Pgex 4t 3

Glutathione S-transferase (GST)-tagged Pirh2, Pirh2-ΔRING, or Pirh2-DN was expressed by pGEX-4T-3 (Amersham Pharmacia Biotech). The recombinant GST-tagged proteins were purified as described previously [37] (link). Briefly, the GST fusions of Pirh2, Pirh2-ΔRING, and Pirh2-DN were expressed in E. coli BL21 (DE3) (Novagene) upon induction with 0.5 mM IPTG for 4 h at 37°C. Bacterial cells were harvested and then resuspended in GST lysis buffer (200 mM Tris-Cl, pH 8.0, 0.5 M NaCl, 100 mM EDTA, 0.1% Triton X-100, and 0.4 mM PMSF). Subsequently, cell lysates were sonicated and clarified by centrifugation. GST fusion proteins were purified using glutathione-Sepharose beads (Amersham Pharmacia Biotech) according to the manufacturer's protocol.

The complete coding region of LcNAC13 was cloned into pET-28a vector (Novagen, Madison, WI, USA) to generate His-LcNAC13, while the LcR1MYB1 cDNA fragment was inserted into pGEX-4T-3 (Amersham Biosciences, Staffanstorpm, Sweden) to fuse in frame with GST. His and GST recombinant fusion proteins were expressed in BL21 (DE3) cells with induction by 1.0 mM isopropyl-b-D-thiogalactoside for 12 h at 16 °C. The recombinant proteins were then purified with Ni²⁺-nitrilotriacetate (Ni-NTA) agarose (Qiagen, Valencia, CA, USA) and Glutathione Sepharose 4B (GE Healthcare, Pittsburgh, PA, USA) according to the manufacturer’s manual, respectively.

The encoding sequence fragments of MaMsrB2, MaAPX1, or MaAPX1 mutants were subcloned into the pGEX-4T-3 (Amersham Biosciences) or pET-28a vector (Novagen), respectively. MaMsrB2-GST, MaAPX1-His, MaAPX1-M36Q-His, and MaAPX1-M36V-His were induced and expressed in the E. coli BL21 (DE3) strain, and then were purified with glutathione sepharose 4B (GE Healthcare) and nickel-nitrilotriacetic acid agarose (Qiagen), respectively, following the manufacturer’s instructions (Figure S1).

The WTAP gene was PCR-amplified from HEK293T cDNA and ligated into the pLVX-Puro vector (Clontech Laboratories) and pcDNA3.1-his-myc-B vector (Invitrogen), named WTAP-pLVX-Puro and pcDNA 3.1-WTAP, respectively. The WTAP-encoding sequence was also inserted into the pGEX-4 T-3 (Amersham biosciences) plasmid, called GST-WTAP thereafter. The BCL6 gene was PCR-amplified from HEK293T cDNA and ligated into the pcDNA3.1-his-myc-B (Invitrogen), named BCL6-His.

All plasmids used in this study were described in earlier studies9 (link)14 (link). VRK2 kinase-dead mutant was generated by site-directed mutagenesis (Lys61 to Ala). Flag-hVRK2-mutants (K311A (Interface 1), D256A (Interface 2), and E66A (Interface 3)) were amplified from Flag-hVRK2-WT plasmid using a forward primer (5′-AAGCGGCCGCAATGCCACCAAAAAGAAATG-3′) and reverse primer (5′-AATCTAGATCAGAGAAAAAATAAAGCAAGAA-3′). The first round of PCR, using forward and reverse primer (5′-GGTTCAAAATTGCCTTGAGGGC-3′ for Interface 1, 5′-AGCCACAGGTGCCTTCAGGTT-3′ for Interface 2, and 5′-CGGGCCATTTGCTTGATATTC-3′ for Interface 3) and another forward (5′-GCCCTCAAGGCAATTTTGAAC-3′ for Interface 1, 5′-AACCTGAAGGCACCTGTGGCT-3′ for Interface 2, and 5′-GAATATCAAGCAAATGGCCCG-3′ for Interface 3) and reverse primer, created two fragments. The two PCR products were mixed together for a second round of PCR. The full-length coding sequence of hVRK2 was digested with NotI/XbaI and cloned into pFlag-CMV2 (Sigma, St. Louis, MO) control vector. The GSK3β constructs were gift from Sang Ki Park (POSTECH, Pohang, South Korea). The HA-tagged cDNA of human GSK3β wild-type and two mutants, catalytically inactive GSK3β (GSK3β K85A) and constitutively active GSK3β (GSK3β S9A), were cloned into pGEX-4T-3 (Amersham, Piscataway, NJ).

GST-ERα (Δ2) was prepared by cutting Flag-ERα (Δ2) with BamHI and XhoI, and the fragment was then inserted into pGEX-4T3 (Amersham Pharmacia). The GST and GST-fusion protein were expressed in BL21(DE3) (Takara), and purified by Pierce GST Spin Purification Kit (Thermo scientific). His-TCF21 was prepared by cutting Flag-TCF21 with EcoRI and XhoI, and then the fragment was inserted into pET32a(+) (Novagen). His-TCF21 protein was expressed in BL21 and purified by Ni-Agarose His-tagged Protein Purification Kit (CW Biotech). GST pull-down assay was performed using a Pierce GST Protein Interaction Pull-Down Kit (Thermo scientific).