Model molecules to identify the signals in Navs that mediate the trafficking regulation. Navs consist of four domains (I, II, III and IV) connected by three intracellular loops (L1–L3); each domain is formed by six transmembrane segments (TM; S1–S6). Both the N-terminus (N) and the C-terminus (C) of Navs are located in the cytoplasm. CD8α and TFR1, which have distinct cell-surface localization, are adapted to detect the roles that particular regions of the Navs have in subcellular distribution. The type I membrane protein CD8α is suitable for testing three intracellular loops, the C-terminus and the transmembrane segments that pass through the membrane in the extracellular to intracellular direction (S2, S4 and S6), whereas the type II membrane protein TFR1 is appropriate for testing the N-terminus and the transmembrane segments that pass through the membrane in the opposite direction (S1, S3 and S5). A Myc tag is inserted to the N-terminus of CD8α or the C-terminus of TFR1, and non-permeabilized immunostaining is performed with Myc antibody in transfected living cells to label these proteins on the plasma membrane. A Flag tag is inserted to the N-terminus of TFR1 for the permeabilized immunostaining with Flag antibody to label the protein in whole cell. The sequence of CD8α or TFR1 is replaced with corresponding region of Nav, such as Myc-CD8α(TMIVS6), Myc-CD8α-C, Flag-TFR1(TMIVS1)-Myc and N-TFR1-Myc. This figure is adapted from Li et al.