phosphorylated stat1  (R&D Systems)

Journal: Signal Transduction and Targeted Therapy

Article Title: Bioengineered iPSC-derived human macrophages with increased angiotensin-converting enzyme (ACE) expression suppress solid tumor growth

doi: 10.1038/s41392-026-02650-3

Figure Lengend Snippet: Determination of M1 vs. M2 polarization in iMac. a Pro-inflammatory M1 cytokines IL-1β, TNFα, IL-6, and IL-8 measured by ELISA. b Anti-inflammatory M2 cytokines IL-4, IL-10, IL-13, CCL5, CCL17, and CCL20 measured by ELISA. For a , b iMac were cultured in 6-well plates (3 × 10 6 cells/well). To challenge with tumor factors, iMac were co-cultured with 50% fresh supernatant from SK-MEL-28 cells every 24 h for 3 days. Supernatants were collected during the final 24 h for cytokine measurement. c Activation (phosphorylation) of NF-kB p65, STAT1, STAT3, and STAT6 in iMac was determined using Western blotting after conditioning with melanoma (SK-MEL-28) supernatant for 72 h. Protein band intensity was quantified using Odyssey version 3.0 software. After adjusting for loading using the β-actin band, data are presented as fold change to the iMac (Dox-) group, which is set as 1. For a – c , one-way ANOVA with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons. Data are presented as means ± SD. Each experiment was performed three times, with triplicate samples each time. * p < 0.05, *** p < 0.001, and **** p < 0.0001

Article Snippet: The polyvinylidene difluoride (PVDF) membranes were incubated with specific primary antibodies against ACE (R&D Systems, MAB9291, 1:1,000), GAPDH (Sigma-Aldrich, SAB5600208, 1:2,000), β-actin (Sigma-Aldrich, A3854; 1:1000), phosphorylated NF-kB p65 (Novus, NB100-82086, 1:500), phosphorylated STAT1 (R&D Systems, AF2894, 1 μg/ml), phosphorylated STAT3 (Novus, NBP2-24463, 0.5 μg/ml), or phosphorylated STAT6 (Millipore, 06-937, 1:1000).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Activation Assay, Phospho-proteomics, Western Blot, Software

Journal: Frontiers in Immunology

Article Title: IFNγ-induced antigen loss in chimeric antigen receptor-T cell therapy

doi: 10.3389/fimmu.2026.1772472

Figure Lengend Snippet: CART-induced GUCY2C loss is mediated by the IFNγ-JAK-STAT1 signaling axis. (A) LS174T cells and orthogonal approaches of cytokine screening (B) and confirmation (C) , IFNγ neutralizing antibody (D) , pharmacologic JAK1/2 blockade with ruxolitinib (E) , and JAK1/2 genetic knockout (F) were used to define the role of the IFNγ-JAK-STAT signaling axis in GUCY2C loss. (B) LS174T cells were treated with GM-CSF (20 ng/mL), TNFα (1 ng/mL), IL-8 (2 ng/mL), MIP-1α (2 ng/mL), MIP-1β (2 ng/mL), or IFNγ (15 ng/mL) for 48 hours, and GUCY2C protein levels were quantified. (C) LS174T cells were treated with 150 ng/mL IFNγ for 48 hours, and pSTAT1 and GUCY2C protein levels were quantified. (D–F) LS174T cells were treated with conditioned media (CM) for 48 hours from control or anti-CD3/CD2/CD28 bead-activated T cells, and pSTAT1 and GUCY2C protein levels were quantified; (D) 13 μg/mL anti-IFNγ neutralizing antibody was included in some conditions; (E) 2.5 μM ruxolitinib was included in some conditions; (F) LS174T cell pools previously treated with control CRISPR/Cas9 or JAK1 + 2 CRISPR/Cas9 were used. Each data point in (B–F) represents average of biological replicates from separate experiments (N = 3–4 experiments). In (C–F) , pSTAT, GUCY2C, and housekeeping control were examined on the same blot. The housekeeping protein is shown only below GUCY2C. A paired t-test was used to compare the two conditions in (C) ; in (B) and (D–F) , one-way ANOVA was used to compare each condition to the control treatment, and additional comparisons are indicated by bars; ns = p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Figure schematics were generated using BioRender.com .

