Glutathione sepharose 4b resin

Equivalent amounts of bacterial lysate expressing GST, GST-E2 full length or GST-E2 TAD were incubated on Glutathione Sepharose 4B resin (GE Healthcare Life Sciences) for 1 h at room temperature. Beads were washed three times with buffer-A containing 0.5% Triton. The beads were further incubated with 50–60 μg of recombinant FADD, which was purified as described earlier22 (link), for 3 h at 4 °C. Post wash, the bound proteins were separated on SDS-PAGE and were probed with 1:800 dilution of anti-his antibody (Abcam, Cambridge, USA).

The coding sequence for the full-length AtPropep1 (residues 1–92) (clone 06-11-N09 from RIKEN, Japan) was subcloned into the BamHI and XhoI sites of pGEX-4T-1 vector (GE Healthcare, Inc.) using standard PCR-based protocols. GST-Propep1 R6A/R7A mutant was produced by PCR. Primers are listed in Supplementary Table 3. Proteins were overexpressed in Escherichia coli BL21 (DE3) pLysS at 16 °C for 20 h induced by addition of 0.2 mM IPTG (final) to the cell culture with an A600 of 0.4–0.6. Harvested cells were resuspended in extraction buffer that contains 25 mM Tris, pH 7.6, 150 mM NaCl, 10 mM DTT, 5% glycerol and protease inhibitors. After cells were lyzed by using an EmulsiFlex-C3 Homogenizer (Avestin, Ottawa, Canada), Triton X-100 (Sigma) was added to lysates to a final concentration of 1% and stirred at 4 °C for 1 h. After centrifugation at 18,000×g for 1 h, the supernatant was purified by using Glutathione Sepharose 4B resin and PD10 column according to the manufacturer’s protocols (GE Healthcare, Inc.). The resins were washed with wash buffer (25 mM HEPES, pH 7.6, 150 mM NaCl, 10 mM DTT, 0.2 mM AEBSF, and 5% Glycerol). Proteins were eluted by wash buffer supplemented with 10 mM glutathione and 0.1% Triton X-100.

Full-length wild-type OGT and mutants (R284P and Δ155–177) were expressed in Escherichia coli as N-terminal His fusion proteins as described previously (45 (link)). The wild-type OGT TPR domain (residues 26–420) and mutants (TPR-R284P and TPR-Δ155–177) were expressed and purified as described previously (11 (link)). HCF1-rep1 (residues 867–1071) was expressed in E. coli as a non-cleavable N-terminal GST and C-terminal His fusion protein. Transformed BL21 (DE3) cells were grown, induced for expression, lysed, and clarified as described previously (45 (link)). Clarified lysate was incubated with 1 ml/liter of culture of glutathione-Sepharose 4B resin (GE Healthcare) for 2 h at 4 °C. The resin was thoroughly washed with base buffer (0.1 m Tris-HCl, pH 7.5, 150 mm NaCl, 0.5 mm TCEP) and eluted using base buffer supplemented with 0.5 m glutathione. Eluted protein was dialyzed overnight at 4 °C in buffer A (0.1 m Tris-HCl, pH 7.5, 25 mm NaCl). Dialyzed protein was passed through 5 ml of HiTrap Q-Sepharose FF anion exchange resin (GE Healthcare), collecting the flow-through, which was then concentrated and passed through a 300-ml Superdex^TM 200 column (GE Healthcare) pre-equilibrated with base buffer. The peak fractions were concentrated to 10 mg/ml, mixed 1:1 with 50% glycerol, snap-frozen, and stored at −80 °C until use.

The coding regions of DDB1 and CUL4A cDNAs were cloned into the pET28a (His tag) and pGEX-6P-1 (GST tag) vectors between BamHI and EcoRI sites, respectively. The pET28a-DDB1 and pGEX-6P-1-CUL4A plasmids were transformed into an Escherichia coli strain BL21 (DE3.0), respectively. The positive colonies were grown in liquid lysogeny broth (LB) medium containing antibiotics to the logarithmic phase. Cells were then induced with 1 mM isopropyl β-D-thiogalactoside (IPTG) for 12 h at 16 °C. Cells expressing GST-CUL4A were lysed in a buffer containing 1×PBS, 1 mM DTT, 0.01% Tween-20, and 1 mM PMSF. GST-CUL4A protein was purified using Glutathione Sepharose 4B resin (GE Healthcare, Chicago, IL, USA, #GE17-0756-01). Cells expressing His-DDB1 were lysed in a buffer containing 50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, 1 mM DTT, 0.01% Tween-20, and 1 mM PMSF. His-DDB1 protein was purified using Ni-NTA resin (ThermoFisher Scientific, Waltham, MA, USA, #88221). Both purified proteins were stored at -80 °C until use.