Fig. 4: Characterization of UBA5 linker.
a ConSeq evolutionary conservation analysis of UBA5(351–390). b SDS–PAGE analysis showing the effect of Y372 mutations on the transfer of UFM1 to UFC1. c Three independent experiments as shown in b were performed to quantify the effect of the indicated mutations on UFM1 transfer. Data are represented by mean ± SD. d ITC experiment of UFC1 binding to UBA5 Y372A. The top graph represents raw data of heat flow versus time. The area under the peaks of the upper panel was integrated and plotted as kcal per mole of UFC1 as a function of binding stoichiometry in the bottom panel. Thermodynamic parameters are summarized in Supplementary Table 2. e UBA5 WT or mutants were incubated with UFC1 that was activated with DPDS and then were subjected to SDS PAGE analysis for UBA5-UFC1 adduct formation. The gel is representative of two independent experiments. f UBA5 WT or mutants were incubated with WT UFC1 and UFM1 then subjected to SDS–PAGE analysis for the formation of UFM1 charged UFC1. The gel is representative of two independent experiments.
