HTRF Human and Mouse Phospho-ATG16L1 Ser278 Detection Kit HTRF®

The HTRF Phospho-ATG16L1 (Ser278) assay measures ATG16L1 when phosphorylated at Serine 278. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.

The HTRF Phospho-ATG16L1 (Ser278) assay uses two labeled antibodies: one with a donor fluorophore, the other with an acceptor. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, and the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving the two labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein’s phosphorylation state under a no-wash assay format.

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of the HTRF Phospho-ATG16L1 (Ser 278) detection reagents.

This protocol enables the cells' viability and confluence to be monitored.

Detection of phospho ATG16L1 (Ser 278) with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required.

This HTS-designed protocol enables miniaturization while maintaining robust HTRF quality.

Human PC-3 cells were plated at different cell densities in a 96-well plate. After 24 h incubation at 37°C, 5% CO2, the cells were treated with 2.5 µM of AZD2014, a mTOR inhibitor, for 3 h at 37°C, 5% CO2. After culture medium removal, cells were then lysed with 25 µl of supplemented lysis buffer #3 (1X) + BR (2X) for 1 h at RT under gentle shaking. After cell lysis, 16 µL of lysate were transferred into a 384-well low volume white microplate, then 4 µL of the HTRF Phospho-ATG16L1 (Ser 278) detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.

The HTRF Phospho-ATG16L1 (Ser278) assay enables correct detection of phospho-ATG16L1 (Ser278) from just 25,000 cells (S/N > 3) PC-3 cells per well. A 100k-cell density was selected for pharmacological validations.

Human U-87 MG  cells  were plated in a 96-well plate (100,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with a dose-response of AZD2014, a mTOR inhibitor, for 3 h at 37 °C, 5% CO2. Following culture medium removal, cells were lysed with 25 µl of supplemented lysis buffer #3 (1X) + BR (2X) for 1 h at RT under gentle shaking. After cell lysis, 16 µL of lysate were transferred into a 384-well low volume white microplate, then 4 µL of the HTRF Phospho-ATG16L1 (Ser 278) or Total ATG16L1 detection reagents were added. The HTRF signal for both kits was recorded after an overnight incubation at room temperature.

As expected, AZD2014, a potent mTOR inhibitor, induced autophagy activation, leading to a dose-dependent increase in ATG16L1 phosphorylation on Serine 278, without any significant effect on the expression level of the total ATG16L1 protein.

Human U-87 MG  cells  were plated in a 96-well plate (100,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with 2.5 µM of AZD2014, a mTOR inhibitor, and  a dose-response of ULK-101, an autophagy kinase ULK1/2 inhibitor, for 3 h at 37°C, 5% CO2. Following culture medium removal, cells were lysed with 25 µl of supplemented lysis buffer #3 (1X) + BR (2X) for 1 h at RT under gentle shaking. After cell lysis, 16 µL of lysate were transferred into a 384-well low volume white microplate, then 4 µL of the HTRF Phospho-ATG16L1 (Ser 278) or Total ATG16L1 detection reagents were added. The HTRF signal for both kits was recorded after an overnight incubation at room temperature.

As expected, ULK-101, a potent and dual autophagy kinase ULK1/2 inhibitor, repressed autophagy activation induced by AZD2014, leading to a dose-dependent decrease in ATG16L1 phosphorylation on Serine 278 without any significant effect on the expression level of the ATG16L1 total protein.

The  human phospho-ATG16L1 (Ser278) expression level was assessed using the HTRF Phospho-ATG16L1 (Ser278) kit in Wild Type (WT) and ATG16 knock-out (KO) HAP1 cells. 

The cell lines were cultured in flasks and incubated 48 h at 37°C, 5% CO2. Then the cells were treated with 100 nM of Calyculin A for 10 min. Following culture medium removal, cells were lysed with supplemented lysis buffer #3 (1X) + BR (2X) for 30 min at RT under gentle shaking.  After cell lysis, 16 µL of lysate were transferred into a 384-well low volume white microplate, then 4 µL of the HTRF Phospho-ATG16L1 (Ser 278) detection reagents were added. In parallel, the GAPDH level was monitored using the GAPDH Housekeeping Cellular Kit (# 64GAPDHPET/G/H). 4 µL of cell lysate were prediluted with 180 µL of diluent #11. Then 16 µL of this diluted material were mixed with 4 µL of the HTRF GAPDH Housekeeping Cellular detection reagents. The HTRF signals for the two kits were recorded after an overnight incubation at room temperature.

