Ubiquitin-Dependent Modification of Skeletal Muscle by the Parasitic Nematode, Trichinella spiralis
Fig 5
Expression, purification and activity of 6His-TsUBE2L3 and 6His-ARIH2 in vitro.
A. Coommassie stain of 6His-TsUBE2L3 from un-induced (UI) and induced (I) E.coli cultures, nickel purification resin flow-through (FT1-2), wash (W) and elutions (E1-2). B and D. Streptavidin-HRP blots of in vitro parkin auto-ubiquitination reactions using human E1 (UBE1A), human Ub-biotin and either no E2, human E2 (HsUBE2L3) or 6His-TsUBE2L3 with (B) human parkin as the E3 or (C) human ARIH2 as the E3. C. Coomassie stain of 6His-HsARIH2Ξ”Ari from un-induced (UI) E.coli cultures, inclusion body supernatants (S1-4) induced inclusion bodies (I) and refolded from inclusion bodies (Rf). E. Surface representation of ARIH2 (grey) and Ub (wheat) with HsARIH2 (green) and TsUBE2L3 (cyan) bound to the RING1 domain of ARIH2. Residue differences between the E2-E3 interfaces are shown as sticks (HsARIH2 in orange and TsUBE2L3 in magenta). Zinc ions are shown as blue spheres, and Val141 of ARIH2 is shown as sticks. F. Zoom of the ARIH2:E2:Ub interface.