Phospho-mTOR (Ser2448) cellular kit
Convenient, fast assay quantifying Phoshpo-mTOR (Ser2448) modulation
This HTRF kit enables the cell-based quantitative detection of ATG14 phosphorylation at Ser29 as a readout of the autophagy pathway.
ATG14, Autophagy related protein 14, or BAKOR for Beclin 1-associated autophagy-related key regulator, is a key player in the autophagosome nucleation step in macroautophagy. Upon cellular stress, the nutrient/energy-sensitive sensors mTOR and AMPK lead to the activation of the ULK1 complex, which allows phosphorylation of ATG14 on Serine 29, in turn enabling the downstream activation of the PIK3C3 autophagosome nucleation complex.
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.
HTRF cellular phospho-protein assays
Physiologically relevant results fo fast flowing research - Flyers
Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences
Insider Tips for successful sample treatment - Technical Notes
Optimize your HTRF cell signaling assays on tissues
HTRF and WB compatible guidelines - Technical Notes
Best practices for analyzing tumor xenografts with HTRF phospho assays
Protocol for tumor xenograft analysis with HTRF - Technical Notes
Key guidelines to successful cell signaling experiments
Mastering the art of cell signaling assays optimization - Guides
HTRF® phospho-protein platform facilitates the dissection of signaling pathways involved in insulin resistance and metabolic disorders
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
HTRF® cell signaling platform combined with iCell® Hepatocytes
A solution for phospho-protein analysis in metabolic disorders - Posters
HTRF phospho-assays reveal subtle drug-induced effects
Detailed protocol and direct comparison with WB - Posters
Implement HTRF phospho-protein assays at every step of the drug discovery process, from in vitro to in vivo models
A single technology for 2D cells, 3D cells, and xenograft models - Posters
Simplify complex pathway dissection by combining the power of HTRF® cellular phospho-assays and the flexibility of the CyBi®-FeliX liquid handling system
PI3K/AKT/mTor translational control pathway - Posters
Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells
Analysis of a large panel of diverse biological samples and cellular models - Posters
From 2D, 3D cell cultures to xenografts: A smart HTRF platform to maximize anticancer drug discovery
One technology across all samples - Application Notes
HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts
Tumor xenograft analysis: HTRF versus Western blot - Application Notes
HTRF cell-based phospho-protein data normalization
Valuable guidelines for efficiently analyzing and interpreting results - Application Notes
HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers
Increased flexibility of phospho-assays - Application Notes
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling
Analyse of PI3K/AKT/mTor translational control pathway - Application Notes
Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening
In collaboration with Bayer - Scientific Presentations
Unleash the potential of your phosphorylation research with HTRF
A fun video introducing you to phosphorylation assays with HTRF - Videos
Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection
Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes
Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)
Learn how to reduce time and sample consumption - Application Notes
Assessment of drug efficacy and toxicity by combining innovative technologies
Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes
Product Insert ATG14 p-S29 Kit / 64ATG14S9PEG-64ATG14S9PEH
64ATG14S9PEG-64ATG14S9PEH - Product Insert
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