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. 2007 Sep 26;2(9):e956. doi: 10.1371/journal.pone.0000956

Figure 3. ESCRT-I is a 1∶1∶1∶1 heterotetrameric complex both in vivo and in vitro. The results presented in each panel are representative of three individual experiments performed. Schematics in the upper right corner of each panel indicate the ESCRT-I components present in each experiment.

Figure 3

(A) TSG-101:6xHIS, VPS-28, and VPS-37 were co-expressed and purified from E. coli extracts using nickel resin. A Coomassie stained gel of the peak elution fractions after fractionation of the recombinant complex on a Superose 6 gel filtration column is shown. The intensities of each band were measured to identify the peak, and a Stokes radius was calculated for each protein based on comparison with the elution profiles of known standards. The asterisk highlights a breakdown product of TSG-101. (B) The recombinant complex described in A, was fractionated on a 5–20% sucrose gradient. A western blot of the fractions for TSG-101 is shown. The asterisk highlights a breakdown product of TSG-101. The intensities of each band were measured to identify the peak, and S-values were calculated for each protein based on the location of known standards run on a parallel gradient. (C) TSG-101:6×HIS, VPS-28, VPS-37 and MVB-12 were co-expressed and purified from E. coli extracts using nickel resin. A Coomassie stained gel of the peak elution fractions after fractionation of the recombinant complex on a Superose 6 gel filtration column is shown. The Stokes radius of the complex, calculated as described for A, is indicated. (D) The recombinant complex described in C, was fractionated on a 5–20% sucrose gradient. A western blot of the fractions for MVB-12 is shown. The S-value of the complex, calculated as described in B is indicated. (E) Western blots of embryo extracts fractionated on a Superose 6 gel filtration column. The peaks corresponding to TSG-101 and MVB-12 are largely overlapping. The Stokes radius of the complex, calculated as described for A, is indicated. (F) Western blots of embryo extracts after fractionation on a 5–20% sucrose density gradient. TSG-101 and MVB-12 co-fractionate, both migrating with an S-value close to 5.0. The S-value of the complex, calculated as described in B is shown.