Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expresssing Myc/DDK-tagged human syntaxin 17 (hSTX17-Myc/DDK; +) and Myc/DDK-tagged human TBK1 (hTBK1-Myc/DDK; +), using Phospho-Syntaxin 17 (Ser202) Antibody (upper), Myc-Tag (71D10) Rabbit mAb #2278 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised June 2016
Protocol Id: 263
Phospho-Syntaxin 17 (Ser202) Antibody recognizes transfected levels of syntaxin 17 protein only when phosphorylated at Ser202.Species Reactivity:
HumanSpecies predicted to react based on 100% sequence homology:
Polyclonal antibodies are produced by immunizing animals with a synthetic phospho-peptide corresponding to residues surrounding Ser202 of human syntaxin 17 protein. Antibodies were purified by peptide affinity chromatography.
Autophagy is a catabolic process for the autophagosomic-lysosomal degradation of bulk cytoplasmic contents (1,2). Autophagy is generally activated by conditions of nutrient deprivation but has also been associated with a number of physiological processes including development, differentiation, neurodegeneration, infection, and cancer (3).
Syntaxin 17/STX17 is a SNARE factor recruited to autophagosomes and required for autophagosome fusion to lysosomes. Syntaxin 17 interacts with SNAP29 (Qbc-SNARE synaptosome-associated protein 29) and the lysosomal factor VAMP8 (R-SNARE vesicle-associated membrane protein 8), as well as BRUCE, an inhibitor of apoptosis (IAP) protein, which is also involved in autophagosome/lysosome fusion (4,5).
Syntaxin 17 promotes initiation of PINK1/Parkin-independent mitophagy, which is regulated by depletion of the mitochondrial outer membrane protein Fis1 (6).
Phosphorylation of Syntaxin 17 at Ser202 by TBK1 promotes the formation of the mammalian pre-autophagosomal structure (mPAS) and interaction with core components Atg13 and FIP200.
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