E coli bl21 codonplus de3 ril

The AtNFXL1ΔNΔZn fragment (2341–3567 bp) was amplified by PCR from cDNA using specific primers (see Supplementary Table S1 at JXB online). The amplified fragment of AtNFXL1ΔNΔZn was cloned into the NdeI and SalI sites of the pET-29a vector (Merck KGaA). The plasmids were transformed into E. coli BL21-CodonPlus (DE3)-RIL (Agilent Technologies). The 6×Histidine (His) tag-labelled AtNFXL1ΔNΔZn protein (His–AtNFXL1ΔNΔZn protein) was purified using a Ni Sepharose High Performance column (GE Healthcare). SDS-PAGE and immunoblotting were carried out as previously described (Asano et al., 2004 (link)). The anti-AtNFXL1C antibody was generated in rabbit and purified using antigen (His–AtNFXL1ΔNΔZn protein)-coupled HiTrap^TM NHS (N-hydroxysuccinimide)-activated HP (high performance; GE Healthcare). Then, an AtNFXL1-containing protein complex was purified using anti-AtNFXL1C antibody coupled to HiTrap^TM NHS-activated HP.

The sequence coding for 6×His tag-labelled ΔMKD1 (ΔMKD1) was amplified by RT-PCR using specific primers (see Supplementary Table S1). The amplified PCR products were introduced into SgfI and PmeI sites of the pF3KWG (BYDV) Flexi plasmid (Promega). The ΔMKD1 protein was synthesized using the TNT SP6 High-Yield Wheat Germ Protein Expression System (Promega). In vitro transcription/translation was performed according to the manufacturer’s instructions. The ΔMKD1 protein was purified using a Ni Sepharose High-Performance column (GE Healthcare). MKK1, MKK2, and MKK5 were amplified from cDNA by PCR using specific primers (Supplementary Table S1). Amplified fragments of MKK1, MKK2, and MKK5 were cloned into SmaI and NotI (blunt ended) sites of the pGEX6p-1 plasmid (GE Healthcare). MKK1, MKK2, and MKK5 plasmids were transformed into E. coli BL21-CodonPlus (DE3)-RIL (Agilent Technologies). The recombinant proteins glutathione S-transferase (GST)–MKK1, GST–MKK2, and GST–MKK5 were digested by PreScission Protease (GE Healthcare). The resulting MKK1, MKK2, and MKK5 proteins were purified using a glutathione Sepharose 4 Fast Flow column (GE Healthcare).

Full-length cDNA of Mst77F was obtained from the Drosophila Gene Collection (DGC) (Clone ID #RH09844). Mst77F cDNA was amplified using a primer set (5′-CGGAATTCATGAGTAATCTGAAACAAAAGG-3′, 5′-AGCTCGAGTTACATCGAGCACTTGGGCTTG-3′), and cloned into the EcoRI and XhoI sites of the pGEX-4T-1 plasmid (GE Healthcare), and into EcoRI and SalI sites of the pMAL-cRI plasmid (New England Biolabs). These plasmids were both transformed into E. coli BL21-CodonPlus (DE3)-RIL (Agilent Technologie). First, E. coli was incubated at 37°C for 3 h, and after adding 1 mM isopropyl beta-d-thiogalactoside (IPTG), incubated at 30°C for more 3 h. Cells were harvested, and the lysis buffer (20 mM Tris-HCL pH 8.0, 150 mM NaCl, 0.1 mM EDTA, 1% Trition X) was added, and briefly sonicated. After centrifugation, the supernatant was collected as a soluble fraction. For glutathione S-transferase (GST) fused Mst77F recombinant protein, the soluble fraction was incubated with glutathione sepharose 4B (GE Healthcare) and eluted by glutathione, and dialysed with 1× PBS buffer, and used as an antigen for the rabbit polyclonal antibody. For maltose-binding protein (MBP) fused Mst77F recombinant protein, the soluble fraction was incubated with amylose resin (New England Biolabs) and eluted by maltose, and dialysed with 1× PBS buffer, and used for the affinity purification.