Figure 7.

Effects of cycloleucine, 5′-dA, and sinefungin on poly-ubiquitination-stimulating activity of viperin. a, flow chart describing the experimental protocol. b, cell lysates were immunoprecipitated with the Myc antibody (magnetic beads), followed by immunoblotting (IB) with anti-ubiquitin antibodies. Expression of transiently transfected proteins was confirmed by immunoblotting with c-Myc antibody. GAPDH served as a loading control. For WT viperin, cycloleucine or 5′-dA inhibit the ubiquitination of IRAK1; in contrast, sinefungin restores the ability of viperinΔ3C to stimulate the ubiquitination of IRAK1. c, quantification of IRAK ubiquitination levels obtained by integrating intensities of bands for blots using ImageJ software.