FIGURE 2.

Ubiquitination and S-palmitoylation of IFITM3 have distinct roles. A, MEFs were treated for 6 h with IFNα before an additional 2 h treatment with IFNα and alkynyl palmitic acid reporter, alk-16, at 50 μm or DMSO as a control. Immunoprecipitated IFITM3 was reacted with az-rho via click chemistry and was visualized by fluorescence gel scanning and α-IFITM3 Western blotting. B, D, E, and F, HEK293T cells were transfected overnight with the indicated plasmids. PalmΔ indicates mutation of Cys-71, Cys-72, and Cys-105 to alanine. UbΔ indicates mutation of Lys-24, Lys-83, Lys-88, and Lys-104 to alanine. B, transfected cells were labeled with alkynyl-palmitic acid reporter, alk-16, for 2 h at 50 μm. Immunoprecipitated proteins were reacted with az-rho via click chemistry and visualized by fluorescence gel scanning and α-HA Western blotting. MEFs treated with IFNα for 8 h (C) or transfected cells (D) were fractionated into membrane and cytosolic compartments. α-Calnexin (CNX), α-GAPDH, and α-caveolin-1 (CAV1) Western blotting provided membrane, cytoplasmic, and intramembrane controls, respectively, for blotting with α-IFITM3 or α-HA antibodies. E, transfected cells were labeled with homopropargylglycine (HPG; 250 mm) or alk-16 (50 μm) for 2 h followed by chase with media containing either 5 mm methionine or 100 μm palmitate, respectively, for the indicated times. Immunoprecipitated proteins were reacted with az-rho via click chemistry and visualized by fluorescence gel scanning and α-HA Western blotting. F, shown are the average results from four experiments performed as in D. Fluorescence signal was normalized to Western blots to control for protein loading, and values were plotted relative to 0 h of chase. Error bars represent S.E.