Article Snippet: Membranes were probed using an anti-human GUCY2C antibody (37517, Cell Signaling Technology), anti-human GAPDH (2118S, Cell Signaling Technology), anti-human STAT1 (14994T, Cell Signaling Technology), anti-human phospho-STAT1 (9167S, Cell Signaling Technology), anti-human CHOP (2895S, Cell Signaling Technology), anti-human β-actin (2128S, Cell Signaling Technology), anti-human EpCAM (2929S, Cell Signaling Technology) anti-human CDH17 (88594T, Cell Signaling Technology), and anti-human HER2 (2165T, Cell Signaling Technology).

Techniques: Knock-Out, Control, CRISPR, Generated

Journal: bioRxiv

Article Title: IFN-I exacerbates the inflammatory response of epithelial cells to Chlamydia trachomatis infection by enhancing TLR3 expression

doi: 10.64898/2025.12.08.692940

Figure Lengend Snippet: PI3K/AKT-mTOR but not STAT1 signaling pathways downstream of IFN-I are implicated in the synergy between IFN-I and C. trachomatis . (A) HeLa cells were incubated with IFNβ and/or C. trachomatis for 24 h followed by the detection of activation and expression of STAT1 via immunoblot. The results are representatives of three independent experiments. (B) siRNA against STAT1 or irrelevant oligonucleotides were transfected into HeLa cells for 24 h prior to incubation with IFNβ alone (left graph) or with IFNβ and C. trachomatis (right graph). The transcriptional level of STAT1 (left graph) was measured by real-time RT-qPCR and normalized to actin transcript following the 2 -ΔΔCt method. The data are presented as relative mRNA levels compared to untreated cells and shown as the mean±SE with individual values of three experiments. P-value of Student’s unpaired t-test is shown (* for p < 0.05). Intracellular IL6 protein (right panel) was determined by flow cytometry using anti-human IL6-PC7 antibody. The results of four independent experiments are shown. C-D) HeLa cells were pre-incubated with wortmannin (5 μM) (C) or SB203580 (10 μM) (D) for 1 h. Cells were then treated with IFNβ and/or C. trachomatis for 24 h in the presence of these inhibitors prior to brefeldin A addition for 6 h. Intracellular IL6 expression was analyzed by flow cytometry using anti-human IL6-PC7 antibody. The histograms are the representatives of two (C) or four experiments (D), respectively. The results of the four independent experiments are shown as a violin plot in (D), with p-value of a Student’s unpaired t-test (* for p< 0.05). (E) Same as in D using mTOR inhibitors, rapamycin and torin1 (1 μM). The results of five independent experiments and p-value of a Student’s unpaired t-test are shown (* for p< 0.05). (F) HeLa cells were treated with pharmacological inhibitors (upper panels) or transfected with siRNA (lower panel) as described above, followed by IFNβ and/or C. trachomatis treatment for 24 h. TLR3 transcripts was measured by rea-time RT-qPCR as above. Data from three independent experiments and p-values of Student’s unpaired t-test are shown (* p< 0.05, ** p<0.01, *** p<0.001 and **** p<0.0001). (G) The cells were incubated with SB203580 (10 μM) for 1 h before addition of IFNβ for 30 min. Phosphorylation of mTOR, AKT and p38 was detected by immunoblot. The results are representatives of three independent experiments.

