Journal: Stem cell research & therapy

Article Title: Intracellular osteopontin potentiates the immunosuppressive activity of mesenchymal stromal cells.

doi: 10.1186/s13287-024-03979-8

Figure Lengend Snippet: Fig. 5 iOPN promoted STAT1-mediated immunosuppression in MSCs. a–d Vector-MSCs and iOPN-MSCs or WT-MSCs and OPN−/−-MSCs were treated with or without TNF-α plus IFN-γ (10 ng/mL) for the indicated times. Cells were harvested, and OPN, NF-κB p65, STAT1, IKBα, phosphorylation of NF-κB p65, phosphorylation of STAT1 at Tyr701 and phosphorylation of IKBα were analyzed by immunoblotting analysis. Full-length blots are presented in Additional file 1: Fig. 5a-d. e–g Vector-MSCs and iOPN-MSCs were stimulated with TNF-α plus IFN-γ (10 ng/mL) for 12 h. The STAT1 inhibitor fludarabine (Flu, 2 μM) was then added to the culture medium of Vector-MSCs and iOPN-MSCs. The expression of iNOS was determined by quantitative real-time PCR (e), immunoblotting analysis (f) and flow cytometry (g). Full-length blots are presented in Additional file 1: Fig. 5f. h Vector-MSCs and iOPN-MSCs were pretreated with DMSO or fludarabine (Flu, 2 μM) for 6 h and then irradiated and cocultured with CFSE-labeled splenocytes activated by anti-CD3/CD28 antibodies for 3 days at a ratio of 1:20. CD4+ T cells and CD8+ T cells were stained for proliferation analysis by flow cytometry at the end of coculture, and the percentages of proliferating T cells are shown. The results are representative of three to six independent experiments. Values are shown as the mean ± SEM and statistical significance is indicated as *P < 0.05, **P < 0.01 and ***P < 0.001

Article Snippet: Antibodies against GAPDH (CAT# 2118, RRID: AB_561053), NF-κB p65 (CAT# 8242, RRID:AB_10859369), phospho-NF-κB p65 (CAT# 3033, RRID:AB_331284), STAT1 (CAT# 14994, RRID:AB_2737027), phospho-STAT1 (Tyr701) (CAT# 7649, RRID:AB_10950970), IκBα (CAT# 4814, RRID:AB_390781), phospho-IκBα (CAT# 2118) and ubiquitin (CAT# 3936, RRID: RRID:AB_331292) were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA).

Techniques: Plasmid Preparation, Phospho-proteomics, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Irradiation, Labeling, Staining

Journal:

Article Title: Adjuvant Activities of Novel Cytokines, Interleukin-23 (IL-23) and IL-27, for Induction of Hepatitis C Virus-Specific Cytotoxic T Lymphocytes in HLA-A*0201 Transgenic Mice

doi: 10.1128/JVI.78.17.9093-9104.2004

Figure Lengend Snippet: Expression and activity of the scIL-12, scIL-23, and scIL-27 fusion proteins. (A) p3XFLAG-IL-12 (IL-12), p3XFLAG-IL-23 (IL-23), p3XFLAG-IL-27 (IL-27), and p3XFLAG (vector) were transiently transfected into 293T cells. Proteins secreted in the supernatants were immunoprecipitated with the anti-FLAG antibody. The immunoprecipitated proteins were separated on by SDS-12% polyacrylamide gel electrophoresis and subjected to Western blotting analysis with the anti-FLAG antibody. The positions of protein molecular mass markers (in kilodaltons) are shown in the figure, and the three arrows indicate the bands of the scIL-12, scIL-23, and scIL-27 fusion proteins. (B to D) STAT tyrosine phosphorylation assay. 293T cells were transiently transfected with either pME18S-IL-12Rβ1 plus pME18S-IL-12Rβ1 (B), pME18S-IL-12Rβ1 plus p3XFLAG-IL-23R (C), or p3XFLAG-WSX-1/TCCR plus p3XFLAG-gp130 (D). After 36 h, the cells were stimulated for 45 min with the culture supernatant containing either scIL-12 (IL-12) (B), scIL-23 (IL-23) (C), or scIL-27 (IL-27) (D) at final concentrations of 0, 0.5, 5, and 50%. As negative controls, the cells were stimulated for 45 min with the culture supernatant containing either scIL-23/scIL-27 (B), scIL-12/scIL-27 (C), or scIL-12/scIL-23 (D) at a final concentration of 50%. The cells were then subjected to Western blotting with anti-STAT1 (Total STAT1) (D), anti-STAT4 (Total STAT4) (B and C), anti-pY-STAT1 (pY-STAT1) (D), and anti-pY-STAT4 (pY-STAT4) (B and C) antibodies.

