pstat1  (Cell Signaling Technology Inc)

Journal: iScience

Article Title: C9ORF72 suppresses JAK-STAT mediated inflammation

doi: 10.1016/j.isci.2023.106579

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Article Snippet: rabbit anti-pSTAT1 , R and D Systems , Cat# AF2894, RRID: AB_2198137.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Bradford Protein Assay, CRISPR, Plasmid Preparation, Software

Journal: Cancer Research

Article Title: PABPC1L Induces IDO1 to Promote Tryptophan Metabolism and Immune Suppression in Renal Cell Carcinoma

doi: 10.1158/0008-5472.CAN-23-2521

Figure Lengend Snippet: Loss of PABPC1L impairs JAK-STAT-IDO1 pathway in RCC. A, Representative Western blot (top) and statistical analysis (bottom) in the immortalized HK-2 renal epithelial cell line and RCC cell lines. GAPDH was used as a loading control. B, KEGG analysis showed the significantly altered signaling pathways after PABPC1L silencing in RCC cells. C, Representative Western blot analysis of STAT1, STAT3, pSTAT1, and pSTAT3 protein expression levels in 769-P cells with PABPC1L knockdown (left) and in Caki-1 cells with PABPC1L overexpression (right). D, mRNA expression of JAK-STAT1 target genes in shCtrl and shPABPC1L 769-P cells with or without IFNγ stimulation. Cells were treated with 100 U/mL IFNγ. E, Representative Western blot analysis showing JAK1, p-JAK1, JAK2, p-JAK2, STAT1, pSTAT1, and IDO1 protein expression levels in 769-P (left) and Caki-1 (right) cells with PABPC1L knockdown. Cells were stimulated with or without 100 U/mL IFNγ. F, Effects of JAK2 overexpression on IDO1 protein expression in shCtrl and shPABPC1L 769-P cells. G, Effects of JAK2 overexpression on IDO1 mRNA expression in shCtrl and shPABPC1L 769-P cells. A – G, All experiments were performed with three independent biological replicates, and data shown are representative of three independent experiments. H, Differently treated cells were cultured for 24 hours without KYNU treatment. KYN and TRP levels in cell supernatants were determined by ELISA. Differently treated cells were injected subcutaneously into BALB/c mice. HPLC/MS-MS analysis was used to generate the TRP metabolomic profiles of mouse plasma, and the KYN/TRP ratio in the mouse plasma was calculated. I, Flow cytometry analysis of GZMB+ in CD8 + T cells. J, Flow cytometry analysis of CD25 + and Foxp3 + Tregs in CD4 + T cells. K, Representative flow cytometry histograms and statistical analysis of cell surface PD-1 with the indicated treatments. L and M, Representative images of tumors ( L ) and growth curves ( M ) of indicated Renca tumors in BALB/c mice ( n = 5 per group). N, Tumor weight measured after surgical dissection ( n = 5 per group). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: The following primary antibodies were utilized in the Western blot and immunoprecipitation analyses: JAK1 (3344, Cell Signaling Technology, CST, RRID:AB_2265054), p-JAK1 (74129, CST, RRID:AB_2799851), JAK2 (3230, CST, RRID:AB_2128522), p-JAK2 (3771, CST, RRID:AB_330403), STAT1 (14994, CST, RRID:AB_2737027), pSTAT1 (9167, CST, RRID:AB_561284), STAT3 (9139, CST, RRID:AB_331757), pSTAT3 (9145, CST, RRID:AB_2491009), anti-human PABPC1L (Abcam, ab233280), EIF4G (2469, CST, RRID:AB_2096028), Flag tag (AE005, ABclonal, RRID:AB_2770401), His tag (AE003, ABclonal, RRID:AB_2728734), mouse control IgG (AC011, ABclonal, RRID:AB_2770414), rabbit control IgG (AC005, ABclonal, RRID:AB_2771930), IDO1 (A12125, ABclonal), and GAPDH(60004-1-Ig, Proteintech, RRID:AB_2107436).

Techniques: Western Blot, Expressing, Over Expression, Cell Culture, Enzyme-linked Immunosorbent Assay, Injection, Tandem Mass Spectroscopy, Flow Cytometry, Dissection

Journal: Journal of Clinical Immunology

Article Title: Patients with STAT1 Gain-of-function Mutations Display Increased Apoptosis which is Reversed by the JAK Inhibitor Ruxolitinib

doi: 10.1007/s10875-024-01684-y

Figure Lengend Snippet: Phosphorylation of STAT1 (pSTAT1) after stimulation with IFNα was assessed by flow cytometry in STAT1-GOF patients (black color) and healthy controls (gray color). In STAT1-GOF patients the expected increase of pSTAT1 was statistically significant compared to healthy controls and was significantly reversed by the JAK inhibitor ruxolitinib with a dose-dependent effect. US , unstimulated

Article Snippet: Intracellular staining of activated STAT1 protein was performed with phycoerythrin (PE)-conjugated mouse anti-pSTAT1-Tyr-701 IgG mAb (BD Pharmigene) and isotype-matched mAb PE (BD Bioscence).

Techniques: Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Lymph node and tumor-associated PD-L1 + macrophages antagonize dendritic cell vaccines by suppressing CD8 + T cells

doi: 10.1016/j.xcrm.2023.101377

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Article Snippet: PE anti-mouse pSTAT1 (clone: pY701) , BD Biosciences , Cat#562069 RRID: AB_11151907.

Techniques: Recombinant, Lysis, Protease Inhibitor, Western Blot, Staining, Stripping, Liposomes, CRISPR, MTS Assay, ATP Assay, Cell Isolation, Enzyme-linked Immunosorbent Assay, Conjugation Assay, Selection, Vaccines, Single-cell Analysis, RNA Sequencing Assay, Mutagenesis, Microarray, Purification, Software