Cells were lysed using RIPA buffer in the presence of protease and phosphatase inhibitors. The centrifuged supernatants were subjected to SDS-PAGE (Criterion™ TGX™) and immunoblotting. Transfer to PVDF membranes was carried out using Trans-Blot Turbo™ Transfer System®. The blots were blocked with 2.5% BSA in PBS containing 0.1% Tween20 before incubating with the following primary antibodies: CD81 (sc-9158, Santa Cruz), CD63 (sc-5275, Santa Cruz), calnexin (ab-22595, Abcam), nSmase2a (sc-166637, Santa Cruz), cGAS (SAB2100310, Sigma-Aldrich), STING (AF6516, R&D), vinculin (V9131, Sigma), HRP-conjugated actin (ab-49906, Abcam). Corresponding peroxidase-conjugated secondary antibodies were used (Jackson ImmunoResearch). Membranes were developed using Super Signal West Dura extended duration substrate (Thermo Scientific, 34076). Original blots are shown in Supplementary Figure 7 .
Sting
Manufactured by R&D Systems
STING is a laboratory equipment product that serves as a cytoplasmic DNA sensor. It is designed to detect the presence of cytoplasmic DNA, which can be an indication of various cellular processes or pathological conditions.
Automatically generated - may contain errors
Lab products found in correlation
Cd63 sc 5275, by Santa Cruz Biotechnology (2 mentions)
Vinculin, by Merck Group (2 mentions)
Hrp conjugated actin, by Abcam (2 mentions)
Nsmase2a sc 166637, by Santa Cruz Biotechnology (2 mentions)
Cgas sab2100310, by Merck Group (2 mentions)
Calnexin, by Abcam (2 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (2 mentions)
Flag epitope, by Merck Group (1 mentions)
Ecl western blotting detection reagent, by Cytiva (1 mentions)
Cin85, by Santa Cruz Biotechnology (1 mentions)
Stim1, by Santa Cruz Biotechnology (1 mentions)
Phospho tbk1 ser172, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Bradford method, by Bio-Rad (1 mentions)
Bovine serum albumin (bsa), by Euromedex (1 mentions)
Ifi16 1g7, by Santa Cruz Biotechnology (1 mentions)
Cgas hpa031700, by Merck Group (1 mentions)
Tbk1 d1b4, by Cell Signaling Technology (1 mentions)
Sting d2p2f, by Cell Signaling Technology (1 mentions)
5 protocols using sting
1
Comprehensive Western Blot Analysis of Exosome Markers
2
Comprehensive Western Blot Analysis of Exosome Markers
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Cells were lysed using RIPA buffer in the presence of protease and phosphatase inhibitors. The centrifuged supernatants were subjected to SDS-PAGE (Criterion™ TGX™) and immunoblotting. Transfer to PVDF membranes was carried out using Trans-Blot Turbo™ Transfer System®. The blots were blocked with 2.5% BSA in PBS containing 0.1% Tween20 before incubating with the following primary antibodies: CD81 (sc-9158, Santa Cruz), CD63 (sc-5275, Santa Cruz), calnexin (ab-22595, Abcam), nSmase2a (sc-166637, Santa Cruz), cGAS (SAB2100310, Sigma-Aldrich), STING (AF6516, R&D), vinculin (V9131, Sigma), HRP-conjugated actin (ab-49906, Abcam). Corresponding peroxidase-conjugated secondary antibodies were used (Jackson ImmunoResearch). Membranes were developed using Super Signal West Dura extended duration substrate (Thermo Scientific, 34076). Original blots are shown in Supplementary Figure 7 .