Article Snippet: Primary antibodies used were rabbit anti-human STAT1 (#9172), Erk (#4695), p38 (#9212), AKT (#9272), rabbit anti-human phosphorylated STAT1 (#9167), Erk (#4370), p38 (#9215), AKT (#9275) and mTOR (#2971), which were purchased from Cell Signaling, rabbit antibodies against the heat shock protein 60 of Chlamydia (obtained by the lab), and mouse anti-human β-actin (Sigma, #A5441).

Techniques: Protein-Protein interactions, Incubation, Activation Assay, Expressing, Western Blot, Transfection, Quantitative RT-PCR, Flow Cytometry, Phospho-proteomics

Journal: bioRxiv

Article Title: IFN-I exacerbates the inflammatory response of epithelial cells to Chlamydia trachomatis infection by enhancing TLR3 expression

doi: 10.64898/2025.12.08.692940

Figure Lengend Snippet: PI3K/AKT-mTOR but not STAT1 signaling pathways downstream of IFN-I are implicated in the synergy between IFN-I and C. trachomatis . (A) HeLa cells were incubated with IFNβ and/or C. trachomatis for 24 h followed by the detection of activation and expression of STAT1 via immunoblot. The results are representatives of three independent experiments. (B) siRNA against STAT1 or irrelevant oligonucleotides were transfected into HeLa cells for 24 h prior to incubation with IFNβ alone (left graph) or with IFNβ and C. trachomatis (right graph). The transcriptional level of STAT1 (left graph) was measured by real-time RT-qPCR and normalized to actin transcript following the 2 -ΔΔCt method. The data are presented as relative mRNA levels compared to untreated cells and shown as the mean±SE with individual values of three experiments. P-value of Student’s unpaired t-test is shown (* for p < 0.05). Intracellular IL6 protein (right panel) was determined by flow cytometry using anti-human IL6-PC7 antibody. The results of four independent experiments are shown. C-D) HeLa cells were pre-incubated with wortmannin (5 μM) (C) or SB203580 (10 μM) (D) for 1 h. Cells were then treated with IFNβ and/or C. trachomatis for 24 h in the presence of these inhibitors prior to brefeldin A addition for 6 h. Intracellular IL6 expression was analyzed by flow cytometry using anti-human IL6-PC7 antibody. The histograms are the representatives of two (C) or four experiments (D), respectively. The results of the four independent experiments are shown as a violin plot in (D), with p-value of a Student’s unpaired t-test (* for p< 0.05). (E) Same as in D using mTOR inhibitors, rapamycin and torin1 (1 μM). The results of five independent experiments and p-value of a Student’s unpaired t-test are shown (* for p< 0.05). (F) HeLa cells were treated with pharmacological inhibitors (upper panels) or transfected with siRNA (lower panel) as described above, followed by IFNβ and/or C. trachomatis treatment for 24 h. TLR3 transcripts was measured by rea-time RT-qPCR as above. Data from three independent experiments and p-values of Student’s unpaired t-test are shown (* p< 0.05, ** p<0.01, *** p<0.001 and **** p<0.0001). (G) The cells were incubated with SB203580 (10 μM) for 1 h before addition of IFNβ for 30 min. Phosphorylation of mTOR, AKT and p38 was detected by immunoblot. The results are representatives of three independent experiments.

Article Snippet: Primary antibodies used were rabbit anti-human STAT1 (#9172), Erk (#4695), p38 (#9212), AKT (#9272), rabbit anti-human phosphorylated STAT1 (#9167), Erk (#4370), p38 (#9215), AKT (#9275) and mTOR (#2971), which were purchased from Cell Signaling, rabbit antibodies against the heat shock protein 60 of Chlamydia (obtained by the lab), and mouse anti-human β-actin (Sigma, #A5441).

