Magnegst glutathione particles

GST-BES1, His-AGB1, and histidine-tagged trigger factor (His-TF) proteins were expressed in Escherichia coli (Rosetta; Kang Wei, Shanghai, China). His-TF was expressed from E. coli harboring the empty pCOLD-TF vector, which acted as negative control. The bait protein GST-BES1 was firstly incubated with 20 μl glutathione beads (MagneGST Glutathione Particles; Promega, Madison, WI, United States) in lysis buffer without EDTA at 4°C for 2 h, and washed three times with the same buffer. Then the beads were resuspended in 1 ml lysis buffer without EDTA and the prey proteins His-AGB1 and His-TF were added to the solution. The mixture was incubated for another 2 h at 4°C then washed with lysis buffer without EDTA three times. Proteins were eluted into the 1× SDS loading buffer, boiled for 5 min, and analyzed by Western blot. The prey proteins were detected by anti-His antibody (NewEast, Wuhan, China) and bait proteins were detected by anti-GST antibody (Sigma). The primer sequences used for plasmid construction for the pull-down assay are listed in Supplementary Table S1.

A total of 10 µl glutathione particles (MagneGST™ Glutathione Particles, Promega) were saturated with 6His-GST tagged Mug105 or Mug105^ZUP1-NT in 200 µl binding buffer (20 mM TRIS pH 7.5, 150 mM NaCl, 20 mM imidazole, and 0.1% NP-40) and incubated for 1 h at 4 °C. Both proteases were inactivated by a C42A mutation. The particles were washed three times with binding buffer and afterward incubated with the twofold molar excess of K63-linked ubiquitin chains for 2 h at 4 °C. The washing steps were repeated and the protein was eluted from the beads by addition of 30 µl Laemmli buffer. The proteins were separated via SDS-PAGE and visualized by coomassie staining or immunostaining with α-ubiquitin P4D1 antibody (1:3000; 05-944; EMD Millipore Corp).

Crude FLAG-GST-GATS recombinant protein (1.5 mL) was synthesized using a wheat germ cell-free reaction. The protein synthetic solution was mixed with 30 µL of MagneGST™ Glutathione Particles (Promega) previously equilibrated with PBS and rotated at 4 °C for 1 h. After the flow-through solvent was removed, the beads were washed thrice with 1 mL PBS. Recombinant proteins were eluted with 55 µL of elution buffer (10 mM reduced GSH, 50 mM Tris–HCl pH 8.0, and 50 mM NaCl).
Purification of GT7-tagged proteins using high-concentration GATS peptides was performed as follows. 10 µg of GATS antibody and 10 µL of protein A Sepharose were added and mixed at 4 °C for 1 h with rotation. Sepharose was transferred to Pierce™ Micro-Spin Columns (Thermo Fisher Scientific) and washed thrice with 1 mL of PBST. Next, 20 µL of PBS containing 150 µM GATS peptide (EEAAGIARPLIATLSVGVQNTF) (GenScript) was added to the column. After incubation at room temperature for 1 h, the eluted fractions were collected via centrifugation. Finally, Sepharose was mixed with 20 μL of SDS-PAGE sample buffer and incubated at 99 °C for 5 min to analyze proteins that were not eluted by peptide treatment. All fractions were confirmed by SDS-PAGE and CBB staining. The original blots are presented in Supplementary Fig. 6.

Rac1 activity was measured as previously described [26 ]. Briefly, 200 μg of total cellular protein was incubated with GST-PAK-CRIB fusion protein beads (donated by James E. Casanora of the University of Virginia) captured on MagneGST Glutathione Particles (Promega) for 4 h at 4 °C. The particles were then washed three times with washing buffer containing 4.2 mM Na₂HPO₄, 2 mM KH₂PO₄, 140 mM NaCl and 10 mM KCl (pH 7.2), resuspended in 2 x SDS sample buffer and subjected to western blotting analysis using a mouse anti-Rac1 antibody (BD Biosciences).