stat1 antibody c 136  (Santa Cruz Biotechnology)

Journal: Redox Biology

Article Title: Hyodeoxycholic acid attenuates atherosclerosis by antagonizing FXR and modulating the PD-1/mTORC1 signaling axis

doi: 10.1016/j.redox.2026.104096

Figure Lengend Snippet: HDCA-treated ameliorate inflammation and modulate lipid metabolism in vivo. (A) Acetyl-CoA levels decreased over time in HDCA-treated Tregs. (B – E) Quantitative real-time PCR analysis of the mRNA expression of key lipogenic genes, including (B) fatty acid synthase (FASN), (C) acetyl-CoA carboxylase (ACC), (D) SCD1, and (E) SREBP-1c, in Tregs with or without HDCA treatment. (F) Functional metabolic flux assay using 13 C-glucose tracing shows decreased enrichment of labeled β-hydroxypalmitate in the HDCA-treated group. (G) Immunoblot analysis of phosphorylated STAT1 in Tregs following HDCA exposure, and quantification statistical analysis results are presented in the bar graphs. (H) Representative IHC images show reduced IL-21 expression in atherosclerotic lesions of mice following HDCA treatment (magnification, 5 × ; scale bar, 50 μm). (I) Plasma cholesterol levels were measured by enzymatic colorimetry assay in FXR-sufficient controls and FXR KO mice ± HDCA. (J) Plasma triglyceride levels in the Vector and FXR KO groups were quantified at 0, 6, 12, and 18 h by a colorimetric assay. (K) Hemodynamic profiling assessed heart rate, systolic blood pressure (SBP), and diastolic blood pressure (DBP) for each group. (L – M) Ratios of monounsaturated to saturated fatty acids (MUFA/SFA) and polyunsaturated to saturated fatty acids (PUFA/SFA) in serum cholesterol esters (CE) and triglycerides (TG) were quantified by lipidomics in FXR-sufficient control and FXR KO mice ± HDCA. Data are presented as the mean ± SD (n = 3-5 biological replicates). ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Proteins were detected using the following antibodies: anti-CPT1a antibody (ab234111, abcam), anti-beta actin antibody (ab8226, abcam), anti-PERK antibody (ab229912, abcam), anti-ERK1+ERK2 antibody (ab184699, abcam), anti-S6K1 antibody (ab14708, abcam), anti-S6K1 (phospho T229) antibody (ab5231, abcam), Rac1/2/3 antibody (G-2) (sc-514583, Santa Cruz), anti-Calpain 1 antibody (ab108400, abcam), anti-MMP2 antibody (ab92536, abcam), anti-IL-10 antibody (ab310329, abcam), anti-ZNF671 antibody (HPA046099, Sigma-Aldrich), anti-MAPK6/ERK3 antibody (ab53277, abcam), SIAH1 recombinant rabbit monoclonal antibody (PSH01-80) (MA5-51926, Thermo Fisher), p-Stat1 antibody (pY701.4A) (sc-136229, Santa Cruz), Stat1 antibody (C-136) (sc-464, Santa Cruz), anti-FXR1 antibody (ab155124, abcam), phospho-Raptor (Ser792) polyclonal antibody (PA5-118730, Thermo Fisher), anti-PD1 antibody (ab214421, abcam), SHP-2 antibody (3752S, Cell Signaling Technology), IL-10R antibody (3F9) (sc-53654, Santa Cruz), GAPDH antibody (6C5) (sc-32233, Santa Cruz), rabbit anti-mouse IgG H&L (HRP) (ab6728, abcam), goat anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody, Alexa FluorTM Plus 488 (A32731, Thermo Fisher).

Techniques: In Vivo, Real-time Polymerase Chain Reaction, Expressing, Functional Assay, Flux Assay, Labeling, Western Blot, Clinical Proteomics, Colorimetric Assay, Plasmid Preparation, Control

Journal: American Journal of Cancer Research

Article Title: STK405759 targets microtubules, modulates STAT1, and enhances ruxolitinib efficacy in myeloproliferative neoplasms

doi: 10.62347/NLYQ6344

Figure Lengend Snippet: STAT1 signaling following STK405759 treatment in JAK2V617F-positive MPN cells. A, B. HEL and SET-2 cells were treated with 100 nM STK405759 for 48 hours. Equal amounts of cellular protein lysates were analyzed using the Human JAK/STAT Pathway Phosphorylation Array Membrane. Representative scanned images are shown. Spot intensities were normalized to reference array spots and then to untreated control. Data are presented as mean ± SE. *** P < 0.005 vs. control. C, D. Representative immunofluorescence images depicting STAT1 expression and subcellular localization in HEL and SET-2 cells treated with increasing concentrations of STK405759. Nuclei were counterstained with Hoescht. Images were acquired using an Olympus Fluoview FV3000 confocal microscope. Scale bar: 100 µm. Magnification: 40×.

