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. 2018 Dec 10;115(52):13246–13251. doi: 10.1073/pnas.1805353115

Fig. 1.

Fig. 1.

Not4 ubiquitin ligase regulates the progression of base assembly. (A) The not4-L35A catalytic mutants affect base assembly, as reflected in the Rpt5-Rpt4 module. Whole-cell extracts (80 μg) were analyzed by 3.5% native PAGE and immunoblotting for Rpt5, a subunit of the base. The not4-L35A allele is integrated into the chromosomal locus of NOT4. The plus (+) indicates the wild-type NOT4. Pgk1, loading control. (B) The not4 catalytic mutants exhibit enhanced progression of base assembly, as indicated by increased RP (base–lid) relative to the Rpt5-Rpt4 module. Assembly intermediates were affinity-purified via 3× FLAG-tagged Nas2 and were analyzed by 3.5% native PAGE and immunoblotting for the indicated Rpt proteins (i and ii). Nas2 serves as a loading control.