Description
Product Usage Information
CST recommends transfection with 100 nM SignalSilence ® ULK1 siRNA I 48 to 72 hours prior to cell lysis. For transfection procedure, follow protocol provided by the transfection reagent manufacturer. Please feel free to contact CST with any questions on use. Each vial contains the equivalent of 100 transfections, which corresponds to a final siRNA concentration of 100 nM per transfection in a 24-well plate with a total volume of 300 μl per well.
Storage
SignalSilence ® siRNA is supplied in RNAse-free water. Aliquot and store at -20ºC.
Background
Two related serine/threonine kinases, UNC-51-like kinase 1 and 2 (ULK1, ULK2), were discovered as mammalian homologs of the gene in which mutants exhibited abnormal axonal extension and growth (1-4). Both proteins are widely expressed and contain an amino-terminal kinase domain followed by a central proline/serine rich domain and a highly conserved carboxy-terminal domain. The roles of ULK1 and ULK2 in axon growth have been linked to studies showing that the kinases are localized to neuronal growth cones and are involved in endocytosis of critical growth factors, such as NGF (5). Yeast two-hybrid studies found ULK1/2 associated with modulators of the endocytic pathway, SynGAP, and syntenin (6). Structural similarity of ULK1/2 has also been recognized with the yeast autophagy protein Atg1/Apg1 (7). Knockdown experiments using siRNA demonstrated that ULK1 is essential for autophagy (8), a catabolic process for the degradation of bulk cytoplasmic contents (9,10). It appears that Atg1/ULK1 can act as a convergence point for multiple signals that control autophagy (11), and can bind to several autophagy-related (Atg) proteins, regulating phosphorylation states and protein trafficking (12-16).~AMPK, activated during low nutrient conditions, directly phosphorylates ULK1 at multiple sites including Ser317, Ser555, and Ser777 (17,18). Conversely, mTOR, which is a regulator of cell growth and an inhibitor of autophagy, phosphorylates ULK1 at Ser757 and disrupts the interaction between ULK1 and AMPK (17).
