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. 2021 Jul 28;10:e70560. doi: 10.7554/eLife.70560

Figure 7. USP36 cleaves linear authentic and artificial UB(L) substrates in vitro.

In vitro processing assays for which 2.5 µM (A) His6-FUBI(WT)-eS30, (B) His6-FUBI(AA)-eS30, (C) His6-FUBI-EGFP, and (D) His6-Ub-EGFP were incubated with 0.5 µM His10-USP36-TEV-St WT or CA mutant at 37°C. Samples taken at the indicated time points (0, 7.5, 15, 30, 60, 120 min) were analyzed on Coomassie brilliant blue-stained gels. Unprocessed substrates are marked with an asterisk (*). Note that the enzyme preparation contains USP36 degradation products (marked with a dagger (†), see Figure 7—figure supplement 1). (E) Quantification of USP36-dependent processing based on the levels of the uncleaved substrates, highlighted by dashed colored boxes in panels (A to D), each normalized to t = 0 min from three technical replicates. Half-lives (t1/2) of fitted one-phase exponential decay curves are indicated for processed substrates.

Figure 7—source data 1. Source data for Figure 7 with relevant areas labeled on the uncropped images of the gels.
Figure 7—source data 2. Unedited image of the gels shown in Figure 7.

Figure 7.

Figure 7—figure supplement 1. Analysis of purified His10-USP36-TEV-St.

Figure 7—figure supplement 1.

Purified WT and CA His10-USP36-TEV-St (0.5 µM) were analyzed by Coomassie brilliant blue (CBB) staining of the gel or by immunoblotting using the indicated antibodies.
Figure 7—figure supplement 1—source data 1. Source data for Figure 7—figure supplement 1 with relevant areas labeled on the uncropped gel and uncropped original blots.
Figure 7—figure supplement 1—source data 2. Unedited image of the gel shown in Figure 7—figure supplement 1.