Peptides were synthesised in a 10 μmol scale (0.25 mmol/g) following the standard solid phase peptide synthesis (SPPS) methodology, using Fmoc-amino acids and Oxyma/DIC as coupling agents. Final deprotection and cleavage from the solid support was performed with 1.5 ml of cleavage cocktail: 94 TFA/1 TIS/ 2.5 DODT/2.5 H2O for 3 h. Obtained peptides were purified at 25°C by preparative reverse phase (RP)-HPLC performed on a PLC 2020 personal purification system (Gilson) with a preparative Nucleodur C18 HTec-column (5 μm, 250 × 16 mm; Macherey Nagel) and a flow rate of 10 ml/min. Detection of the signals was achieved with a UV detector at 220 nm wavelength. The eluents were MilliQ H2O and MeCN with addition of 0.1% TFA applied at a gradient of 5-40% MeCN.

Peptides were diluted and concentrations were determined according to [49]. The following concentrations were used in interaction assays: 3.5 μM, 7.0 μM, 14.0 μM (FOG1 peptides) and 17.5 μM, 35 μM (Ush peptides) in GST pulldown assays; 1.0 μM, 2.0 μM, 3.0 μM FOG1 peptides in immunoprecipitation assays.

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