Article Snippet: The cells were then subjected to Western blotting with anti-STAT1, anti-STAT4 (Santa Cruz Biotechnology, Santa Cruz, Calif.), anti-pY-STAT1 (Cell Signaling Technology, Inc., Beverly, Mass.), or anti-pY-STAT4 (Zymed Laboratories, Inc., South San Francisco, Calif.) antibody.

Techniques: Expressing, Activity Assay, Plasmid Preparation, Transfection, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Western Blot, Phosphorylation Assay, Concentration Assay

Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: bioRxiv

Article Title: Bclaf1 critically regulates the type I interferon response and is degraded by alphaherpesvirus US3

doi: 10.1101/392555

Figure Lengend Snippet: (A) IB analysis of phosphorylated(P)-STAT1, P-STAT2, STAT1, STAT2 and Bclaf1 in HeLa WT and HeLa Bclaf1-KO cells treated with human IFNα (500U/mL) for the indicated time. Data were quantified and shown as the ratio of P-STAT1 to STAT1 and P-STAT2 to STAT2. (B) IB analysis of P-STAT1, P-STAT2, STAT1, STAT2 and Bclaf1 in HEp-2 cells transfected with si-control or si-Bclaf1 followed by PBS or human IFNα (500U/mL) treatment for the indicated time. Data were quantified and shown as the ratio of P-STAT1 to STAT1 and P-STAT2 to STAT2. (C) IB analysis of P-STAT1, P-STAT2, STAT1, STAT2 and Bclaf1 in cytoplasmic and nuclear extracts of HEp-2 cells transfected with si-control or si-Bclaf1 followed by PBS or human IFNα (500U/mL) treatment for the indicated time. α-Tubulin and Histone H3 were used as the cytoplasmic and nuclear controls, respectively.

Article Snippet: The following antibodies were used for immunoblot analysis: anti-Bclaf1 (1:500, sc-135845, Santa Cruz), anti-Flag (1:2000, F1804, Sigma), anti-α-Tubulin (1:8000, PM054, MBL), anti-HA (1:1000, sc-805, Santa Cruz), anti-GFP (1:1000, sc-9996, Santa Cruz), anti-ISG15 (1:500, sc-166755, Santa Cruz), anti-PKR (1:1000, 12297, Cell Signaling Technology), anti-STAT1 (1:1000, 14995, Cell Signaling Technology), anti-STAT2 (1:1000, 72604, Cell Signaling Technology), anti-P-STAT1 (Tyr701) (1:1000, 9167, Cell Signaling Technology), anti-P-STAT2 (Tyr690) (1:1000, 88410, Cell Signaling Technology), anti-IRF9 (1:1000, 76684, Cell Signaling Technology), anti-JAK1 (1:500, 3344, Cell Signaling Technology), anti-TYK2 (1:1000, 14193, Cell Signaling Technology), anti-Histone H3 (1:2000, 17168-1-AP, Proteintech), and anti-caspase3 p17 (1:1000, sc-166589, Santa Cruz).

Techniques: Transfection

Journal: bioRxiv

Article Title: Bclaf1 critically regulates the type I interferon response and is degraded by alphaherpesvirus US3

doi: 10.1101/392555

Figure Lengend Snippet: (A) ChIP analysis of STAT1/STAT2 DNA-binding in promoters of IFIT1 and IFIT2 in HeLa WT and HeLa Bclaf1-KO cells simulated with PBS or human IFNα (500U/mL) for 1h. (B) IB analysis of Bio-ISRE pull-down STAT1, STAT2, IRF9 and Bclaf1. Unlabeled ISRE was used for control. (C) IB analysis of ISRE-binding Bclaf1. Unlabeled ISRE and Bio-GFP were used for control. (D) ChIP analysis of Bclaf1 DNA-binding in promoters of ISG15, IFIT1 and IFIT2 in HeLa cells simulated with PBS or human IFNα (500U/mL) for 1h. An amplicon located in IFIT1 exon2 was also tested for control. (E) IB analysis of WT or mutated (1–3) Bio-ISRE pull-down Bclaf1.

