Bl ac40ts c2 cantilevers

To investigate the protein coverage and protein height, atomic force micrographs of the SLB surface before and after protein addition were taken. ENTH^WT and ENTH^R114A (1 µM) were added for 2 h at RT. The solution was mixed with a stirring bar to ensure homogenous distribution of the protein. Afterwards 10 × 10 µm² and 1 × 1 µm² areas of the substrates were imaged in contact mode with BL-AC40TS-C2 cantilevers (f = 85.4–139.1 kHz, k = 0.03–0.12 N m⁻¹, Olympus, Tokio, Japan). Measurements were performed using a JPK Nanowizard 4 (JPK Instruments, Berlin, Germany) equipped with a CCD camera (Fire Wire CCD color camera, The Imaging Source, NC, USA). The protein height and cluster surface coverage were calculated with a MATLAB routine.

We fixed the cell-cultured MPM onto the MPM holder dish using a perfusion system (custom-made by Nagata Industry Co.) with the membrane facing up. The MPM was bathed in Leibovitz’s L-15 medium (Thermo Fisher Scientific) supplemented with 1% penicillin/streptomycin (Fig. 1f). Then, the MPM holder dish was set on the stage of an inverted fluorescence microscope (Eclipse Ti2, Nikon) coupled to a JPK NanoWizard 4 BioAFM (Bruker). The temperature was kept at 37 °C using the heater equipped with this microscope to observe live cells. All AFM imaging was performed using BL-AC40TS-C2 cantilevers (Olympus, spring constant approximately 0.1 N/m). We used the QI settings with following parameters: topography Imaging: 2.5 × 2.5 or 0.5 × 0.5 μm scale, 64 × 64 pixels, Z-length 1 μm, setpoint 0.1 nN, speed 166 μm/s; imaging at the 100 × 100 nm scale: 64 × 64 pixels, Z-length 50 nm, setpoint 0.1 nN, speed 166 μm/s; for Young’s modulus measurement: 100 × 100 nm scale, 64 × 64 pixels, Z-length 2 μm, setpoint 0.1 nN, speed 166 μm/s; for the experiments of Fig. 4: 3.5 × 3.5 μm scale, 512 × 512 pixels, Z-length 1 μm, setpoint 0.1 nN, speed 166 μm/s.