Defect in the internalization of transferrin in Sac2 KO MEFs. Internalization of biotinylated transferrin over time was analyzed biochemically in WT and Sac2 KO MEFs using an ELISA-based assay (see Materials and methods for details). Data were expressed as mean ± SD (error bars) of three independent experiments (Student’s t test, **, P < 0.01; ***, P < 0.001).