anti human pstat1  (Cell Signaling Technology Inc)

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Persistent tailoring of MSC activation through genetic priming

doi: 10.1016/j.omtm.2024.101316

Figure Lengend Snippet: MSC IRF1 avoids STAT1 phosphorylation and subsequent signaling present in MSC IFN-γ in favor of nuclear localization of unphosphorylated STAT1 (A) Activation of STAT1 transcription by IFN-γ stimulation and IRF1 transgene overexpression. (B) Western blotting confirms STAT1 phosphorylation occurs in MSC IFN-γ , but not MSC IRF1 or unprimed MSCs. (C) Immunocytochemical imaging of STAT1 and pSTAT1 localization. Images are contrasted equivalently. Scale bar, 100 μm. (D) Summary quantification of nuclear localization of pSTAT1. One-way ANOVA. Error bars represent the SD.∗∗∗∗ p < 0.0001.

Article Snippet: Cells were washed thoroughly with 0.2-μm filtered 5% BSA in PBS (blocking solution), and permeabilized by incubation in 0.2% Triton X-100 in PBS at room temperature for 5 min. After washing and incubating with blocking solution for 1 h, cells were immuno-stained overnight at 4°C–8°C with rabbit anti-human IRF1 antibody (1:1,000, ab243895, Abcam) or rabbit anti-human pSTAT1 (1:400, MA5-15071, Thermo Fisher Scientific) and mouse anti-human STAT1 (1:800, 66545-1, Proteintech) diluted in blocking buffer.

Techniques: Phospho-proteomics, Activation Assay, Over Expression, Western Blot, Imaging

Journal: iScience

Article Title: C9ORF72 suppresses JAK-STAT mediated inflammation

doi: 10.1016/j.isci.2023.106579

Figure Lengend Snippet:

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: bioRxiv

Article Title: Human DUX4 and mouse Dux interact with STAT1 and broadly inhibit interferon-stimulated gene induction

doi: 10.1101/2022.08.09.503314

Figure Lengend Snippet: ( A ) Western blot showing input and immunoprecipitated proteins from either 3xFLAG-iDUXB (DUXB) or a truncation series of the 3x-FLAG-iDUX4-CTD cells (iDUX4) precipitated with anti-FLAG and probed with the indicated antibodies. Serial deletions of the iDUX4-CTD were assayed as indicated. All samples were treated with IFNγ. An asterisk indicates the correct band for each FLAG-tagged construct. See Source Data for uncropped/raw Western blots. ( B ) Input and anti-FLAG immunoprecipitation from 3xFLAG-iDUXB or 3x-FLAG-iDUX4-CTD cells co-expressing doxycycline inducible 3xMYC-iSTAT1, -iSTAT1-Y701A, or -iSTAT1-S727A with or without IFNγ treatment and probed with the indicated antibodies. An “x” indicates the endogenous (non-MYC tagged) STAT1 band. See Source Data for uncropped/raw Western blots. ( C ) Proximity-ligation assay (PLA) showing co-localization of endogenous STAT1 and pSTAT1 701 with the iDUX4-CTD compared to the interaction with the DUXB-CTD, in the nuclear compartment of IFNγ- and doxycycline-treated MB135 cells. Mean fluorescent intensity (MFI) of the nuclei in the PLA channel was measured for 10 images per cell line and treatment and plotted (**** p<0.0001, unpaired t-test). An unpaired t-test was used because the samples are biologically independent.

Article Snippet: Antibodies against STAT1 [EPR4407] (ab109320, RRID:AB_10863383), STAT1 [EPR21057-141] (ab234400), STAT1 [EPR23049-111] (ab239360), pSTAT1 Y701 [M135] (ab29045, RRID:AB_778096), pSTAT1 S727 [EPR3146] (ab109461, RRID:AB_10863745), YBX1 [EP2708Y] (ab76149, RRID:AB_2219276), PABPC1 (ab21060, RRID:AB_777008), hnRNPM [EPR13509(B)] (ab177957, RRID:AB_2820246), and PRKDC [Y393] (ab32566, RRID:AB_731981) were purchased from Abcam.

Techniques: Western Blot, Immunoprecipitation, Construct, Expressing, Proximity Ligation Assay

Journal: bioRxiv

Article Title: Human DUX4 and mouse Dux interact with STAT1 and broadly inhibit interferon-stimulated gene induction

doi: 10.1101/2022.08.09.503314

Figure Lengend Snippet: ( A, left panel ) RT-PCR of the indicated genes in MB135-iDux cells untreated or treated with IFNγ ±doxycycline. Ct values were normalized to the housekeeping gene RPL27 , then normalized to the IFNγ-only treatment to set the induced level to 100%. Data represent the mean ±SD of three biological replicates with three technical replicates each. ( A, right panel ) Western blot showing input and immunoprecipitated proteins from either 3xFLAG-iDux or 3x-FLAG-iDUXB cells ±IFNγ precipitated with anti-FLAG and probed with the indicated antibodies. See Source Data for uncropped/raw Western blots. ( B ) A model supported by the data showing how the DUX4-CTD might prevent STAT1 ISG induction. (Top) In the absence of the DUX4-CTD, pSTAT1 Y701 (black “P”) dimerizes, translocates to the nucleus, binds its GAS motif in the ISG promoter, acquires secondary phosphorylation at S727 (grey “P”), and recruits a stable transcription complex that includes Pol II to drive transcription of ISGs. (Bottom) In the presence of the DUX4-CTD, STAT1 is phosphorylated, translocates to the nucleus, and binds its GAS motif as evidenced by the pSTAT1 S727 in complex with the CTD. However, diminished steady-state occupancy of STAT1 at the ISG promoters and absence of Pol-II recruitment indicate that the STAT1-DUX4-CTD complex does not stably bind DNA and fails to recruit Pol-II and the pre-initiation complex. The (L)LXXL(L) motifs (black bars in DUX4-CTD) are necessary to interfere with transcription suppression and likely prevent STAT1 from interacting with a factor in the pre-initiation complex or recruit a co-repressor.

Article Snippet: Antibodies against STAT1 [EPR4407] (ab109320, RRID:AB_10863383), STAT1 [EPR21057-141] (ab234400), STAT1 [EPR23049-111] (ab239360), pSTAT1 Y701 [M135] (ab29045, RRID:AB_778096), pSTAT1 S727 [EPR3146] (ab109461, RRID:AB_10863745), YBX1 [EP2708Y] (ab76149, RRID:AB_2219276), PABPC1 (ab21060, RRID:AB_777008), hnRNPM [EPR13509(B)] (ab177957, RRID:AB_2820246), and PRKDC [Y393] (ab32566, RRID:AB_731981) were purchased from Abcam.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunoprecipitation, Phospho-proteomics, Stable Transfection