Fig. 3: Ub4a binding protects tri-Ub and longer chains against disassembly by DUBs. | Nature Communications

Fig. 3: Ub4a binding protects tri-Ub and longer chains against disassembly by DUBs.

From: Mechanism of selective recognition of Lys48-linked polyubiquitin by macrocyclic peptide inhibitors of proteasomal degradation

Fig. 3

SDS PAGE gels of the indicated time points of IsoT disassembly reactions for a mixture of K48-linked polyUb chains of length n ≥ 3, and b mixture of Ub2, Ub3 and Ub4 chains, alone (left) and in complex with Ub4a (right). SDS PAGE gels of select time points of OTUB1 disassembly reactions for K48-linked c Ub3, Ub4, and Ub5; d Ub3 and Ub4; e [Ub]3-UbR74, f [Ub]2-UbR74, and g [Ub]2-UbR72 alone and in complex with Ub4a. Molecular weight (MW) values are shown in kDa. The plot in e (right) shows normalized intensities of the Ubn gel bands at the indicated time points for [Ub]3-UbR74:Ub4a disassembly by OTUB1 (see details in Methods). The sum of normalized Ub1, Ub3, and Ub4 band intensities equals one. The data for Ub1, Ub3, and Ub4 are colored blue, red, and black, respectively. Shown are the mean values obtained in three attempts (n = 3) to quantitate gel bands using ImageJ; the error bars, centered on the mean values, represent standard deviations among these measurements. The gray dots depict the normalized intensities obtained in each of these attempts. The cartoon drawings depict the anticipated modes of peptide binding to and protection of the respective chains. In all the assays, Ub4a was added in equimolar ratio to polyUb. Source data are provided as a Source Data file.

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