FIGURE 5.

EHD1 interacts with CD44 and enhances its recycling, thus preventing its lysosomal degradation. (A) Amino acid sequences of peptides specifically associated with the EHD1 protein (top) or CD44 protein (bottom) were identified using MS. (B) IF staining was used to detect the colocalisation of EHD1 and CD44 in LUAD cells. Endogenous (C and D) and exogenous (E) IP experiments validated the interaction of EHD1 and CD44. (F) Western blot analysis was performed to examine the effect of EHD1 knockdown on the regulation of CD44 protein expression. (G) The MFI of relative surface CD44 was determined by FACS. Representative flow cytometry data and statistical analysis of cell surface CD44 in A549 (left) and H1299 cells (right) are shown. The relative surface level is related to the amount of the receptor that undergoes ligand fixation, stimulation, internalisation and recycling to the cell surface. (H) The biotinylation and recycling assay of CD44 by ELISA showed CD44 recycling in NC and EHD1^KD A549 cells. (I) A CHX chase assay was performed to analyse the half‐life of the CD44 protein in NC and EHD1^KD A549 cells. Cells were incubated in the presence of CHX (20 μg/ml) for 0, 1, 2, 3 or 4 h. (J) Western blot analysis of CD44 expression in NC and EHD1^KD treated with the proteasome inhibitor MG132 for 8 h. (K) Western blot analysis of CD44 expression in NC and EHD1^KD cells treated with NH₄Cl, an inhibitor of the lysosomal pathway, for 8 h. (L) CD44 ubiquitination assays in LUAD cells transfected with the Total‐Ub, K63‐Ub or K48‐Ub plasmid after treatment with NH₄Cl for 8 h. The data are shown as the mean ± SD values. *p < .05, **p < .01 and ***p < .001