3
Cell Lysis and Protein Detection
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Cells were solubilized in triple detergent buffer (50 mM Tris-HCl [pH 8], 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% Nonidet P-40, 0.5% sodium deoxycholate, 100 mg/mL of phenylmethylsulfonyl fluoride) supplemented with phosphatase inhibitors (10 mM NaF, 10 mM b-glycerophosphate, 0.1 mM sodium vanadate) and protease inhibitor cocktail (Sigma) and briefly sonicated. Protein concentration was determined with the Bradford method (Bio-Rad Laboratories). The mouse monoclonal antibodies to ICP8, CD63, VP16, gD, CIN85 (Santa Cruz), β-actin, Flag epitope (Sigma), ARF6 (Thermo Fisher Scientific), STING (R&D systems), Us11 (kindly provided by Dr. Roizman-University of Chicago), and STIM1 (Santa Cruz) were used in a 1:1,000 dilution. The rabbit polyclonal phospho-STING (Ser-366), TBK1, and phospho-TBK1 (Ser-172) antibodies (Cell Signaling Technology) were used in a 1:500 dilution. The anifrolumab (Invitrogen) was used at 1 µg/mL. Proteins were visualized with 5-bromo-4-chloro-3 indolylphosphate-nitroblue tetrazolium) or with ECL western blotting detection reagents (Amersham Biosciences).
4
Quantification of IFI16, cGAS, and STING
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1.5 million sorted naive and central memory or activated CD4+ T cells were lysed in sample buffer (4% SDS, 20% glycerol, 0.1 M Tris-HCl, pH 6.8, 0.05% bromophenol blue, and 0.1 M dithiothreitol). Cellular protein lysates were resolved on 4–20% precast SDS-PAGE gels (Bio-Rad Laboratories) and transferred on a nitrocellulose membrane. The membrane was blocked in 1× Tris-buffered saline, 0.1% Tween 20 (AMRESCO), and 5% BSA (Euromedex). Proteins were blotted with antibodies as follow: IFI16 (1G7; Santa Cruz Biotechnology, Inc.), cGAS (HPA031700; Sigma-Aldrich), STING (D2P2F; Cell Signaling Technology), and actin (C4; EMD Millipore) in 1× Tris-buffered saline, 0.1% Tween 20 (AMRESCO), and 5% BSA (Euromedex). For immunoprecipitation experiments, proteins were blotted with antibodies as follow: TBK-1 (D1B4; Cell Signaling Technology), STING (D2P2F; Cell Signaling Technology), or STING (clone 723505; R&D Systems). Membranes were washed three times in 1× PBS and 0.1% Tween 20 (AMRESCO). Enhanced chemiluminescence signal was recorded on the ChemiDoc XRS Imager (Bio-Rad Laboratories). Data were analyzed and quantified with Image Lab software (Bio-Rad Laboratories).
5
STING Dimerization and GSDMD Cleavage Analysis
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STING dimerization was assayed under semi-native conditions. Five hundred thousand THP1 MΦ after stimulation with µRNA of S. aureus (SA), ∆agr and in-vitro-transcribed RNAIII (3 µg ml -1 , 1 µg ml -1 ) were lysed in 30 µl of 1X cell lysis buffer (Cell Signaling) supplemented with 1X protease cocktail inhibitor (Roche) and 1X sample buffer. Whole cell lysate were sonicated for 7 min with at intensity before loading onto gel without heating. Separation was done using 4-12% SDS-PAGE gel electrophoresis were each gel was run initially for 10 min at 70 V, and then at 120 V for 1 h 30 min. GSDMD cleavage in human plasma of S. aureus sepsis patients and ICU controls was assayed through Western blot. 1 µg plasma with 1X sample buffer was heated at 95°C for 5 min before loading on the gel. Separation was performed by 4-12% SDS-PAGE gel electrophoresis with each gel run at 70 V.
Proteins were blotted onto PVDF membrane, blocked in bovine serum albumin for anti-GSDMD and in skim-milk for anti-STING, respectively with indicated primary and secondary antibodies. Chemiluminescent signals were recorded with a CCD camera.
Antibodies used: STING (R&D systems), GAPDH (Thermo Fisher), GSDMD (Proteintech).
Proteins were blotted onto PVDF membrane, blocked in bovine serum albumin for anti-GSDMD and in skim-milk for anti-STING, respectively with indicated primary and secondary antibodies. Chemiluminescent signals were recorded with a CCD camera.
Antibodies used: STING (R&D systems), GAPDH (Thermo Fisher), GSDMD (Proteintech).
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