Article Snippet: Cells were then incubated overnight at 4°C with either an anti-β-tubulin Alexa Fluor ® 488 conjugated antibody (Merck) or a STAT1 (C-136) Alexa Fluor ® 488 conjugated antibody (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Phospho-proteomics, Membrane, Control, Immunofluorescence, Expressing, Microscopy

Journal: American Journal of Cancer Research

Article Title: STK405759 targets microtubules, modulates STAT1, and enhances ruxolitinib efficacy in myeloproliferative neoplasms

doi: 10.62347/NLYQ6344

Figure Lengend Snippet: STK405759 cytotoxicity and mechanistic effects in BCR-ABL1-positive MEG-01 cells. A. Dose- and time-dependent cytotoxicity after STK405759 treatment measured by XTT assay, values expressed as % viability relative to control. B. Representative images of apoptosis detected by Annexin V (green) and propidium iodide (red) staining after treatment for 72 hours, images acquired using Operetta CLS™ and quantified with Harmony 4.5 software. Total cell death = Annexin V + propidium iodide positive cells. C. Cell cycle distribution following STK405759 treatment, analyzed by propidium iodide and flow cytometry. D. Polymerized and soluble tubulin fractions analyzed by immunoblotting. E. Representative confocal images of β-tubulin (green) and nuclei (Hoescht, blue) after 48 hours of treatment; captured using Zeiss LSM780 confocal microscope. F. CD41 expression levels following treatment. Scale bar: 20 µm. Magnification: 60×. G. Representative immunofluorescence images showing STAT1 (green) expression and subcellular localization after STK405759 exposure; nuclei counterstained with Hoescht (blue). Scale bar: 100 µm. Magnification: 40×. Data represent mean ± SE. **** P < 0.001, *** P < 0.005, ** P < 0.01 vs. control.

Article Snippet: Cells were then incubated overnight at 4°C with either an anti-β-tubulin Alexa Fluor ® 488 conjugated antibody (Merck) or a STAT1 (C-136) Alexa Fluor ® 488 conjugated antibody (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: XTT Assay, Control, Staining, Software, Flow Cytometry, Western Blot, Microscopy, Expressing, Immunofluorescence

Journal: Theranostics

Article Title: UBR5 promotes tumor immune evasion through enhancing IFN-γ-induced PDL1 transcription in triple negative breast cancer

doi: 10.7150/thno.74989

Figure Lengend Snippet: UBR5 is crucial for IFN-γ-induced activation of STAT1 and IRF1 transcription. (A-B) The mRNA (A) and protein (B) levels of UBR5, STAT1, pSTAT1 and IRF1 were detected in WT, Ubr5 -/- and h UBR5 -reconstituted Ubr5 -/- 4T1 cells with or without IFN-γ stimulation. (C-D) The mRNA (C) and protein levels (D) of STAT1 and IRF1were detected in IFN-γ-treated UBR5-knockdown BT549 (C and D) and MDA-MB-231 (D) stable cell lines. (E-G) BT549, MDA-MB-231 and MCF7 cells were transfected with either an empty vector or UBR5 plasmids. 24 hours later, the cells were treated with IFN-γ for 24 h. Then the mRNA and protein levels of STAT1 (E and G) and IRF1 (F and G) were measured by qPCR and western blot, respectively. (H) Surface PD-L1 levels were detected in IFN-γ-treated GFP, Ubr5 -/- 4T1 cells and GFP 4T1 cells treated with siNC, siSTAT1 or siIRF1. GAPDH was used for normalization. (I) Luciferase reporter vectors containing either STAT1 or IRF1 promoter regions were cotransfected with an empty vector or UBR5 plasmids into the indicated cells. After 24 h, the transfected cells were treated with IFN-γ stimulation for 24 h, the cells were lysed to perform luciferase assay. The results are presented as the mean ± SEM from three individual experiments. *P < 0.05, **P < 0.01, ***P< 0.001, ****P < 0.0001. (J) The IRF1 and STAT1 binding sites in the m Pdl1 promoter region were predicted using the ALGGEN website. (K) Summary of the results of a ChIP assay using anti-IRF1, STAT1, H3K4me1 and H3K27ac antibodies in WT, Ubr5 -/- or h UBR5 -reconstituted Ubr5 -/- 4T1 cells after treatment with or without IFN-γ.

Article Snippet: Proteins were resolved on a 10% SDS PAGE gel and transferred to the NC membrane, blocked with 5% milk and probed for monoclonal antibodies against UBR5 (Santa Cruz, sc-515494), PD-L1 (Proteintech) #66248-1-lg, EIF2AK2 (Beyotime) #AF2125, STAT1 p84/91 (C-136) (Santa Cruz, sc-464), STAT1 (D1K9Y) Rabbit (Cell Signal Technology) mAb #14994, Phospho-STAT1 (Tyr701) Rabbit (Beyotime) #AF5935, IRF1 (E-4) (Santa Cruz, sc-514544) or anti-GAPDH (Proteintech).

