Western Blot, Activation Assay

Journal: Cell

Article Title: Human IRF1 governs macrophagic IFN-γ immunity to mycobacteria.

doi: 10.1016/j.cell.2022.12.038

Figure Lengend Snippet: Figure 2. IRF1 mRNA and protein levels in cells from the two patients (A–J) qPCR for IRF1 normalized against GUSB and the mean value for controls (CTLs) for cDNA from (A) primary fibroblasts, (B) SV40-fibroblasts, (C) EBV-B cells, (D) iPSC-derived macrophages (iPSC-MF cells), (E) HSV-T cells, (F) monocytes-derived macrophages (MDMs), and (G) T cell blasts. Bars represent the mean and SD. Western blot for indicated protein in total lysate from (H) SV40-fibroblasts, (I) EBV-B cells, or (J) iPSC MF, with and without IFN-g stimulation. Data from 2 to 3 independent experiments are shown. (K) IRF1 staining and intracellular flow cytometry on SV40-fibroblasts with and without IFN-g stimulation. (L) Flow cytometry with intracellular IRF1 staining on SV40-fibroblasts retrotransduced with an empty vector (EV) or WT IRF1 cDNA. The data shown are representative of 2–3 independent experiments. Statistical analysis by Mann-Whitney tests. ns = not significant, p > 0.05; *p < 0.05; **p < 0.01; ****p < 0.0001. See also Figure S2.

Article Snippet: Membranes were probed with antibodies directed against IRF1 (unconjugated, clone D5E4, #8478, Cell Signaling; or unconjugated, polyclonal, #11335-1-AP, Proteintech), IRF8 (unconjugated, goat polyclonal, #sc-6058, Santa Cruz; or unconjugated, clone D20D8, #5628, Cell Signaling), IRF9 (unconjugated, rabbit polyclonal, #sc-496, Santa Cruz; or unconjugated, rabbit polyclonal, #14167-1-AP, ProteinTech), IRF3 (unconjugated, clone D9J5Q, #10949, Cell Signaling), STAT1 (unconjugated, clone 1, #610115, BecktonDickinson), pSTAT1 (unconjugated, clone 4a, #612232, Beckton-Dickinson), STAT2 (unconjugated, clone B-3, #sc-514193, Santa Cruz), MX1 (unconjugated, polyclonal, #13750-1-AP, ProteinTech), ISG15 (HRP-conjugated, clone F-9, sc-166755, Santa-Cruz), DDK-tag (HRP-conjugated, clone M2, #A8592, Sigma-Aldrich), GBP1 (unconjugated, clone 1B1, #sc-53857, Santa-Cruz, #A8592, Sigma-Aldrich), APOL3 (unconjugated, clone EPR8238(2), #ab154869, Abcam), RARRES3 (unconjugated, rabbit polyclonal, #12065-1-AP, ProteinTech), vinculin (unconjugated, clone EPR8185, #ab129002, Abcam; or HRP-conjugated, clone 7F9, #sc73614-HRP, Santa Cruz), and lamin A/C (HRP-conjugated, clone E 1, #sc-376248-HRP, Santa Cruz).

Techniques: Derivative Assay, Western Blot, Staining, Cytometry, Flow Cytometry, Plasmid Preparation, MANN-WHITNEY

Journal: Cell

Article Title: Human IRF1 governs macrophagic IFN-γ immunity to mycobacteria.

doi: 10.1016/j.cell.2022.12.038

Figure Lengend Snippet: Figure 4. Production of IFN-g by the lymphoid cells of IRF1-deficient patients (A) Induction of IFN-g secretion in a whole-blood assay, for controls (CTLs), IL-12Rb1-deficient patients, and patients. Bars represent the mean. (B) Intracellular flow cytometry on IFN-g+ PBMCs after stimulation with IL-12, IL-23, or BCG. Bars represent the mean. Technical duplicates of the same experiment are shown for P1 and P2. (C) UMAP analysis of intracellular T-bet and IFN-g expression by intracellular spectral flow cytometry across various PBMC subsets. Lymphoid subsets based on surface marker expression (upper panel), and their levels of IFN-g and T-bet expression (lower panel). (D) Cytokine levels in the supernatant of naive CD4+ T cells in polarizing conditions. Bars represent the median. See also Figure S4.

