Ni beads

Co-IP assays were carried out as described [9 (link), 14 (link)]. The antibodies used are as follows: Ajuba (#4897, Cell Signaling Technology), SP1 (#sc-420, SCBT), Flag (Sigma-Aldrich, F7425), Myc (9B11, #2276, SCBT), normal IgG (#2729, Cell Signaling Technology). GST-tagged SP1 and His tagged Ajuba were expressed in BL21 respectively, and purified by Glutathione Sepharose beads (17–0756-01, GE Healthcare) or Ni-beads (17–5318-06, GE Healthcare). For the in vitro binding assays, the purified proteins of GST-SP1 were mixed with His-Ajuba was added into the mixture for 12 h.

19-MBP was purified using amylose resin (New England Biolabs) and 20-His₆ was purified using Ni beads (GE Healthcare, Sweden). The substrate was supercoiled pMAL-c5x plasmid DNA, which was extracted using a TIANprep Mini Plamid Kit (Tiangen, China). Ten micrometers of purified pMF1.20 protein and 0.5 μg DNA were incubated for 30 and 60 min at 37°C in a reaction buffer (20 mM Tris-HCl, 200 mM NaCl and 5% glycerol, pH 8.0). The reaction was stopped by the addition of phenol/chloroform/isoamyl-alcohol, and the solutions were examined using 0.8% agarose gel electrophoresis. A supercoiled DNA ladder marker (Takara, China) was used.

His-TPR, His-CRM1 and His-p53 recombinant proteins were expressed in HEK293T cells and purified with Ni beads (17–5318-06, GE Healthcare). Experiments with recombinant proteins were performed in the presence of a synthetic peptide comprising the nuclear export signal (NES) derived from PKI (ELALKLAGLDIN) to strengthen the protein-protein interactions [21 (link)].

GST-TRIB2 fusion proteins and His-AP4 proteins were expressed in BL21 and purified using Glutathione Sepharose 4B beads (Amersham Pharmacia, Piscataway, NJ, USA) or Ni beads (GE Healthcare, CA, USA), respectively. Purified His-AP4 protein was incubated with GST or GST-TRIB2 fusion proteins bound to Glutathione Sepharose beads at 4 °C overnight. Beads-associated proteins were detected by western blot. Expression of GST fusion proteins was confirmed by Coomassie Blue staining.

The co-immunoprecipitation (co-IP), Western blotting, and glutathione S-transferase (GST) pull-down assays were described previously (Jia et al., 2017 (link)). In brief, cells were lysed 48 h of post-transfection in buffer containing 20 mM/L Tris-HCl (pH 8.0), 150 mM/L NaCl, 2.5 mM/L EDTA, 0.5% NP40, 0.1 mM/L phenylmethylsulfonyl fluoride (PMSF), and protease inhibitor cocktail. Lysates were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot assays. Lysates were pre-incubated with protein A/G PLUS-agarose beads for 1 h and the beads were removed by centrifugation. The resulting supernatants were then incubated with antibodies against the indicated epitope tags followed by incubation with protein A/G PLUS-agarose (SC-2003, Santa Cruz). Beads were washed three times with cell lysis buffer and the co-eluted proteins were analyzed by SDS-PAGE and Western blot assays.
In the GST pull-down assay, GST-Snail and His-Flag-USP37 were expressed in Escherichia coli BL21 (DE3) cells, which were induced by isopropyl-β-d-thiogalactoside (IPTG). The GST-tagged Snail protein was purified by Glutathione Sepharose beads (17-0756-01, GE Healthcare) and His-Flag-USP37 was purified with Ni beads (17-5318-06, GE Healthcare).

FAM35A_C (381–904 aa) was fused with MBP-tag and His₆-tag at C-terminus and N-terminus, respectively. Protein was expressed in E. coli (Transetta cells, TransGen) using pCPD9 vector. Cells were grown at 37 °C until OD600 = 0.8, and were induced with 0.05% Isopropyl β-D-1-thiogalactopyranoside at 16 °C for 12 h. The cell pellet from 12-liter culture was lysed by French Press in 200 ml lysis buffer (40 mM Tris Cl, pH 8.0, 500 mM NaCl, 25 mM imidazole, 7% glycerol, 0.2 mM DTT and 1 mM PMSF, 1 μg ml⁻¹ leupeptin and 1 μg ml⁻¹ aprotinin). The lysate was centrifuged at 35,000 × g for 30 min and the supernatant was incubated with 3 ml Ni-beads (GE Healthcare) at 4 °C for 1 h. The beads were washed four times with lysis buffer. The proteins were eluted with 15 ml His-Elution Buffer (20 mM Tris Cl, pH 8.0, 160 mM NaCl, 400 mM imidazole, 7% glycerol, 1 mM DTT, 1 mM PMSF, 1 μg ml⁻¹ leupeptin and 1 μg ml⁻¹ aprotinin). The eluted proteins were incubated with 0.5 ml Amylose resins (NEB) at 4 °C for 1 h. After washing four times with washing buffer (20 mM Tris-HCl, 500 mM NaCl, 0.1% Triton X-100, 1 mM DTT), protein was eluted with MBP-Elution Buffer (20 mM Tris Cl, pH 8.0, 160 mM NaCl, 7% glycerol, 1 mM DTT and 30 mM maltose). The protein was then concentrated, flash-frozen and stored at –80 °C.