Imaging of Myc-tagged Protein Internalization

Briefly, J774 cells stably expressing Myc-tagged proteins were incubated at 37°C for 10 min in the presence of opsonized Texas Red-conjugated zymosan A (Fujifilm Wako Pure Chemical Industries), as described previously (Sakurai et al., 2018 (link)). The cells were washed in PBS and fixed with 100% methanol for 7 min at −20°C. Subsequently, the cells were incubated with 2% bovine serum albumin (BSA)/PBS for 30 min at 25°C, treated with anti-Myc antibodies, and stained with goat antimouse IgG antibodies conjugated to Alexa 488 (Thermo Fisher Scientific, Waltham, MA). Images were obtained using an LSM780 confocal laser-scanning microscope with a Plan-Apochromat 63 × /1.40 oil DIC M27 objective lens (Carl-Zeiss, Oberkochen, Germany).