Identification of ARF4 and VCP as regulators of NIS activity. A, Western blot analysis of whole-cell lysate and PM fraction in MDA-MB-231 (NIS+) cells used in MS/MS. B, Top hits for putative NIS interactors identified by MS/MS (peptides ≥ 6). C and D, Western blot analysis and RAI uptake of MDA-MB-231 (NIS+) cells transfected with esiRNA specific for indicated NIS interactors. NT, nontransfected cells. E and F, Western blot analysis and RAI uptake in MDA-MB-231 (NIS+) cells, TPC-1 (NIS+) cells, and human primary thyrocytes transfected with ARF4 siRNA (E) or ARF4 (F). G and H, Same as E and F, but cells transfected with VCP siRNA (G) or VCP (H). NS, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.