Alexa flour 488 goat anti rabbit igg

The MTP‐LRS‐UNE‐L or M12‐LRS‐UNE‐L were added to C2C12 myoblasts or HEK293 cells for the specified time in growth media (Dulbecco's Modified Eagle Medium containing 4.5 g/L glucose with 10% foetal bovine serum and 1% penicillin–streptomycin), as indicated in figure legends. For FACS analysis, the cells were washed twice with PBS and then incubated with APC anti‐DYKDDK tag (637308; Biolegend, San Diego, CA, USA) for 60 min at room temperature without or with permeabilization of the cell membrane using a Cell Fixation and Permeabilization kit (ab185917; Abcam, Cambridge, UK). After washing with PBS, the cells were analysed by flow cytometry (BD FACS Calibur; BD Biosciences, San Jose, CA, USA), and intracellular levels of MTP‐LRS‐UNE‐L or M12‐LRS‐UNE‐L were analysed. For M12‐LRS‐UNE‐L staining in C2C12 cells, the cells were washed twice with PBS, fixed in 10% formaldehyde, and permeabilized in 0.1% Triton X‐100. After the cells were blocked with 3% bovine serum albumin in PBS, the cells were subjected to intracellular staining with anti‐Flag at 4°C overnight. The cells were incubated with Alexa Flour 488 goat anti‐rabbit IgG (A11034, Molecular Probes, Thermo Fisher Scientific). Fluorescent images of cells were captured using a Laser Scanning Confocal Microscope 700 (LSM 700, Carl Zeiss) equipped with an LSM T‐PMT camera (Carl Zeiss, LSM 700).