Article Snippet: The following antibodies were used for immunoblot analysis: anti-Bclaf1 (1:500, sc-135845, Santa Cruz), anti-Flag (1:2000, F1804, Sigma), anti-α-Tubulin (1:8000, PM054, MBL), anti-HA (1:1000, sc-805, Santa Cruz), anti-GFP (1:1000, sc-9996, Santa Cruz), anti-ISG15 (1:500, sc-166755, Santa Cruz), anti-PKR (1:1000, 12297, Cell Signaling Technology), anti-STAT1 (1:1000, 14995, Cell Signaling Technology), anti-STAT2 (1:1000, 72604, Cell Signaling Technology), anti-P-STAT1 (Tyr701) (1:1000, 9167, Cell Signaling Technology), anti-P-STAT2 (Tyr690) (1:1000, 88410, Cell Signaling Technology), anti-IRF9 (1:1000, 76684, Cell Signaling Technology), anti-JAK1 (1:500, 3344, Cell Signaling Technology), anti-TYK2 (1:1000, 14193, Cell Signaling Technology), anti-Histone H3 (1:2000, 17168-1-AP, Proteintech), and anti-caspase3 p17 (1:1000, sc-166589, Santa Cruz).

Techniques: Binding Assay, Amplification

Journal: bioRxiv

Article Title: Bclaf1 critically regulates the type I interferon response and is degraded by alphaherpesvirus US3

doi: 10.1101/392555

Figure Lengend Snippet: (A) IB analysis of STAT1, STAT2, P-STAT1, P-STAT2 and Flag-Bclaf1 in cytoplasmic or nuclear immunoprecipitates of a HEp-2-Flag-Bclaf1 cell line treated with PBS or human IFNα (500U/mL) for 2h. IgG was used for control immunoprecipitation. (B) IB analysis of IRF9 and Flag-Bclaf1 in cytoplasmic or nuclear immunoprecipitates of a HEp-2-Flag-Bclaf1 cell line treated with PBS or human IFNα (500U/mL) for 4h. (C) IB analysis of immunoprecipitates of HEK293T cells co-transfected with Flag-tagged Bclaf1 truncations and Ha-tagged STAT1/STAT2IRF9 expression plasmids. (D) qRT-PCR analysis of IFIT1 mRNA levels in HEp-2 cells transfected with Flag-tagged EV, full-length Bclaf1 or its truncations expression plasmids followed by PBS or human IFNα (500U/mL) treatment for 3h. IB analyzed the expression of Bclaf1. Data are shown as mean ± SD of three independent experiments. Statistical analysis was performed by the one-way ANOVA test. ***p<0.001

Article Snippet: The following antibodies were used for immunoblot analysis: anti-Bclaf1 (1:500, sc-135845, Santa Cruz), anti-Flag (1:2000, F1804, Sigma), anti-α-Tubulin (1:8000, PM054, MBL), anti-HA (1:1000, sc-805, Santa Cruz), anti-GFP (1:1000, sc-9996, Santa Cruz), anti-ISG15 (1:500, sc-166755, Santa Cruz), anti-PKR (1:1000, 12297, Cell Signaling Technology), anti-STAT1 (1:1000, 14995, Cell Signaling Technology), anti-STAT2 (1:1000, 72604, Cell Signaling Technology), anti-P-STAT1 (Tyr701) (1:1000, 9167, Cell Signaling Technology), anti-P-STAT2 (Tyr690) (1:1000, 88410, Cell Signaling Technology), anti-IRF9 (1:1000, 76684, Cell Signaling Technology), anti-JAK1 (1:500, 3344, Cell Signaling Technology), anti-TYK2 (1:1000, 14193, Cell Signaling Technology), anti-Histone H3 (1:2000, 17168-1-AP, Proteintech), and anti-caspase3 p17 (1:1000, sc-166589, Santa Cruz).

Techniques: Immunoprecipitation, Transfection, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Bclaf1 critically regulates the type I interferon response and is degraded by alphaherpesvirus US3

doi: 10.1101/392555

Figure Lengend Snippet: (A) GST pulldown analysis of the interaction between His-STAT2 and GST-Bclaf1 F2. (B) IB analysis of immunoprecipitates of HEK293T cells co-transfected with Flag-tagged Bclaf1, Ha-tagged STAT1 or STAT2/IRF9 expression plasmids. (C) IB analysis of immunoprecipitates of HEK293T cells co-transfected with Flag-tagged Bclaf1, Ha-tagged IRF9 or STAT2/STAT1 expression plasmids. (D) IB analysis of STAT1, STAT2, IRF9 and Flag-Bclaf1 in nuclear immunoprecipitates of a HEp-2-Flag-Bclaf1 cell line transfected with si-control or si-STAT2 followed by PBS or human IFNα (500U/mL) treatment for 3h. (E) IB analysis of Bio-ISRE pull-down STAT1, STAT2, IRF9 and Bclaf1.

