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. Author manuscript; available in PMC: 2013 May 22.
Published in final edited form as: Biochemistry. 2012 May 14;51(20):4175–4187. doi: 10.1021/bi300058m

Figure 6.

Figure 6

Interaction of UbcH5~Ub conjugate oligomers with the U-box-type E3 CHIP. (A) Full-length His6-CHIP, bound to Ni-coated magnetic beads, was used to pull down backside-mediated oligomerization deficient HsUbcH5b(C85S/S22R)~Ub (A) or backside-mediated oligomerization competent HsUbcH5b(C85S)~Ub (B) at the indicated molar equivalents. Eluted CHIP and UbcH5b~Ub in (A) and (B) were analyzed by SDS-PAGE. Gels were stained with IRDye Blue Protein Stain (LI-COR) and detected by fluorescence imaging at 700nm. (C) LI-COR quantified UbcH5~Ub band intensities were compared to the eluted CHIP intensity within the same lane, calibrated against input bands and plotted as a function of the molar equivalents of UbcH5b~Ub added. Data points represent the average of 3 separate trials with error bars indicating the standard deviation.