Article Snippet: Membranes were probed with antibodies directed against IRF1 (unconjugated, clone D5E4, #8478, Cell Signaling; or unconjugated, polyclonal, #11335-1-AP, Proteintech), IRF8 (unconjugated, goat polyclonal, #sc-6058, Santa Cruz; or unconjugated, clone D20D8, #5628, Cell Signaling), IRF9 (unconjugated, rabbit polyclonal, #sc-496, Santa Cruz; or unconjugated, rabbit polyclonal, #14167-1-AP, ProteinTech), IRF3 (unconjugated, clone D9J5Q, #10949, Cell Signaling), STAT1 (unconjugated, clone 1, #610115, BecktonDickinson), pSTAT1 (unconjugated, clone 4a, #612232, Beckton-Dickinson), STAT2 (unconjugated, clone B-3, #sc-514193, Santa Cruz), MX1 (unconjugated, polyclonal, #13750-1-AP, ProteinTech), ISG15 (HRP-conjugated, clone F-9, sc-166755, Santa-Cruz), DDK-tag (HRP-conjugated, clone M2, #A8592, Sigma-Aldrich), GBP1 (unconjugated, clone 1B1, #sc-53857, Santa-Cruz, #A8592, Sigma-Aldrich), APOL3 (unconjugated, clone EPR8238(2), #ab154869, Abcam), RARRES3 (unconjugated, rabbit polyclonal, #12065-1-AP, ProteinTech), vinculin (unconjugated, clone EPR8185, #ab129002, Abcam; or HRP-conjugated, clone 7F9, #sc73614-HRP, Santa Cruz), and lamin A/C (HRP-conjugated, clone E 1, #sc-376248-HRP, Santa Cruz).

Techniques: Whole Blood Assay, Cytometry, Expressing, Marker

Journal: Cell

Article Title: Human IRF1 governs macrophagic IFN-γ immunity to mycobacteria.

doi: 10.1016/j.cell.2022.12.038

Figure Lengend Snippet: Figure 5. Response to IFN-g of IRF1-deficient fibroblasts (A) HOMER de novo motif analysis of the genes differentially expressed in the primary fibroblasts after IFN-g stimulation. (B) qRT-PCR for GBP4 (normalized against GUSB) in SV40-fibroblasts with or without retrotransduction with EV or WT IRF1 cDNA and with or without stimulation IFN-g. Data from 2 to 6 independent experiments are shown. Bars represent the mean and SD. (C) Immunoblots in SV40-fibroblasts with and without stimulation with IFN-g. (D) Mass spectrometry on lysates of primary fibroblasts with and without IFN-g stimulation. On the right, heatmaps for proteins (1) positively induced after stimulation with a log2FC > 1 over the mean in the non-stimulated state for controls (2) and a log2FC < 0.5 over the mean in the non-stimulated state for patients. On the left, 10th–90th percentiles for all proteins positively induced after stimulation with a log2FC > 1 over the mean value in the non-stimulated state for controls. See also Figure S5 and Table S7.

Article Snippet: Membranes were probed with antibodies directed against IRF1 (unconjugated, clone D5E4, #8478, Cell Signaling; or unconjugated, polyclonal, #11335-1-AP, Proteintech), IRF8 (unconjugated, goat polyclonal, #sc-6058, Santa Cruz; or unconjugated, clone D20D8, #5628, Cell Signaling), IRF9 (unconjugated, rabbit polyclonal, #sc-496, Santa Cruz; or unconjugated, rabbit polyclonal, #14167-1-AP, ProteinTech), IRF3 (unconjugated, clone D9J5Q, #10949, Cell Signaling), STAT1 (unconjugated, clone 1, #610115, BecktonDickinson), pSTAT1 (unconjugated, clone 4a, #612232, Beckton-Dickinson), STAT2 (unconjugated, clone B-3, #sc-514193, Santa Cruz), MX1 (unconjugated, polyclonal, #13750-1-AP, ProteinTech), ISG15 (HRP-conjugated, clone F-9, sc-166755, Santa-Cruz), DDK-tag (HRP-conjugated, clone M2, #A8592, Sigma-Aldrich), GBP1 (unconjugated, clone 1B1, #sc-53857, Santa-Cruz, #A8592, Sigma-Aldrich), APOL3 (unconjugated, clone EPR8238(2), #ab154869, Abcam), RARRES3 (unconjugated, rabbit polyclonal, #12065-1-AP, ProteinTech), vinculin (unconjugated, clone EPR8185, #ab129002, Abcam; or HRP-conjugated, clone 7F9, #sc73614-HRP, Santa Cruz), and lamin A/C (HRP-conjugated, clone E 1, #sc-376248-HRP, Santa Cruz).

Techniques: Quantitative RT-PCR, Western Blot, Mass Spectrometry