Cfi apo tirf 100x na 1

Cells were incubated with Alexa Fluor 647-conjugated transferrin (T23366, Life Technologies, distributed by ThermoFisher Scientific, Waltham, USA) for 15 min, washed with PBS and fixed with paraformaldehyde (PFA). Images were acquired on a confocal microscope (Nikon Eclipse T1, Nikon Tokyo, Japan) with spinning disk (3I, Denver, CO, USA) with a 100x oil objective (Nikon CFI Apo TIRF 100x NA 1.4). z-stack images in a total range of 10 µm were acquired and maximum projections obtained with ImageJ. The quantification of the fluorescence signal was performed in ImageJ by measuring the mean grey value for the transferrin channel (far red) per pixel in the area of the cells.

Genome-edited SK-MEL-2 cells expressing the endogenous dynamin2 with a GFP tag (kind gift from David Drubin lab, UC Berkeley, USA; [Doyon et al., 2011 (link)]) were transfected using 0.5 µg of plasmids containing human endophilin A2 with C-terminal TagRFP-T under the control of the CMV promoter with X-tremeGENE HP DNA Transfection Reagent (06366236001, Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s protocol. The endophilin plasmid was kindly provided by Emmanuel Boucrot, University College London, UK. For the recovery experiments, human dynamin2 cherry (kindly provided by Christien Merrifield, LEBS, Gif-Sur-Yvette, France) was overexpressed together with a GFP version of human endophilinA2 (kindly provided by Emmanuel Boucrot [Boucrot et al., 2015 (link)]). Images of the cells were acquired on a microscope (Nikon Eclipse T1, Nikon Tokyo, Japan) with a 100x oil objective (Nikon CFI Apo TIRF 100x NA 1.4) using total internal reflection fluorescence (TIRF).