5‐bromo‐4‐chloro‐3‐indolyl B3‐D‐galactopyranoside (X‐α‐Gal) was purchased from Takara Bio Inc. Isopropyl-1-thio-β-D-galactoside (IPTG) was purchased from Sangon Biotech. pGADT7-Nrf2, pGADT7-Neh2, pGADT7-Neh2-4, pGADT7-Neh2-4-5, pGADT7-Neh7, pGADT7-Neh7-6, pGADT7-Neh7-6-1, pGADT7-Neh7-6-1-3, pGADT7-Neh1, pGADT7-Neh3, pGADT7-Neh1-3 and pGBKT7-SIRT6 were generated by inserting cDNA of Nrf2 or its different domains and SIRT6 into the pGADT7 and pGBKT7 vectors respectively. pET21a (+)-His-Nrf2, pET21a (+)-Nrf2ΔNeh1, pET21a (+)-Nrf2ΔNeh3 and pET21a (+)-Nrf2ΔNeh1-3 were generated by inserting cDNA of Nrf2 or its mutants missing Neh1, Neh3 or Neh1-3 into the PET21a (+) vector. pET21a (+)-SIRT6-Flag was generated by inserting SIRT6 cDNA fragment into pET21a (+) vector.
Saccharomyces cerevisiae strain Y2H Gold was cultured in YPDA medium (1% yeast extract, 2% polypeptone and 2% glucose). Plasmids include pGADT7-Nrf2/pGBKT7-SIRT6, pGADT7-Neh2/pGBKT7-SIRT6, pGADT7-Neh2-4/pGBKT7-SIRT6, pGADT7-Neh2-4-5/pGBKT7-SIRT6,pGADT7-Neh7/pGBKT7-SIRT6,pGADT7-Neh7-6/pGBKT7-SIRT6, pGADT7-Neh7-6-1/pGBKT7-SIRT6, pGADT7-Neh7-6-1-3/pGBKT7-SIRT6, pGADT7-Neh1/pGBKT7-SIRT6, pGADT7-Neh3/pGBT-SIRT6, pGADT7-Neh1-3/pGBKT7-SIRT6 were transformed by LiAc method. The cells were cultured in a complete minimum medium lacking tryptophan (Trp) or both tryptophan and leucine (leu) for positive selection of plasmid-transformed yeast clones. For plate assays, yeast were spotted onto triple dropout medium (TDO) plates lacking Trp, Leu, and histidine (His), and quadruple dropout medium (QDO) which in addition lacks adenine (Ade), containing 2% glucose and the chromogenic substrate 5‐bromo‐4‐chloro‐3‐indolyl-α‐D‐galactopyranoside (X‐α‐Gal) for identification of the interaction of various proteins expressed from the plasmids.
Recombinant plasmids pET21a (+)-His-Nrf2, pET21a (+)-His-Nrf2ΔNeh1, pET21a (+)-His-Nrf2ΔNeh3 and pET21a (+)-His-Nrf2ΔNeh1ΔNeh3 were transferred in Escherichia coli strain BL21, and cultured at 37℃ until OD600 value reached 0.6. Isopropyl-β-D- Thiogalactopyranoside (IPTG) at 1 mM was added to induce protein expression, and cells were collected after incubated at 37℃ for 5 h. The proteins were purified by Ni-NTA agarose beads (Qiagen) in buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0). The expressed protein was immobilized onto Ni-NTA agarose beads and washed with washing buffer (50 mM NaH2PO4, 300 mM NaCl, 30 mM imidazole, pH 8.0). The SIRT6 protein with the Flag tag was expressed, and the total protein was extracted by cell lysis buffer. Cell lysates were incubated with Ni-NTA agarose beads containing immobilized bait protein for 3 h at 4°C. Then, rinse thoroughly with washing buffer (50 mM NaH2PO4 300 mM NaCl, 30 mM imidazole, pH 8.0). Finally, the bound protein was eluted with eluent (50 mM NaH2PO4 300 mM NaCl, 250 mM imidazole, pH 8.0) and the proteins pulled down were analyzed by Western blot.
The proteins were resolved by SDS-PAGE and transferred onto nitrocellulose membranes which were blocked with 5% non-fat milk in blocking bffer (10 mM Tris-HCl, 100 mM NaCl, 25 mM NaF, 8 mM NaN3, and 0.1% Tween 20, pH 7.4), and incubated for 2h at room temperature. The membranes were incubated overnight with primary antibodies including mouse monoclonal anti-6×His Tag antibody (Santa Cruz Biotechnology) and rabbit polyclonal anti-Flag antiboday (Sangon Biotech) at 4℃. After washing, membranes were incubated with secondary antibodies (goat polyclonal antibody to mouse IgG H&L (Alexa Fluor® 488, Abcam) and goat polyclonal antibody to rabbit IgG H&L (Alexa Fluor® 647, Abcam.), incubate at room temperature in the dark, detected and analyzed by Typhoon FLA 9500 system (GE Healthcare, USA).