Quantifying Protein Synthesis and Amino Acid Uptake

Protein synthesis rate was measured using the Click-iT Plus OPP Protein Synthesis Assay (Thermo Fisher, C10456) according to manufacturer’s protocol. Geometric mean fluorescence intensity (compared to vehicle) was used as an indicator of the relative translation rate. AA uptake in autophagy-deficient HSCs was measured by the flow cytometry-based kynurenine (Kyn) assay as previously described [27 (link)], and cystine uptake was assessed using BioTracker Cystine-FITC Live Cell Dye (Sigma-Aldrich, SCT047).

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Borsa M., Obba S., Richter F.C., Zhang H., Riffelmacher T., Carrelha J., Alsaleh G., Jacobsen S.E, & Simon A.K. (2023). Autophagy preserves hematopoietic stem cells by restraining MTORC1-mediated cellular anabolism. Autophagy, 20(1), 45-57.

Publication 2023

Click-iT Plus OPP Protein Synthesis Assay BioTracker Cystine-FITC Live Cell Dye

Corresponding Organization :

Other organizations : University of Oxford, MRC Weatherall Institute of Molecular Medicine, MRC Human Immunology Unit, Karolinska Institutet, Max Delbrück Center

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Variable analysis

independent variables

Autophagy-deficiency in HSCs

dependent variables

Protein synthesis rate
Amino acid (AA) uptake (kynurenine assay)
Cystine uptake (BioTracker Cystine-FITC Live Cell Dye)

control variables

Vehicle treatment (for protein synthesis assay)

controls

Positive control: Not specified
Negative control: Vehicle treatment (for protein synthesis assay)

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