Human STING/TMEM173 Antibody

Detection of STING/TMEM173 in U937 Human Cell Line by Flow Cytometry.

U937 human histiocytic lymphoma cell line was stained with Mouse Anti-Human STING/TMEM173 Monoclonal Antibody (Catalog # MAB7169, filled histogram) or isotype control antibody (MAB0041, open histogram), followed by Allophycocyanin-conjugated Anti-Mouse IgG Secondary Antibody (F0101B). To facilitate intracellular staining, cells were fixed with paraformaldehyde and permeabilized with saponin.

STING/TMEM173 in U937 Human Cell Line.

STING/TMEM173 was detected in immersion fixed U937 human histiocytic lymphoma cell line using Mouse Anti-Human STING/TMEM173 Monoclonal Antibody (Catalog # MAB7169) at 3 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (red; NL007) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Non-adherent Cells.

Detection of Mouse STING/TMEM173 by Western Blot

CYLD interacts with STING in a ubiquitination-dependent manner.(A) HEK293T cells were transfected with the indicated plasmids. Thirty-six hours after transfection, the cell lysates were immunoprecipitated with an anti-Myc antibody and then immunoblotted with the indicated antibodies. (B) HEK293T cells were transfected with the indicated plasmids. Thirty-six hours after transfection, cell lysates were immunoprecipitated with an anti-HA antibody and then immunoblotted with the indicated antibodies. (C and D) HEK293T cells were transfected with the indicated plasmids. Thirty-six hours after transfection, the cell lysates were immunoprecipitated with an anti-Myc antibody (C) or an anti-HA antibody (D) and then immunoblotted with the indicated antibodies. (E) HEK293T cells were transfected with the indicated plasmids. Thirty-six hours after transfection, the cell lysates were immunoprecipitated with an anti-Myc antibody and then immunoblotted with the indicated antibodies. (F) After stimulation with HSV-1 (MOI = 10) for the indicated time periods in the presence of MG132 (10 μM), lysates of MEFs were immunoprecipitated with an anti-STING antibody and then immunoblotted with the indicated antibodies. (G) HeLa cells were transfected with empty vector or Flag-tagged RNF5 for 24 h and then stained with the indicated antibodies before imaging by confocal microscopy. STING (Green) (R&D Systems); CYLD (Red) (Abcam); Nucleus (Blue). Scale bars represent 50 μm. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/30388174), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human STING/TMEM173 by Western Blot

HCT116 cells surviving PARP1 depletion activate innate immune signaling.(A-B) RNA-Seq data from HCT116EV and HCT116PARP1-/- cells (clones C2 and C4) was analyzed using Gene Set Enrichment Analysis (GSEA). The top category of differentially expressed genes was “Interferon Alpha Response”. The enrichment plot is shown in (A) and the heat map for the 97 mRNAs in this category in shown in (B). Map was generated using Cufflinks software (version 2.2.1) and shows absolute expression values independently normalized and analyzed for each comparison pair (EV/C2 and EV/C4). (C) The same RNA-Seq dataset was analyzed using Ingenuity Pathway Analysis (IPA). A representative plot highlighting enrichment for Interferon-Stimulated Genes (ISGs) is shown. (D) The induction of multiple ISGs observed by RNA-Seq was confirmed by q-RT-PCR. Bars represent the average and standard deviation of quadruplicate samples in each experiment and data is representative of 2–4 independent experiments. (E-F) The induction of factors involved in the sensing/signaling of cytoplasmic nucleic acids observed by RNA-Seq was confirmed by Q-RT-PCR (E). Bars represent the average and standard deviation of quadruplicate samples in each experiment and data is representative of 2–3 independent experiments. Protein expression for the same factors was assessed by immunoblotting (F). Blots are representative of 2–3 independent experiments. (G) Fixed cells were stained with an antibody to IRF3 and counterstained with DAPI. Images are representative of 5 random fields per slide. The experiment was repeated twice with similar results. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/29590171), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human STING/TMEM173 by Western Blot

ISG induction in cells surviving PARP1 depletion is dependent on RIG-I and MAVS.(A-E) To assess the efficiency of RNA silencing to STING (A), RIG-I (B), MDA-5 (C), TLR3 (D) or MAVS (E), cells transfected with the specific pooled siRNAs and control cells transfected with a scrambled siRNA or untreated were harvested 4 days after transfection and probed with the indicated antibodies. GAPDH (A, B, C, E) or alpha-tubulin (D) served as loading controls. In blots where multiple bands are observed (A, D, E), black arrows point to specific bands and asterisks to unspecific bands. (F) The expression of ISGs OAS1 and IFIT3 was quantified by qRT-PCR after knock down. Data is normalized to the expression in HCT116EV cells (= 1). Bars represent the average and standard deviation of quadruplicates. Data is representative of two independent experiments. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/29590171), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of STING/TMEM173 by Immunoprecipitation.

PMA-treated THP-1 lysates were prepared, and immunoprecipitation was performed using 2.0 μg of Mouse Anti-Human STING/TMEM173 Monoclonal Antibody (Catalog # MAB7169) pre-coupled to Dynabeads protein G. Immunoprecipitated STING/TMEM173 was detected with a Rabbit Anti-STING/TMEM173 antibody. For western blot, the Rabbit Anti-STING/TMEM173 antibody was used at 1/1000. The Ponceau stained transfers of each blot are shown. SM=4% starting material; UB=4% unbound fraction; IP=immunoprecipitate; HC=antibody heavy chain. *=monoclonal antibody, **=recombinant antibody. Image, protocol and testing courtesy of YCharOS Inc. (ycharos.com).