The measured level of GAPDH was equivalent under the two conditions tested. In contrast, while the HTRF Phospho-ATG16L1 (Ser278) signal was significantly positive in HAP1 WT cells, it had dramatically decreased (equivalent to the negative signal) in HAP1 ATG16 KO cells, demonstrating the specificity of the HTRF Phospho-ATG16L1 (Ser278) assay for the detection of the ATG16L1 protein.

HAP1 ATG16 KO (10bp deletion) cell line from Horizon Discovery # HZGHC000296c010

HAP1 WT cell line from Horizon Discovery # C631

Human (PC-3), mouse (C2C12), and rat (H4-II-E) cell lines were plated in a 96-well plate (100,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with 2.5 µM of AZD2014, an mTOR inhibitor, for 3 h at 37°C, 5% CO2. Following culture medium removal, cells were lysed with 25 µL of supplemented lysis buffer #3 (1X) + BR (2X) for 1 h at RT under gentle shaking. After cell lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate, then 4 µL of the HTRF Phospho-ATG16L1 (Ser 278) detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.

The HTRF Phospho-ATG16L1 (Ser278) assay efficiently displayed significant positive signals in the cell lines from the 3 species tested, demonstrating its ability to detect human, mouse, and rat versions of the protein, and its suitability for translational research.

HCT116 cells were cultured in flasks and incubated 48 h at 37°C, 5% CO2. Then the cells were treated with 100 nM of Calyculin A for 10 min. After culture medium removal, cells were lysed with supplemented lysis buffer #3 (1X) + BR (2X) for 30 min at RT under gentle shaking.  Soluble supernatants were collected after a 10 min centrifuging. Equal amounts of lysates were used for a side-by-side comparison of WB and HTRF.

The HTRF Phospho-ATG16L1 (Ser278) assay was 4 times more sensitive than the Western Blot assay.

Autophagy is an evolutionary conserved cellular process that occurs in virtually all eukaryotic cells, ranging from yeast to mammals. Autophagy is crucial in the maintenance of homeostasis, being involved in numerous physiological processes including stress responses (e.g. starvation, hypoxia, high temperature), cell growth, and aging. Conversely, dysfunctions in autophagic mechanisms have been associated with diseases such as cancer, neurodegenerative diseases, infectious diseases, and cardiac and metabolic diseases.

There are 3 types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy.

Macroautophagy (hereafter referred to as autophagy) starts with the formation of a bud from lipid phosphatidylinositol 3-phosphate rich membranes such as reticulum endoplasmic, Golgi, or plasma membranes, and known as a pre-autophagosomal structure (PAS). This cup-shaped structure requires the ULK1/Atg1 complex formed by Atg13, Atg101, FIP200, and ULK1 proteins. The transmembrane protein Atg9, contained in vesicles, is involved in the recruitment of Atg2-Atg18 to the PAS. With its phospholipid transfer activity, the Atg2 protein supplies phospholipids necessary for membrane elongation. ULK1 phosphorylates and activates Beclin1, which partners with Vsp15, Vsp34, Atg14, and NRFB2/Atg38 to form a class III Phosphatidylinositol 3-kinase (PI3KC3-Complex 1). This complex leads to the production of phospho-inositol triphosphate (PIP3), which further directs the recruitment of PI3P binding Atg18/WIPI 1–4 proteins and the Atg12-Atg5-Atg16 complex. The latter complex, along with Atg4 and Atg7, is implicated in the LC3B lipidation (LC3-I to LC3-II conversion) on the phagophore and contributes to autophagosome maturation, which subsequently fuses with lysosomes forming an autolysosome where trapped cellular components are degraded and building blocks recycled.

Phosphorylated ATG16L1 at Ser278 is only present on newly-forming autophagosomes, making its quantification an ideal surrogate for autophagic vesicle formation and autophagy induction. Importantly, its levels are not affected by prolonged stress or late-stage autophagy blocks, which can jeopardize autophagy analysis. Moreover, ATG16L1 phosphorylation by the autophagy kinase ULK1 is a conserved indicator of autophagy induction, which is activated by multiple stimuli.

Taken together, analysis of ATG16L1 phosphorylation represents an excellent readout when studying autophagy induction.