Figure 4. Phosphorylation stimulates ubiquitin-specific protease-14 (USP14) activity towards both K48 and K63 ubiquitination.
(A) Dimeric Ub cleavage assay. USP14 S432E cleavage of Lys 48 and Lys 63 Ub chain linkages was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). (B) Cleavage of Lys 48, Lys 63 and linear dimeric Ub chain types in the presence of USP14 S432E was measured over time and analyzed using SDS-PAGE. Quantification of the amount of dimer remaining from the data was shown below. (C) Dimeric Ub cleavage analysis of immunoprecipitates derived from HEK293T cells transfected with wild type HA-USP14 or S432A mutant plasmids.
Figure 4—figure supplement 1. Phosphorylation of ubiquitin-specific protease-14 (USP14) promotes both K48 and K63 deubiquitination activity.
(A) USP14 S432E does not cleave linear di-Ub. USP14 S432E cleavage of linear dimeric Ub chain types and analyzed using SDS-PAGE. Cleavage of Lys48 dimeric Ub chain types was used as a positive control. (B, C) USP14 immunoprecipitated from transfected HEK293T cells and eluted with HA-peptide has high deubiquitination activity towards both K48 and K63 di-Ub but not linear di-Ub.