c-Kit Internalization Assay

The internalization assay was carried out as previously described [34 (link)]. Briefly, the cells were incubated with cycloheximide (75 µg/mL) to prevent the transport of de novo synthesized c-Kit to the cytoplasmic membrane. Then, the cells were blocked with human BD Fc block™ (BD Biosciences, San Diego, CA, USA), to inhibit the Fc receptor-mediated internalization of antibodies, and 5% BSA (CellNest, Gyeonggi-do, Korea) at RT for 10 min. The cells were washed with PBS and incubated in the presence or absence of the 4C9 antibody (1 µg/mL) at 4 °C or 37 °C for 1–4 h. The cells were then incubated with FITC-conjugated anti-human IgG (0.3 μg/mL, Invitrogen) for 1 h, washed three times with PBS, and analyzed using a CyFlow^® Cube 6 (PARTEC).