Techniques: Protein-Protein interactions, Incubation, Activation Assay, Expressing, Western Blot, Transfection, Quantitative RT-PCR, Flow Cytometry, Phospho-proteomics

Journal: Cell Reports Medicine

Article Title: Therapeutic stress triggers tumor STAT1 acetylation to disarm immunotherapy

doi: 10.1016/j.xcrm.2025.102448

Figure Lengend Snippet: STAT1 Lys637 acetylation correlates with poor response to ICB therapy (A) Schematic of a syngeneic murine oral cancer model receiving anti-PD1 injection. The murine oral squamous cell carcinoma cell line MOC-L2-1 with Stat1 knockdown (shmStat1) and reconstituted with human STAT1 (hSTAT1(WT) or hSTAT1(K637Q) or hSTAT1(K637R)) was inoculated subcutaneously into C57BL/6J mice until tumors reached a volume of 100 mm 3 . Five doses of anti-PD1 or isotype IgG were administered to tumor-bearing mice. n = 9–10 per group. (B) Tumor growth inhibition (TGI, %) calculated as the relative change in tumor volume between day 0 and day 38 in different groups. Data presented as mean ± SEM. ∗∗∗ p < 0.001. (C) Tumor weight in the mouse experiments. Data presented as mean ± SEM. ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, not significant. (D) Kaplan-Meier overall survival curves for HNSCC patients ( n = 63) stratified by H-score cutoff of 166 with median follow-up of 8.0 months (range 0.5–45.1). (E) Kaplan-Meier overall survival curves for GC patients ( n = 46) stratified by H-score cutoff of 166 with median follow-up of 9.7 months (range 1.97–60.2). (F) Kaplan-Meier overall survival curves for hepatocellular carcinoma (HCC) patients ( n = 39) stratified by H-score cutoff of 166 with median follow-up of 15.5 months (range 3.1–81.1). (G) Comparison of STAT1 Lys637 acetylation levels between HNSCC responders ( n = 27) and non-responders ( n = 36) to ICB treatment. Statistical analyses were performed using an unpaired Student’s t test. ∗∗ p < 0.01. (H) Comparison of STAT1 K637 acetylation levels between HNSCC disease control patients ( n = 42) and those with progressive disease ( n = 21) following ICB therapy. Statistical analyses were performed using an unpaired Student’s t test. ∗∗∗ p < 0.001. See also , , , , and .

Article Snippet: To generate the pLV-STAT1(Y701F), pLV-STAT1(S727A), pLV-STAT1(Y701F/S727A), pLV-STAT1(K637R), and pLV-STAT1(K637Q) plasmids, human STAT1 cDNA was amplified from p-LV-STAT1 (RRID: Addgene_71454).

Techniques: Injection, Knockdown, Inhibition, Comparison, Control

Journal: Cell Reports Medicine

Article Title: Therapeutic stress triggers tumor STAT1 acetylation to disarm immunotherapy

