EGFR and ATG9 Trafficking upon PFL Treatment

The cellular distribution of EGFR upon PFL treatment was observed by immunofluorescence microscopy as described previously [6 (link)]. Confluent MKN28 cells growing on coverslips in a 6-well plate were treated with 30 μg/mL Alexa488-conjugated-PFL in RPMI-1640 and incubated for various periods of time. The cells were fixed with 80 % acetone and incubated with mouse monoclonal anti-EGFR antibody (Thermo Scientific, UK) at 37 °C for 1 h. Subsequently, the cells were incubated with Alexa568-conjugated goat anti-mouse IgG antibody (Life technologies, Japan) at 37 °C for 1 h. The cells were mounted using Vectashield with DAPI (Vector Laboratories) and were observed using a confocal laser scanning microscope (IX70; Olympus, Japan). The cellular distribution of EGFR together with ATG9 was examined in a similar way using mouse anti-EGFR antibody and rabbit anti-ATG9 antibody. Accordingly, a distinct secondary antibody was employed to visualize EGFR and ATG9, using Alexa488-conjugated goat anti-mouse IgG antibody and Alexa568-conjugated goat anti-rabbit IgG antibody, respectively. The cellular distribution of α2 integrin following PFL treatment was observed as above after incubation with 10 μM PFL for 24 h.