Phospho-ATG14 Ser29 ELISA Kit

Sample

cell lysates

Species Reactivity

Human

Intended Use

The Phospho-ATG14 Ser29 ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Atg14 protein phosphorylated at Ser29.

Sensitivity

The Phospho-ATG14 Ser29 ELISA Kit detects endogenous levels of Atg14 protein phosphorylated at Ser29. The kit sensitivity is shown in Figure 1. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.


Figure 1. Atg14 is phosphorylated at Ser29 in 293T cells that were co-transfected with constructs expressing human Atg14 and mouse ULK1, but is not phosphorylated in cells transfected with only an Atg14 expression construct. The relationship between lysate protein concentration from Atg14/ULK1 co-transfected and Atg14-only transfected cells and the absorbance at 450 nm using the Phospho-Atg14 Ser29 ELISA Kit is shown in the upper figure. The corresponding western blots using phospho-Atg14 (Ser29) antibody (left panel) and Atg14 antibody (right panel) are shown in the lower figure. 293T cells were either co-transfected with constructs expressing GFP-tagged human Atg14 protein (hAtg14-GFP) and mouse ULK1 (mULK1) or just transfected with a construct expressing GFP-tagged human Atg14 protein (hAtg14-GFP), and then lysed.

General Description

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 is also associated with a number of physiological processes including development, differentiation, neurodegeneration, infection, and cancer (3). The molecular machinery of autophagy was largely discovered in yeast and is directed by a number of autophagy-related (Atg) genes. These proteins are involved in the formation of autophagosomes, cytoplasmic vacuoles that are delivered to lysosomes for degradation. The class III type phosphoinositide 3-kinase (PI3K) Vps34 regulates vacuolar trafficking and autophagy (4,5). Multiple proteins associate with Vps34, including p105/ Vps15, Beclin-1, UVRAG, Atg14, and Rubicon, to determine Vps34 function (6-12). Atg14 and Rubicon were identified based on their ability to bind to Beclin-1 and participate in unique complexes with opposing functions (9-12). Rubicon, which localizes to the endosome and lysosome, inhibits Vps34 lipid kinase activity; knockdown of Rubicon enhances autophagy and endocytic trafficking (11,12). In contrast, Atg14 localizes to autophagosomes, isolation membranes and ER, and can enhance Vps34 activity. Knockdown of Atg14 inhibits starvation-induced autophagy (11,12).
The serine/threonine kinase ULK1 phosphorylates Atg14 at Ser29 to promote autophagosome formation (13).