E2 enzyme

In vitro ubiquitination assays were done as previously described (68 (link)), but modified for 48-well plates. Briefly, the ubiquitination reaction mixture contained 125 ng of E1 enzyme (Boston Biochem), 250 ng of E2 enzyme (Boston Biochem), immunopurified Cul3–KLHL15 E3 complex, 10 μg of Myc-ubiquitin (Boston Biochem), 1 μM ubiquitin aldehyde (Boston Biochem), 10 mM MgCl₂, 2 mM DTT, 10 mM creatine phosphate (Sigma), 0.5 mg/ml creatine phosphokinase (Sigma), and 20 μg of purified proteins including DCX-HaloTag-His₆ (WT or Y259L) or Halo-His₆ as control protein. The ubiquitination reactions were diluted to a final volume of 0.25 ml in 50 mM HEPES, pH 7.5, initiated upon addition of 5 mM ATP (Thermo Scientific), and incubated for up to 2 h at 37 °C on a titer plate shaker (constant speed 3, Lab-Line Instruments, Inc). Thirty-microliter reaction mixtures were sampled at the indicated times and immediately terminated in 4× Laemmli buffer. Proteins were then resolved on 8% gels by SDS-PAGE, and ubiquitinated proteins were detected by immunoblotting with Myc-Tag antibody.
Relative DCX ubiquitination in vitro was quantified by dividing Myc-Tag signals by DCX signals and GFP (KLHL15) signals in the same lane and normalizing to the zero time point (set to 1). The enzymatic kinetics was plotted in Michaelis–Menten model using GraphPad Prism.

Ubiquitination was analyzed with an ubiquitination kit (Boston Biochem) following protocols recommended by the manufacturer. Recombinant proteins were mixing with 20X E1 Enzyme, 10X Mg2+-ATP Solution, 10X Ubiquitin Solution, 1ug E2 Enzyme (UbcH7, Boston Biochem; UBE2D1, Sino Biological Inc.) in a final volume of 20 µl reaction buffer. The reaction was carried out at 37 °C for 1 h and products were analyzed by western-blot assays with anti-YAP antibody (#14074, Cell signaling, 1:1000).

For the in vitro ubiquitination assay, purified eIF4E-6His was added to a reaction mixture consisting of 9 µg purified Ubiquitin-6His, 0.5 µg E1 enzyme (BostonBiochem, Cambridge, MA), and 10 µg purified E2 enzyme (GST-Ubc5a), in the presence or absence of purified GST-cIAP1 or GST-CHIP as an E3 ligase. All reactions were incubated at 37 °C, stopped by adding 1× SDS sample buffer, and analyzed by WB with the indicated antibodies. For the in vivo ubiquitination assay, cells were transfected with HA-tagged ubiquitin (HA-Ub) and the indicated plasmids. Cells were treated 24 h after transfection with 20 µM MG132 for 6 h, then lysed in lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol, protease inhibitor cocktail). Co-IPs were performed using the appropriate antibody and analyzed by WB.