ARF4 and VCP bind NIS in vitro and modulate PM NIS. A, Co-IP assays in MDA-MB-231 (NIS+) cells showing specific interaction between NIS and ARF4 (left) or VCP (right). B, Same as A, but in TPC-1 (NIS+) cells. C, PLA demonstrating specific interaction between NIS-MYC and ARF4 (top) or VCP (bottom) in MDA-MB-231, TPC-1, and HeLa cells. Red fluorescent spots, specific interactions. Blue, DAPI nuclear staining. Magnification, ×100. Scale bars, 10 μm. D and E, Western blot analysis of NIS protein levels at the PM relative to Na+/K+ ATPase following the CSBA in TPC-1 (NIS+) cells (D) and MDA-MB-231 (NIS+) cells (E) after ARF4 transfection. F and G, Same as D and E, but after VCP transfection. *, P < 0.05; ***, P < 0.001.