Article Snippet: The following antibodies were used for immunoblot analysis: anti-Bclaf1 (1:500, sc-135845, Santa Cruz), anti-Flag (1:2000, F1804, Sigma), anti-α-Tubulin (1:8000, PM054, MBL), anti-HA (1:1000, sc-805, Santa Cruz), anti-GFP (1:1000, sc-9996, Santa Cruz), anti-ISG15 (1:500, sc-166755, Santa Cruz), anti-PKR (1:1000, 12297, Cell Signaling Technology), anti-STAT1 (1:1000, 14995, Cell Signaling Technology), anti-STAT2 (1:1000, 72604, Cell Signaling Technology), anti-P-STAT1 (Tyr701) (1:1000, 9167, Cell Signaling Technology), anti-P-STAT2 (Tyr690) (1:1000, 88410, Cell Signaling Technology), anti-IRF9 (1:1000, 76684, Cell Signaling Technology), anti-JAK1 (1:500, 3344, Cell Signaling Technology), anti-TYK2 (1:1000, 14193, Cell Signaling Technology), anti-Histone H3 (1:2000, 17168-1-AP, Proteintech), and anti-caspase3 p17 (1:1000, sc-166589, Santa Cruz).

Techniques: Transfection, Expressing

Journal: bioRxiv

Article Title: Bclaf1 critically regulates the type I interferon response and is degraded by alphaherpesvirus US3

doi: 10.1101/392555

Figure Lengend Snippet: Upon IFN-I binding with receptors, Bclaf1 facilitates the phosphorylation of STAT1/STAT2 in an indirect manner. Phosphorylated STAT1 and STAT2 associate with IRF9 to format a complex called ISGF3 and translocate to the nucleus. Bclaf1 is acting as a mediator attracting ISGF3 to ISGs promoters for efficient transcription. On the one hand, Bclaf1 interacts with STAT2 directly to associate with ISGF3. On the other hand, Bclaf1 binds with ISRE. During PRV or HSV-1 infection, US3 is dispatched to degrade Bclaf1 to inhibit IFN signaling.

Article Snippet: The following antibodies were used for immunoblot analysis: anti-Bclaf1 (1:500, sc-135845, Santa Cruz), anti-Flag (1:2000, F1804, Sigma), anti-α-Tubulin (1:8000, PM054, MBL), anti-HA (1:1000, sc-805, Santa Cruz), anti-GFP (1:1000, sc-9996, Santa Cruz), anti-ISG15 (1:500, sc-166755, Santa Cruz), anti-PKR (1:1000, 12297, Cell Signaling Technology), anti-STAT1 (1:1000, 14995, Cell Signaling Technology), anti-STAT2 (1:1000, 72604, Cell Signaling Technology), anti-P-STAT1 (Tyr701) (1:1000, 9167, Cell Signaling Technology), anti-P-STAT2 (Tyr690) (1:1000, 88410, Cell Signaling Technology), anti-IRF9 (1:1000, 76684, Cell Signaling Technology), anti-JAK1 (1:500, 3344, Cell Signaling Technology), anti-TYK2 (1:1000, 14193, Cell Signaling Technology), anti-Histone H3 (1:2000, 17168-1-AP, Proteintech), and anti-caspase3 p17 (1:1000, sc-166589, Santa Cruz).

Techniques: Binding Assay, Infection

Journal: PLoS ONE

Article Title: Activation of Protein Tyrosine Phosphatase Non-Receptor Type 2 by Spermidine Exerts Anti-Inflammatory Effects in Human THP-1 Monocytes and in a Mouse Model of Acute Colitis

doi: 10.1371/journal.pone.0073703

Figure Lengend Snippet: Representative Western blots and densitometry (n = 3) show phosphorylation status of ( a and b ) STAT1, ( c ) STAT3 and ( d ) p38 MAPK in THP-1 cells treated with IFN-γ (1000 U/ml) and/or spermidine (100 µM) for ( a , c , and d ) 30 min or ( b ) 36 h. Blots were probed for β-actin to show equal protein loading. Significant differences compared to untreated cells (* = p<0.05, ** = p<0.01, *** = p<0.001) or compared to cells treated with IFN-γ only ( ## = p<0.01, ### = p<0.001) are indicated.

Article Snippet: Rabbit anti-phospho-STAT1 (Tyr701), rabbit anti-STAT1, rabbit anti-phospho-STAT3 (Tyr705), rabbit anti-STAT3, mouse anti-phospho-p38 (Thr180/Tyr182) and rabbit anti-p38 antibodies were obtained from Cell Signaling Technologies (Danvers, MA).

Techniques: Western Blot, Phospho-proteomics