Confocal immunofluorescent analysis of Raw 264.7 cells, transfected with poly(dA:dT) (5 ug/ml, 3 hr; left) or mock transfected (right), using Phospho-STING (Ser365) (D1C4T) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).Learn more about how we get our images
Achieve higher quality immunofluorescent images using the efficient and cost-effective, pre-made reagents in our #12727 Immunofluorescence Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted November 2006
revised November 2013
Protocol Id: 24
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
Phospho-STING (Ser365) (D1C4T) Rabbit mAb recognizes endogenous levels of STING protein only when phosphorylated at Ser365.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser365 of mouse STING protein.
Stimulator of interferon genes (STING, TMEM173, MITA) is a transmembrane adaptor protein that is a critical component of the cellular innate immune response to pathogenic cytoplasmic DNA (1,2). STING is a ubiquitously expressed protein found predominantly in the ER (1). The enzyme cGAMP synthase (cGAS) produces the second messenger cyclic-GMP-AMP (cGAMP) in response to cytoplasmic DNA (3,4). cGAMP binds and activates STING (3,4). In addition, detection of cytoplasmic DNA by nucleic acid sensors, including DDX41 or IFI16, results in STING activation (5,6). Following activation, STING translocates with TBK1 to perinuclear endosomes (7). The TBK1 kinase phosphorylates and activates interferon regulatory factors (IRFs) and NF-κB, which leads to the induction of type I interferon and other immune response genes (1,2,7).
Following activation and trafficking, STING gets phosphorylated by ULK1 at Ser366 (Ser365 in mouse), which leads to STING inactivation and eventually lysosomal degradation (8).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. DRAQ5 is a registered trademark of Biostatus Limited. DyLight is a trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.