Fig. 5.

ISG15 expression weakens the interaction of VPS4 with ESCRT-III proteins. (A) 293E cells were cotransfected with plasmids expressing HA-VPS4 and FLAG-tagged CHMP1B, CHMP2A, or CHMP3 and 3 μg of pHis-ISG15 where indicated. At 48 h posttransfection, HA-VPS4 was immunoprecipitated from cell extracts with an anti-HA serum, and samples were resolved by 10% SDS-PAGE. For the top panel, precipitation of CHMP1B-FLAG (lanes 1 to 3), CHMP2A-FLAG (lanes 4 to 6), and CHMP3-FLAG (lanes 7 to 9) was determined by Western blotting with anti-FLAG antibody. The bottom panel shows a Western blot of the 10% input of total cell lysates used in the coimmunoprecipitation assay to verify even expression of HA-VPS4 (lanes 2 and 3, 5 and 6, and 8 and 9) and CHMP1B-FLAG (lanes 1 to 3), CHMP2A-FLAG (lanes 4 to 6), and CHMP3-FLAG (lanes 7 to 9), respectively. β-Actin served as a loading control. (B) 293E cells were transfected as described in the legend to panel A except that expression vectors of CHMP4B-FLAG (lanes 1 and 2) and CHMP6-FLAG (lanes 3 and 4) were substituted for the other ESCRT-III proteins. These experiments were repeated three different times with similar results.