doi: 10.1016/j.xcrm.2025.102448

Figure Lengend Snippet: Impaired IFN-γ response and reduced STAT1 protein in cetuximab-resistant HNSCC (A) RT-qPCR of IFN-γ response-associated gene expression, including tumor immunology-related genes (upper), antiviral-related genes (middle), and antigen processing and presentation genes (lower) in OECM-1-WT and OECM-1-Ctx R cells. n = 3 (each with two technical replicates). The cells were then treated with IFN-γ (100 ng/mL) for 24 h. Data are presented as mean ± SD. Statistical significance was determined using unpaired Student’s t test. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, not significant. (B) RT-qPCR of IFN-γ signaling-associated components in OECM-1-WT/CAL-27-WT and OECM-1-Ctx R /CAL-27-Ctx R cells. n = 3 (each with two technical replicates). Data are presented as mean ± SD. Statistical significance was determined using unpaired Student’s t test. ∗ p < 0.05; ∗∗∗ p < 0.001; ns, not significant. (C) Representative western blot analysis of IFN-γ signaling-related proteins in OECM-1-WT/OECM-1-Ctx R and CAL-27-WT/CAL-27-Ctx R cells. GAPDH was the loading control. The experiments were performed in triplicate. (D) Heatmap showing STAT1 and STAT3 protein levels from mass spectrometry in OECM-1 cells after cetuximab treatment (500 μg/mL) across different passages. (E) Representative western blot analysis of STAT family in OECM-1-WT/OECM-1-Ctx R and CAL-27-WT/CAL-27-Ctx R cells. α-tubulin was used as the loading control. The experiments were performed in triplicate. (F) Representative western blot analysis of STAT1 protein levels in OECM-1 cells across different passages of cetuximab treatment (500 μg/mL). GAPDH was used as a loading control. The experiments were performed in triplicate. (G) Left: Schematic of the mouse experiment. Murine oral squamous cell carcinoma MOC-L2-1 cells were transduced with a doxycycline (DOX)-inducible vector for the knockdown of Stat1 (shStat1) or a scramble control (shScr) and were then inoculated subcutaneously into C57BL/6 mice. Doxycycline administration was initiated on day 18 to induce vector expression in syngeneic tumors. Mice were treated with either isotype IgG or murine anti-PD1 (200 μg) for 8 doses at specified time points. Right: Tumor growth curves are presented as mean ± SD. n = 3 per group. Statistical significance was determined using unpaired Student’s t test. ∗∗ p < 0.01. (H) Upper: Histogram showing weights of shScr and shStat1 MOC-L2-1 tumors. n = 3 per group. Statistical significance was determined using unpaired Student’s t test. ∗ p < 0.05. Lower: Representative images of tumors. See also .

Article Snippet: To generate the pLV-STAT1(Y701F), pLV-STAT1(S727A), pLV-STAT1(Y701F/S727A), pLV-STAT1(K637R), and pLV-STAT1(K637Q) plasmids, human STAT1 cDNA was amplified from p-LV-STAT1 (RRID: Addgene_71454).

Techniques: Quantitative RT-PCR, Gene Expression, Western Blot, Control, Mass Spectrometry, Transduction, Plasmid Preparation, Knockdown, Expressing

Journal: Cell Reports Medicine

Article Title: Therapeutic stress triggers tumor STAT1 acetylation to disarm immunotherapy

doi: 10.1016/j.xcrm.2025.102448

Figure Lengend Snippet: Tyrosine 701 phosphorylation promotes STAT1 degradation in cetuximab-resistant HNSCC (A) Upper: Representative western blot analysis of STAT1 protein levels in OECM-1-WT/OECM-1-Ctx R (left) and CAL-27-WT/CAL-27-Ctx R (right) cells following treatment with cycloheximide (20 μg/mL) for the indicated times. β-actin was the loading control. Lower: Quantification of STAT1 protein levels. Data are presented as the mean ± SD. n = 3 per group. Statistical significance was determined using unpaired Student’s t test. ∗ p < 0.05; ∗∗∗ p < 0.001; ns, not significant. (B) Upper: Representative western blot analysis of STAT1 protein levels in OECM-1-Ctx R (left) and CAL-27-Ctx R (right) cells transfected with STAT1 (OECM-1-Ctx R -STAT1 and CAL-27-Ctx R -STAT1) and treated with proteasome inhibitor (MG132, 20 μM) for 18 h. Snail was the positive control for proteasomal degradation. Lower: Representative western blot analysis of STAT1 protein levels in OECM-1-Ctx R (left) and CAL-27-Ctx R (right) cells transfected with STAT1 (OECM-1-Ctx R -STAT1 and CAL-27-Ctx R -STAT1) and treated with lysosomal inhibitor (bafilomycin A1, 100 nM) or autophagic degradation inhibitor (hydroxychloroquine [HCQ], 20 μM). LC3B is a marker for monitoring autophagy. GAPDH was the loading control. The experiments were performed in triplicate. (C) Representative immunoprecipitation and western blot analyses of polyubiquitinated STAT1 in OECM-1-WT/OECM-1-Ctx R (left) and CAL-27-WT/CAL-27-Ctx R (right) cells transfected with STAT1. The cells were treated with MG132 (20 μM) for 6 h to inhibit proteasome degradation. The experiments were performed in triplicate. (D) Representative western blot analysis of total STAT1, Tyr701-phosphorylated STAT1, and Ser727-phosphorylated STAT1 in OECM-1-WT/OECM-1-Ctx R (left) and CAL-27-WT/CAL-27-Ctx R (right) cells transfected with STAT1. The cells were treated with MG132 (10 μM) for 16 h to inhibit proteasome degradation. GAPDH was the loading control. The experiments were performed in triplicate. (E) Representative immunoprecipitation and western blot analyses of polyubiquitinated STAT1 in OECM-1-Ctx R cells transfected with wild-type (WT) or Tyr701-unphosphorylatable mutant (Y701F) STAT1. Cells were treated with MG132 (10 μM) for 6 h to inhibit proteasomal degradation. The experiments were performed in triplicate. See also .

