Clathrin heavy chain

Cells were grown to 50-70% confluency on coverslips in 24-well plates and subjected then to different experimental immunofluorescence analyses. To assess the intracellular uptake and trafficking of nanosystems, cells were treated with 0.44 mg mL -1 PSN-pDNA (TopFluor-labelled PSN/Cy5-labelled pDNA) for different time points. Specifically, to test the intracellular trafficking, cells were treated with PSN-pDNA for 4 h then medium was replaced, and cells were cultured on complete medium for 2 and 4 h. After, cells were fixed with 4% paraformaldehyde for 20 min at RT followed by 5 min permeabilization with 0.1% Triton™ X-100. Then, coverslips were incubated with corresponding primary antibodies, caveolin-1 (3267, Cell Signalling Technology, Massachusetts, USA), clathrin heavy chain (ab21679, Abcam, Cambridge, UK), EEA1
(3288, Cell Signaling Technology), LAMP1 (9091, Cell Signaling Technology), calreticulin (PA3-900, Invitrogen) or GM130 (610822, BD Biosciences). After, incubation with secondary antibody was carried out using Alexa Fluor 546-labelled (A-11035, Invitrogen) for 1 h at RT. Cell nuclei were stained with DAPI (D1306, Invitrogen). Images were captured on a LSM710 confocal microscope (ZEISS, Oberkochen, Germany). The analysis and quantification were done using ImageJ software.