Figure 2. GRIM-1 isoforms interact with endogenous NAF1 in mammalian cells.

HeLa cells transiently expressing Myc-tagged GRIM-1 isoforms were analyzed for interaction with NAF1 using the indicated antibodies. A) Expression levels of GRIM-1 isoforms (top), NAF1 (middle) and actin (bottom) in the indicated transfectants. GRIM-1γ levels are low compared to GRIM-1α/β when normalized to actin levels. No significant change in NAF1 levels are observed upon GRIM-1 expression. B & C) Total cell lysates (signal-normalized lysate amount) were subjected to IP with the indicated antibodies and subsequently analyzed by WB with the indicated antibodies. All GRIM-1 isoforms interact with NAF1. More NAF1 in GRIM-1γ-expressing cells is due to more lysate used for IP. D–E) GRIM-1 and NAF1 co-localize in situ. D) Confocal images of Myc-tagged GRIM-1 isoforms (green channel) and NAF1 (red channel). Nuclei (blue channel) were visualized using DAPI. GRIM-1 isoforms and NAF1 complexes (yellow) can be seen in the merged channel indicative of interaction in situ. Lobed nuclei appear with a high frequency in GRIM-1γ-expressing cells. E) GRIM-1 is necessary for delocalizing NAF1. Immunofluorescent images of GRIM-1 and NAF1 in the indicated cell lines at steady state and RA/IFN-stimulated state. In both cell lines, NAF1 is nuclear at steady state. Upon RA/IFN stimulation, NAF1 is delocalized in SC cells, but not in GS cells. F) Confocal images of endogenous GRIM-1. RA/IFN stimulation increases GRIM-1 that accumulates more in the cytoplasm. G) NAF1 levels are not influenced by IFN/RA treatment. Total cell lysates were prepared at the indicated time point after RA/IFN treatment and analyzed using the indicated antibodies in WB. Numbers to the side in WB indicate positions of protein markers.