The cell lysates were run on a 10% SDS polyacrylamide gel and transferred to a PVDF-Star transfer membrane (AppliChem). The amount of IRF3 and IRF7 in the cell lysates was detected using a mouse anti-V5 antibody (1:5000, Invitrogen), MAVS and cGAS was detected with a mouse anti-FLAG antibody (1:2000, Sigma Aldrich), STING was detected with a rabbit anti-STING antibody (1:1000, Cell signaling), pTBK-1 was detected with a rabbit anti‐pTBK1 (Ser172) (1:1000, Cell Signaling), vinculin was detected with anti-vinculin (Sigma) and the amount of GAPDH was detected using a rabbit anti-GAPDH antibody (1:1000, Santa Cruz Biotechnology). The primary antibodies were followed by either HRP-conjugated anti-mouse antibody (Jackson Immuno research) for the anti-V5 antibody or HRP-conjugated anti-rabbit (Dako Cytomation) for the anti-GAPDH antibody. The western blot was developed using SuperSignal West Dura extended Duration substrate (Thermo Scientific) and visualized on an X-ray film (Konica Minolta).
Rabbit anti sting antibody
Manufactured by Cell Signaling Technology
Rabbit anti-STING antibody is a primary antibody that recognizes the STING (Stimulator of Interferon Genes) protein. STING is a critical component of the innate immune response pathway that senses the presence of cytosolic DNA and triggers the production of type I interferons.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Merck Group (2 mentions)
Modified dynabeads, by BD (2 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Mouse anti v5 antibody, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated anti mouse antibody, by Jackson ImmunoResearch (1 mentions)
X ray film, by Konica Minolta (1 mentions)
Rabbit anti gapdh antibody, by Santa Cruz Biotechnology (1 mentions)
Anti vinculin, by Merck Group (1 mentions)
Mouse anti flag antibody, by Merck Group (1 mentions)
Odyssey clx imager, by LI COR (1 mentions)
Rabbit anti gapdh antibody, by ABclonal (1 mentions)
4 protocols using rabbit anti sting antibody
1
Immunoblotting for IRF3, IRF7, MAVS, cGAS, STING, and pTBK-1
2
STING Binding Interaction with PC7A Copolymer
3
STING Binding Interaction with PC7A Copolymer
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To investigate the STING interaction with PC7A copolymer, we labeled the PC7A copolymer with biotin (2–3 biotins per polymer chain). For THP-1 cell pulldown assay, PC7A-biotin (200 μg/ml) was incubated with THP-1 cells for 8 hrs, and then cells were collected and lysed in RIPA buffer (Sigma R0278). Lysates were precipitated with streptavidin-modified dynabeads (BD 557812). Samples were analyzed using SDS-PAGE and Western blots by rabbit anti-STING antibody (Cell Signaling, Cat # 13647). For STING protein pull down assay, human STING CTD (139–379) expression and purification had been described before39 (link), PC7A-biotin(50 μg/mL) was incubated with STING CTD(1 μg/ml) for 3hrs in PBS (pH=6.8), PEPA-biotin in PBS (pH=6.8) and PD5A-biotin in PBS (pH=4.4) with the same concentration were used as control groups, and then precipitated with streptavidin-modified dynabeads. Samples were analyzed using SDS-PAGE and Western blots.
4
Comprehensive Protein Extraction and Analysis
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Snap-frozen hearts or isolated cardiomyocytes were lysed in the RIPA buffer (20 mM Tris-HCl, 150 mM NaCl, 1% IGEPAL, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM dithiothreitol, protease and phosphatase inhibitors) or the Urea buffer (20 mM HEPES pH 7.4, 1 M NaCl, 8 M urea, protease and phosphatase inhibitors). Proteins were extracted using pestle homogenization and sonication. Cell lysates were centrifuged at 13,000 rpm for 5 minutes at 4°C, and proteins in the supernatant were separated by SDS-PAGE. Proteins were transferred to a PVDF membrane. Membranes were blocked with nonfat milk. The primary antibodies were rabbit anti-Lamin A/C antibody (Santa Cruz, sc-20681, 1:1000), rabbit anti-cGAS antibody (Cell Signaling, #31659, 1:500), rabbit anti-STING antibody (Cell Signaling, #13647, 1:500), and rabbit anti-GAPDH antibody (ABclonal, AC001, 1:1000); Secondary antibodies: anti-rabbit/mouse IgG (H+L) DyLight 680 and 800 (Cell Signaling, 1:5000). Signals were detected and quantified in the Odyssey CLx Imager (LI-COR). The gel after protein transfer was counter-stained by Coomassie to evaluate the loaded protein amount.
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