Article Snippet: To generate the pLV-STAT1(Y701F), pLV-STAT1(S727A), pLV-STAT1(Y701F/S727A), pLV-STAT1(K637R), and pLV-STAT1(K637Q) plasmids, human STAT1 cDNA was amplified from p-LV-STAT1 (RRID: Addgene_71454).

Techniques: Phospho-proteomics, Western Blot, Control, Transfection, Positive Control, Marker, Immunoprecipitation, Mutagenesis

Journal: Cell Reports Medicine

Article Title: Therapeutic stress triggers tumor STAT1 acetylation to disarm immunotherapy

doi: 10.1016/j.xcrm.2025.102448

Figure Lengend Snippet: Reduced transcriptional activity of STAT1 in cetuximab-resistant HNSCC via Lys637 acetylation (A) Representative western blot analysis of the indicated proteins in OECM-1-WT/OECM-1-CtxR (left) and CAL-27-WT/CAL-27-Ctx R (right) cells transfected with STAT1 and treated with or without IFN-γ (100 ng/mL) for 24 h. α-tubulin was the loading control. The experiments were performed in triplicate. (B) Mass spectrometric analysis of CAL-27-Ctx R cells, identifying acetylation at Lys637 of STAT1. (C) Sequence alignment showing the conservation of STAT1 Lys637 across various species. (D) Representative western blot analysis of CAL-27-Ctx R and OECM-1-Ctx R cells transfected with wild-type or unacetylatable mutant STAT1(K637R), treated with or without IFN-γ (100 ng/mL) for 24 h. GAPDH was the loading control. The experiments were performed in triplicate. (E) Representative co-immunoprecipitation and western blot analyses detecting lysine-acetylated STAT1 in CAL-27-Ctx R and OECM-1-Ctx R cells transfected with wild-type STAT1 or STAT1(K637R). The cells were treated with MG132 (10 μM) for 16 h. The experiments were performed in triplicate. (F) Representative electrophoretic mobility shift assay assesses the DNA binding of wild-type STAT1 or STAT1(K637R) in CAL-27-Ctx R cells. The cells were transfected with the corresponding vectors, treated with MG132 (10 μM, 16 h) and IFN-γ (100 ng/mL, 30 min). (G) Representative western blot analysis of the indicated proteins in U3A cells transfected with STAT1(K637R) or STAT1(K637Q) mutants and treated with IFN-γ (100 ng/mL) for 24 h. GAPDH was a loading control. The experiments were performed in triplicate. (H) Representative blot detecting dimerized STAT1 and Tyr701-phosphorylated STAT1 in U3A cells transfected with STAT1(K637R) or STAT1(K637Q) mutants treated with IFN-γ (100 ng/mL) with or without disuccinimidyl suberate (DSS) (2.5 μM) for 10 min. The experiments were performed in triplicate. See also .

Article Snippet: To generate the pLV-STAT1(Y701F), pLV-STAT1(S727A), pLV-STAT1(Y701F/S727A), pLV-STAT1(K637R), and pLV-STAT1(K637Q) plasmids, human STAT1 cDNA was amplified from p-LV-STAT1 (RRID: Addgene_71454).