Techniques: Activation Assay, Knockdown, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Luciferase, Binding Assay

Journal: Theranostics

Article Title: UBR5 promotes tumor immune evasion through enhancing IFN-γ-induced PDL1 transcription in triple negative breast cancer

doi: 10.7150/thno.74989

Figure Lengend Snippet: UBR5-mediated transactivation of PDL1 is independent of its E3 ubiquitin ligase. (A-D) MDA-MB-231 cells were cotransfected with WT, HECT-mutation (UBR5-C2768A) or PABC-deletion UBR5 (A) together with luciferase reporters containing the human PDL1 WT promoter or a promoter with 2 deletions, including the STAT1/3 or IRF1 binding sites (B), and then stimulated with IFN-γ for 24 h. Luciferase activity was measured in cell lysates of MDA-MB-231 (C) and 4T1 cells (D) by dual luciferase assay. (E-F) The protein levels of UBR5 (E) and PD-L1 (F) were measured in IFN-γ-treated GFP, Ubr5 -/- , h UBR5 -reconstituted Ubr5 -/- 4T1 cells and Ubr5 -/- 4T1 cells reconstituted with either h UBR5 -C2768 or h UBR5 -ΔPABC. The results are presented as the mean ± SEM from three individual experiments. ns, no significance, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Proteins were resolved on a 10% SDS PAGE gel and transferred to the NC membrane, blocked with 5% milk and probed for monoclonal antibodies against UBR5 (Santa Cruz, sc-515494), PD-L1 (Proteintech) #66248-1-lg, EIF2AK2 (Beyotime) #AF2125, STAT1 p84/91 (C-136) (Santa Cruz, sc-464), STAT1 (D1K9Y) Rabbit (Cell Signal Technology) mAb #14994, Phospho-STAT1 (Tyr701) Rabbit (Beyotime) #AF5935, IRF1 (E-4) (Santa Cruz, sc-514544) or anti-GAPDH (Proteintech).

Techniques: Ubiquitin Proteomics, Mutagenesis, Luciferase, Binding Assay, Activity Assay

Journal: Theranostics

Article Title: UBR5 promotes tumor immune evasion through enhancing IFN-γ-induced PDL1 transcription in triple negative breast cancer

doi: 10.7150/thno.74989

Figure Lengend Snippet: The transactivation of UBR5 to STAT1 and PDL1 is mediated by protein kinase RNA-activated. (A-B) The mRNA (A) and protein levels (B) of EIF2AK2 in WT, Ubr5 -/- , h UBR5 -reconstituted Ubr5 -/- 4T1 cells and Ubr5 -/- 4T1 cells reconstituted with either h UBR5 -C2768A or h UBR5 -ΔPABC were measured by qPCR and western blot analysis after cells were treated with or without IFN-γ. (C-D) The mRNA (C) and protein levels (D) levels of EIF2AK2 in WT and UBR5-knockdown BT549 cells were measured by qPCR and western blot analysis after cells were treated with IFN-γ for 24 h. (E-G) The mRNA (E) and protein levels of UBR5, STAT1, IRF1 and EIF2AK2 (F) and relative fold changes of cell-surface PD-L1 levels (G) in IFN-γ-treated WT, Ubr5 -/- , and 4T1 cells with stable knockdown by shEif2ak2 #1 and shEif2ak2 #1. Shscramble served as the control. (H-J) The mRNA levels (H) of Eif2ak2 , Ubr5 , Pdl1 , Stat1 and Irf1 , the protein levels of UBR5, EIF2AK2, STAT1 and IRF1 (I) and the surface levels of PD-L1 (J) were measured in IFN-γ-treated 4T1 WT, Ubr5 -/- and m Eif2ak2 -reconstituted Ubr5 -/- cell lines. The results are presented as the mean ± SEM from three individual experiments. ns, no significance, *P< 0.05, **P < 0.01, ***P< 0.001, ****P < 0.0001. (K) The correlations of EIF2AK2 with UBR5 , PDL1 and STAT1 mRNA levels were assessed in the TCGA BRCA database (1104 samples) by starBase database. (L) The summary correlations of EIF2AK2 with UBR5 , PDL1 , STAT1 and IRF1 mRNA levels (normalizated to GAPDH) were analysed in the TCGA BRCA database by starBase and GEPIA database respectively.

Article Snippet: Proteins were resolved on a 10% SDS PAGE gel and transferred to the NC membrane, blocked with 5% milk and probed for monoclonal antibodies against UBR5 (Santa Cruz, sc-515494), PD-L1 (Proteintech) #66248-1-lg, EIF2AK2 (Beyotime) #AF2125, STAT1 p84/91 (C-136) (Santa Cruz, sc-464), STAT1 (D1K9Y) Rabbit (Cell Signal Technology) mAb #14994, Phospho-STAT1 (Tyr701) Rabbit (Beyotime) #AF5935, IRF1 (E-4) (Santa Cruz, sc-514544) or anti-GAPDH (Proteintech).

Techniques: Western Blot, Knockdown, Control