Eea1 antibody

Immunofluorescence staining was used to identify early endosome in PC9 cells. After FMSN incubated with PC9 cells for 15, 30 and 60 min, the PC9 cells were washed with PBS and fixed with 1% paraformaldehyde for 10 min. The PC9 cells were then permeabilized with 0.1% NP-40 for 20 min, and then the cells were subsequently incubated overnight with the early endosome marker EEA1 antibody (Abcam, ab70521). The cells were then washed with PBS and treated with the fluorescence-conjugated secondary antibody (Jakson Immunoreseatch, 111-545-003, 315-545-003) and DAPI. The stained PC9 cells were observed using a fluorescence microscope.

Cells were seeded onto poly-l-lysine-coated glass cover-slides. For binding experiments, cells were incubated with biotinylated nanocages in DMEM 10% FBS for 1 h at 4 °C, washed in PBS, fixed in 4% paraformaldehyde, and incubated for 5 min with NaBH₄. For uptake experiments, cells were incubated with nanocages at 37 °C for different time periods, fixed with 4% paraformaldehyde, and permeabilized for 4 min with Tris/Triton (Tris HCl 0.1 M, Triton 0.1%, pH 7.7). Rabbit polyclonal anti-flotillin-1 antibody (Santa Cruz Biotechnology Inc., Dallas, TX, USA) was used to detect the flotillin-1 protein. Early endosomes were visualized with polyclonal EEA1 antibody (Abcam Inc., Toronto, ON, Canada) and lysosomes with mouse monoclonal anti-LAMP-1 antibody (Abcam Inc., Toronto, ON, Canada). Donkey anti-rabbit IgG and donkey anti-mouse IgG, both Rhodamine Red-X-conjugated AffiniPure (Jackson ImmunoResearch, Cambridgeshire, UK) were used as secondary antibodies [24 (link)]. Biotinylated cages were detected by using streptavidin–FITC (Jackson ImmunoResearch, Cambridgeshire, UK). The nuclei were stained with DAPI (Invitrogen, Carlsbad, CA, USA). Images were obtained with a laser confocal fluorescent microscope Olympus FV1000 at 60× magnification, and the fluorescence signal was evaluated with the IMARIS software. Co-localization events were evaluated as previously described [24 (link)].