*2.9. Immunofluorescence Staining*

Cells were grown on coverslips, fixed in 4% paraformaldehyde (Sigma-Aldrich) solution for 10 min. Then, samples were permeabilized in 0.2% Triton X-100/PBS for 5 min. After blocking with 3% BSA (Sigma-Aldrich) in PBS, cells were probed with the indicated primary antibodies and with Alexa Fluor 488-conjugated anti-rabbit IgG (Thermo Fisher Scientific, #A11034) or Alexa Fluor 555-conjugated anti-rabbit IgG (Thermo Fisher Scientific, #A-21428). Nuclei were counterstained with Hoechst (Fluka Biochemika, Buchs, Switzerland). Fluorescently labelled samples were imaged using a confocal LEICA TCS SP5 microscope (Leica, Heidelberg, Germany) equipped with an argon/krypton laser. Confocal sections were acquired at 0.4 μm intervals.

Excitation/emission wavelengths were 346/460 nm for Hoechst, 488/520 nm for Alexa Fluor 488 and 555/580 nm for Alexa Fluor 555. Images are shown as three-dimensional (3D) maximal projections reconstructed from z-stacks unless otherwise indicated. Magnification: 63 × zoom 5 (bar = 5 μm), 63 × (bar = 25 μm) and 20 × (bar = 100 μm).
