HTRF Human and Mouse Phospho-ATG14 Ser29 Detection Kit HTRF®

The HTRF Phospho-ATG14 (Ser29) assay measures ATG14 when phosphorylated at Ser 29. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.

The Phospho-ATG14 (Ser29) assay uses 2 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 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 2 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 Phospho-ATG14  Ser 29 HTRF detection reagents.

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

Detection of phospho ATG14 Ser 29 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 HCT116 cells  were plated in a 96-well culture-treated plate (200,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with a dose-response of MRT68921 for 4h at 37 °C, 5% CO2. After culture medium removal, cells were then lysed with 25 µl of supplemented lysis buffer #4 (1X) for 30 min at RT under gentle shaking. After cell lysis, 14 µL of lysate were transferred into a 384-well low volume white microplate and 2 µL of Activation Buffer then 4 µL of the HTRF Phospho-ATG14 (Ser 29) or Total-ATG14 detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.

As expected, MRT68921, a potent and dual autophagy kinase ULK1/2 inhibitor, repressed ULK1 activation, reducing the autophagy initiation machinery and leading to a dose-dependent decrease in ATG14 phosphorylation without any effect on the expression level of the ATG14 total protein.

Human HCT116 cells  were plated in a 96-well culture-treated plate (200,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with a dose-response of AZD2014 (Vistusertib) for 4h at 37°C, 5% CO2. After culture medium removal, cells were then lysed with 25 µl of supplemented lysis buffer #4 (1X) for 30 min at RT under gentle shaking. After cell lysis, 14 µL of lysate were transferred into a 384-well low volume white microplate. 2 µL of Activation Buffer, then 4 µL of the HTRF Phospho-ATG14 (Ser 29) or Total-ATG14 detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.

As expected, AZD2014, a potent mTOR inhibitor, alleviated ULK1 inhibition, increasing autophagy initiation machinery, and leading to an increase in ATG14 phosphorylation on Serine 29 without any effect on the expression level of the ATG14 total protein.

Human basal phospho (Ser29) ATG14 expression level was assessed with the Phospho (Ser29) HTRF kit in HAP1 cells with both Wild Type (WT) and ATG14 knock-out (KO). Normalization was done using the GAPDH Housekeeping HTRF kit.

The cell lines were cultured in flasks at 37°C, 5% CO2 and after culture medium removal, cells were then lysed with 1 mL of supplemented lysis buffer #4 (1X) per 10 million cells, for 30 min at RT under gentle shaking. After cell lysis, 14 µL of lysate were transferred into a 384-well low volume white microplate, and 2 µL of Activation Buffer, then 4 µL of the HTRF Phospho-ATG14 (Ser 29) detection reagents were added. For GAPDH, 8 µL of lysate was prediluted with 60 µL diluent before mixing 16 µL of the diluted material with 4 µL of the GAPDH detection reagents. The HTRF signal was recorded after an overnight incubation at room temperature. In HAP1 KO ATG14 cells, the HTRF signal was equivalent to the non-specific signal (not shown) indicating a complete ATG14 gene silencing and thus demonstrating the specificity of the HTRF Phospho(Ser29) ATG14 kit for the ATG14 protein. GAPDH normalization was applied to take into account slight growth differences between parental and KO cells (the phospho-ATG14 over GAPDH ratio is presented here).

HAP1 ATG14 KO (1bp del) cell line from Horizon Discovery # HZGHC024663c012

HAP1 WT from Horizon Discovery # C631

Cell lysates from various human, mouse, and rat cell lines were cultured at different densities and lysed in supplemented LB4 lysis buffer.

After culture medium removal, cells were then lysed with appropriate volumes of supplemented lysis buffer #4 (1X) for 30 min at RT under gentle shaking. After cell lysis, 14 µL of lysates were transferred into a 384-well low volume white microplate and 2 µL of Activation Buffer, then 4 µL of the HTRF Phospho-ATG14 (Ser 29) detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.

As expected, the phospho-ATG14 (Ser29) assay detected human, mouse, and rat versions of the protein, as is shown by significant positive signals in the cell lines tested from these 3 species. This demonstrates the versatility of the assay and its suitability for translational research.

HCT116 cells were cultured in a T175 flask in complete culture medium at 37°C, 5% CO2. After incubation, the cells were lysed with 3 mL of supplemented lysis buffer #4 (1X) for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 14 µL of each dilution were transferred into a low volume white microplate before the addition of 2µL of Activation Buffer, then 4 µL of HTRF Phospho-ATG14 (Ser29) detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

The side by side comparison of Western Blot and HTRF demonstrates that the HTRF assay is 16-fold more sensitive than the Western Blot, at least under these experimental conditions.

Cellular Autophagy is a specialized degradation and recycling process that is instrumental for cell homeostasis, being activated in response to several different stresses. There are 3 types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy. Macroautophagy pathways involve several key steps: initiation, nucleation, elongation of phagophore complexes, then sequestration of cytoplasmic cargos with LC3-PE recruitment followed by fusion with lysosome yielding to cargo degradations.

Biogenesis of the autophagosome is controlled by sequential and concerted actions of the so-called autophagy related proteins, ATGs, which are activated and recruited to the ER and autophagosome membranes. The recruitment of the ULK1 complex is the first event in the initiation step. ULK1 is a Ser/Thr kinase which forms a complex with the Atg13, Atg101 and FIP200 proteins. This complex is the most upstream component of the core autophagy machinery and is therefore the key initiator of autophagy in mammalian cells. ULK1 is regulated by the key nutrient/energy-sensitive kinases mTOR and AMPK, which are both able to phosphorylate ULK1 on Serine 317 and Serine 556, and directly regulate its kinase activity.

The Activated ULK1 complex then binds ATG14 via ATG13, and phosphorylates ATG14 on Serine 29. The kinase activity of phosphorylated and activated ATG14 stimulates other proteins of the PIK3C3 complex (nucleation) that is responsible for the critical step of phosphorylation of phosphatidylinositols (PI) into phosphatidylinositol-3-phosphate (PI3P). This in turn is responsible for the formation of the initial phagosomal membrane structure and later allows fixation of LC3-II using other ATG proteins (ATG16/12/5 complex), thus generating a support for elongation and closing steps.

This means tha phosphorylation of ATG14 protein on serine 29 is a key early marker of the nucleation step in the engagement of the macroautophagic process.