Opaque 384 well plate

The protein buffers were exchanged for anisotropy buffer (20 mM Hepes/KOH pH 7.5, 200 mM KCl, 5 mM MgCl₂, 1 mM DTT) using Zeba spin columns (Thermo Scientific). 1 µM BSA was added to prevent unspecific binding of the fluorescently labelled peptide to the microplate (Greiner Bio-One opaque 384 well plate). Fluorescence anisotropy measurements were performed using the LP-peptide (MFSLPTL) or the NR-peptide (NRLLLTG) labelled at the N terminus with fluorescein (Peptide Specialty Laboratories GmbH, Heidelberg). Two-fold serial dilutions of each protein (ScSsz1-Zuo1N or ScSsz1-Zuo1NΔLP) were mixed in a 1:1 ratio with 40 nM peptides in anisotropy buffer in 384-well opaque plates (Greiner Bio One) and were incubated at room temperature for 30 min. Measurements were performed in triplicate using a plate reader (SpectraMax M5e Multi Mode Microplate reader, Molecular Devices). To determine the binding constant (K_D) the data were fitted to a one-site binding equation using Python⁵⁷ .

Cell cultures grown on 12 mm coverglasses were transferred to custom‐made cups (inner diameter = 13 mm, wall thickness = 0.5 mm) and rested in culture medium for 1 h in the incubator. This was followed by two washes with HBSS and incubation with HBSS (265 µL) containing iron oxide particles for 1 h at room temperature. At the beginning of the experiment, a pre‐stimulation sample (80 µL) was drawn and immediately frozen on dry ice. Then the experimental manipulation was carried out and a post‐stimulation sample (80 µL) was collected and frozen on dry ice.
ATP concentration in the cell culture medium samples was measured using an assay (CellTiter‐Glo, Promega) based on the luciferin‐luciferase reaction. Specifically, each sample (20 µL) as well as a series of ATP standard solutions (20 µL, 0–80 nm) were added to an opaque 384‐well plate (Greiner Bio One International), and each of them was mixed with the luciferin‐luciferase reagent (20 µL). The bioluminescence was recorded using an IVIS Lumina imaging system (PerkinElmer), and the photon count was converted to ATP concentration using the standard curve.