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. 2018 Jan 3;4(1):e1701393. doi: 10.1126/sciadv.1701393

Fig. 5. In vitro assay to verify that E4B and CHIP catalyzed the ubiquitination of the substrates.

Fig. 5

(A) Ubiquitination of PRMT1, MAPK3, PPP3CA, PGAM5, and OTUB1 by wt UB through the wt Uba1-UbcH5b-fE4B cascade (lane 1). In lanes 2, 3, 4, and 5, wt E4B, Uba1, UbcH5b, and wt UB were missing from the reaction mixture, respectively. GST, glutathione S-transferase. (B) Ubiquitination of PRMT1, MAPK3, PPP3CA, β-catenin, and CDK4 by wt UB through the wt Uba1-UbcH5b-CHIP cascade (lane 1). In lanes 2, 3, 4, and 5, wt CHIP, Uba1, UbcH5b, and wt UB were missing from the reaction mixture, respectively. Bands marked with asterisks were likely generated by monoubiquitinated substrates.