E1 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.

The FLAG (1:2,000, M20008L), GST (1:2,000, M20007L), Myc (1:1,000, M20002M) and mouse GFP (1:1,000, M20004M) antibodies were purchased from Abmart (Shanghai, China), and the Xpress (1:2,000, R910-25) antibody was purchased from Invitrogen (Carlsbad, CA). The GFP (1:1,000), Ufd2 (1:1,000), and Pgk1(1:5,000) polyclonal antibodies were generated in rabbits using the corresponding recombinant proteins as antigens. The E1 enzyme, FLAG-ubiquitin, K48-linked di-ubiquitin, K63-linked di-ubiquitin and methylated ubiquitin were purchased from Boston Biochem (Cambridge, MA).

The polyubiquitination assay was performed as previously described by Kitada et al. [9 ]. Briefly, a 100 μL reaction mixture including E1 enzyme 100 nM (Boston Biochem, Minneapolis, MN, USA, Cat#E-304-050), UbcH7 5 µM (Boston Biochem, Minneapolis, MN, USA, Cat# E2-640-100), recombinant human Parkin 1.5 µM (Boston Biochem, Minneapolis, MN, USA, Cat#E3-160-025), recombinant human phospho-ubiquitin (S65) (Boston Biochem, Minneapolis, MN, USA, Cat# U-102), and final of 1× E3 ligase buffer (Boston Biochem, Minneapolis, MN, USA, Cat# B-71). FLAG-FAF1 protein purified through immunoprecipitation was added at 3 different ratios (Parkin: FAF1 1:0, 1:2, 1:3, 1:6). The samples were incubated for 60 min at 37 °C, and the reaction was terminated by adding 1× loading buffer, and samples were separated using SDS gel for the Western blot (WB) analysis using anti-Parkin, anti-FLAG, and anti-Ubi antibodies following standard protocol. All the reagents and antibodies used in the experiment are listed in Table 1, Table 2 and Table 3.

The proteins were over-expressed in HEK293 cells and immunoprecipitated with antibodies. 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 ul reaction buffer. The reaction was carried out at 37 °C for 1 h and products were analyzed by western-blot assays with anti-TAZ antibody.