Cells, grown on coverslips, were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde (PFA) and quenched with 50 mM NH4Cl. Then, cells were permeabilised with 0.2% Triton X-100 for 5 or 7 min (depending on the antibody) and blocked for 30 min in PBS containing 10% FBS and 1% bovine serum albumin (BSA). Primary antibodies were detected with Alexa Fluor-conjugated secondary antibodies.
For lysosome staining, cells were incubated for 1 h with Lysotracker (1:1000) in complete medium before fixing. Images were collected using a laser scanning confocal microscope (LSM 510; Carl Zeiss MicroImaging, Inc.) equipped with a planapo 63× oil-immersion (NA 1.4) objective lens by using the appropriate laser lines. Images were acquired with the confocal pinhole set to one Airy unit, taking Z-slices from the top to the bottom of the cell by using the same setting (laser power, detector gain), as well as the same threshold of fluorescence intensity in all experimental conditions (control and silenced cells). Quantification and colocalisation analyses were carried out using LSM 510 software as previously described58 (link),59 (link). The mean fluorescence intensities were measured by drawing regions of interest (ROIs) around the entire cell and corrected for background. The number and size of fluorescent puncta were carried out by using ImageJ software59 (link). For GFP–EGFR internalisation experiments, the mean fluorescence intensity of surface and intracellular GFP signals was measured by drawing ROIs around plasma membrane labelled by a specific marker (CD55) and around areas, which excluded surface signals, respectively.
For lysosome staining, cells were incubated for 1 h with Lysotracker (1:1000) in complete medium before fixing. Images were collected using a laser scanning confocal microscope (LSM 510; Carl Zeiss MicroImaging, Inc.) equipped with a planapo 63× oil-immersion (NA 1.4) objective lens by using the appropriate laser lines. Images were acquired with the confocal pinhole set to one Airy unit, taking Z-slices from the top to the bottom of the cell by using the same setting (laser power, detector gain), as well as the same threshold of fluorescence intensity in all experimental conditions (control and silenced cells). Quantification and colocalisation analyses were carried out using LSM 510 software as previously described58 (link),59 (link). The mean fluorescence intensities were measured by drawing regions of interest (ROIs) around the entire cell and corrected for background. The number and size of fluorescent puncta were carried out by using ImageJ software59 (link). For GFP–EGFR internalisation experiments, the mean fluorescence intensity of surface and intracellular GFP signals was measured by drawing ROIs around plasma membrane labelled by a specific marker (CD55) and around areas, which excluded surface signals, respectively.
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