His6 e1

UbcH7 (10 μg) was charged with N-terminal fluorescein-labelled Ub (10 μg FluoUb) using 0.02 μg His₆-E1 (Boston Biochem Inc.), 0.5 mM ATP and 10 mM MgCl₂ in a 100 μl reaction (in 50 mM Tris/HCl pH 7.4, 120 mM NaCl and 1 mM TCEP). After 60 min incubation at 30 °C, 0.8 μg of UbcH7∼FluoUb was mixed with 1.2 μg of WT or mutant RnParkin (in 50 mM Tris/HCl pH 7.4, 120 mM NaCl and 1 mM TCEP). Mixtures were incubated for 20 min at 30 °C for discharging. Reactions were stopped with the addition of SDS–PAGE sample buffer containing 12 mM TCEP, resolved on SDS–PAGE and analysed using the Typhoon fluorescent scanner (GE) and Coomassie staining. Fluorescence strength of UbcH7∼FluoUb was measured for quantification using ImageJ.

The in vitro ubiquitination assay was performed as described with some modifications (Lu et al., 2011 (link)). The reactions contain 1 μg of purified MBP–FLS2CD, 1 μg of purified His₆-E1 (AtUBA1), 1 μg of purified His₆-E2 (AtUBC8), 1 μg of His₆-ubiquitin (Boston Biochem), 1 μg of purified GST–PUB, and 1–16 μg of purified GST or GST–ARM with 6 μl of 5× ubiquitination buffer (0.1M TRIS-HCl, pH 7.5, 25mM MgCl₂, 2.5mM DTT, 10mM ATP) to a final volume of 30 μl. The reactions were incubated at 30 °C for 2h, and then stopped by adding SDS sample buffer and boiled at 100 °C for 5min. The samples were then separated by 7.5% SDS–PAGE and the ubiquitinated substrates were detected by western blot analysis.

Ten micrograms of ubiquitin-conjugating enzyme, UbcH7, were charged with 10 µg ubiquitin or N-terminally fluorescein-labelled Ub (FluoUb, Boston Biochem) using 0.02 µg ubiquitin-activating enzyme (His6-E1, Boston Biochem Inc.), 0.5 mM ATP and 10 mM MgCl₂ in 50 mM Tris-HCl pH 7.4, 120 mM NaCl and 1 mM TCEP, in a 100 µl reaction. Reactions were performed at 37°C at different time points to a maximum of 60 min.
Ubiquitin-loaded UbcH7 (0.8 µg UbcH7–Ub or UbcH7–FluoUb) was mixed with 1.2 µg of RnParkin (in 50 mM Tris-HCl pH 7.4, 120 mM NaCl and 1 mM TCEP). Mixtures were incubated at 30°C for up to 40 min to monitor discharging (figure 2a,c). Reactions were stopped with the addition of SDS-PAGE sample buffer containing 12 mM TCEP, resolved on SDS-PAGE and analysed using the Typhoon fluorescent scanner (GE), Coomassie staining, silver staining (Thermo Pierce kit) or transferred to a nitrocellulose membrane and probed with mouse anti-ubiquitin (Covance, 1 : 10 000) and detected with Clarity Lightning ECL (Bio Rad). Images acquired with an ImageQuant LAS 500 (GE Healthcare). Densitometry and fluorescence intensity analysis was performed using Fiji [83 (link)].