Fig. 3.

ISG15 expression results in release of LIP5 from the budding complex in membranes into the cytosol. 293E cells were transfected with plasmids expressing FLAG-LIP5 and, where indicated, pHis-ISG15, E1, and E2. Forty-eight hours posttransfection, cell lysates were prepared and fractionated into soluble and pellet fractions as described in Materials and Methods. Distribution of FLAG-LIP5 and endogenous CHMP2A or CHMP5 in the resulting soluble (S) (lanes 1 and 3) and membrane pellet (P) (lanes 2 and 4) fractions were visualized by Western blotting with anti-FLAG serum to detect LIP5 or anti-CHMP2A and anti-CHMP5 sera to detect endogenous CHMP2A and CHMP5, respectively. β-Actin served as a loading control.