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. 2022 Sep 5;12(17):e4497. doi: 10.21769/BioProtoc.4497

Figure 3. Split-CAT assay for protein-protein interactions, ubiquitylation, and small molecule modulator.

Figure 3.

Split-CAT spot assay reporting Rpn10 ubiquitylation and Rpn10:Ub non-covalent binding on selective agar media supplemented with 12 μg/mL CAM. (A) Representative scan at 24 h post seeding. (B) Relative growth curves acquired with the time series analyzer V3 plugin. Quantification of the scans during 36 h represented in A, with standard deviation (n=3). (C) Relative cumulative growth of the curves in B. Single values of the integrals (area under curves) shown in B, presented as a bar-plot with standard deviation (n=3). (D) The E1 inhibitor PYR-41 (50 μM), arrests Rpn10 ubiquitylation dependent growth in liquid medium (24 μg/mL CAM).