Techniques: Activity Assay, Western Blot, Transfection, Control, Sequencing, Mutagenesis, Immunoprecipitation, Electrophoretic Mobility Shift Assay, Binding Assay

Journal: Cell Reports Medicine

Article Title: Therapeutic stress triggers tumor STAT1 acetylation to disarm immunotherapy

doi: 10.1016/j.xcrm.2025.102448

Figure Lengend Snippet: IFN-β and TNF-α as potential upstream regulators of STAT1 inactivation in cetuximab-resistant HNSCC (A) Schematic representation of the identification of upstream regulators using Ingenuity Pathway Analysis in OECM-1-Ctx R and CAL-27-Ctx R cells (left). Expression levels of the indicated genes based on RNA sequencing in OECM-1-Ctx R and CAL-27-Ctx R cells compared to parental cells (right). (B) ELISA of IFN-β (left) and TNF-α (right) concentrations in conditioned media from CAL-27 and CAL-27-Ctx R cells ( n = 3, with two technical replicates each). Data are presented as mean ± SD. Statistical analyses were performed using unpaired Student’s t test. ∗∗∗ p < 0.001. (C) Representative western blot of the indicated proteins in CAL-27-Ctx R cells transfected with STAT1 (CAL-27-Ctx R -STAT1) and treated with MG132 (10 μM) combined with JAK1 (left), JAK2 (middle), and TYK2 inhibitors (right) at the indicated concentrations for 16 h. GAPDH was a loading control. The experiments were performed in triplicate. (D) Representative western blot of the indicated proteins in CAL-27-Ctx R (left) and OECM-1-Ctx R (right) cells transfected with STAT1 (CAL-27-Ctx R -STAT1 and OECM-1-Ctx R -STAT1) and treated with MG132 (10 μM) and IFN-β-neutralizing antibody at indicated concentrations for 16 h. GAPDH was the loading control. The experiments were performed in triplicate. (E) Representative western blot of STAT1 Tyr701 phosphorylation in OECM-1-Ctx R (left) and CAL-27-Ctx R (right) cells transfected with STAT1 (CAL-27-Ctx R -STAT1 and OECM-1-Ctx R -STAT1) and treated with MG132 (10 μM) combined with an IFN-α-neutralizing antibody at indicated concentrations for 16 h. α-tubulin was the loading control. Experiments were duplicated. (F) Representative co-immunoprecipitation and western blot analyses to investigate the interaction between STAT1 and histone acetyltransferases in the CAL-27-Ctx R and OECM-1-Ctx R cells transfected with STAT1 (CAL-27-Ctx R -STAT1 and OECM-1-Ctx R -STAT1). The cells were then treated with MG132 (10 μM) for 16 h. The experiments were performed in triplicate. (G) Representative in vitro acetylation assay. Biotin-labeled synthetic peptides, corresponding to the sequence encompassing STAT1 lysine 637 (K637) or a mutant variant where K637 was substituted with arginine (K637R), were utilized. These peptides were incubated in the presence or absence of the histone acetyltransferase (PCAF) and with acetyl-coenzyme A (acetyl-CoA). Following the incubation, the reaction products were analyzed by dot blot for assessing acetylation levels. The experiments were performed in triplicate. See also .

Article Snippet: To generate the pLV-STAT1(Y701F), pLV-STAT1(S727A), pLV-STAT1(Y701F/S727A), pLV-STAT1(K637R), and pLV-STAT1(K637Q) plasmids, human STAT1 cDNA was amplified from p-LV-STAT1 (RRID: Addgene_71454).

Techniques: Expressing, RNA Sequencing, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Control, Phospho-proteomics, Immunoprecipitation, In Vitro, Acetylation Assay, Labeling, Sequencing, Mutagenesis, Variant Assay, Incubation, Dot Blot