Nickel nitrilotriacetic acid agarose resin

For ARzD17-His pull downs, HEK293T cells expressing the corresponding EGFP-tagged proteins in 6-well plates were lysed by addition of 600 μl of lysis buffer (20 mm Tris-HCl, pH 8, 150 mm NaCl, 1% Triton X-100, 20 mm imidazole); 200 μl of the corresponding lysate was then diluted in 1 ml with lysis buffer without Triton, and diluted lysates were incubated with 25 μl of nickel-nitrilotriacetic acid-agarose resin (Qiagen, Manchester, UK), and 31 μl (50 μg) of ARzD17-His (or equivalent volume of ARzD17-His buffer) for 2 h at 4 °C. After extensive washing with washing buffer (20 mm Tris-HCl, pH 8, 300 mm NaCl, 1% Triton, 40 mm imidazole), bound proteins were eluted by boiling in 50 μl of Laemmli sample buffer. 7.5% of total inputs and 40% of total bound fractions were run on 12% SDS-polyacrylamide gels, and following electrophoresis and transfer to nitrocellulose, bound ARzD17-His was detected by Ponceau S staining and EGFP-tagged proteins by Western blotting using a GFP antibody.

Expression constructs were generated by a standard PCR-based cloning method. The full length human Ubc9 and E1 UFD domain (residues 447–547) were cloned to pET28a tagged with 6x His at the N-terminal. Escherichia coli BL21(DE3) plysS containing the expression vector were grown in Luria Bertani medium with chloramphenicol (17 μg/mL) and kanamycine (50 μg/mL) at 37 °C until the OD600 reached to 0.8. Expression was induced by 0.1 mM IPTG, followed by overnight culturing at 28 °C. Recombinant proteins were purified by nickel-nitrilotriacetic acid agarose resin (Qiagen) and dialyzed against 250 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM β-mercaptoethanol in the presence of thrombin protease overnight at 4 °C to remove the 6x His tag. Proteins were further purified by gel filtration chromatography on a Superdex75 column (GE Healthcare), which was pre-equilibrated in 250 mM NaCl, 20 mM Tris-HCl pH 7.5, 1 mM β-mercaptoethanol.

A plant microsomal fraction was prepared and tested by immunoblotting as described previously (Bultreys et al., 2009 (link)). The primary antibodies used were rabbit polyclonal antibodies against the whole plasma-membrane H⁺-ATPase family (Morsomme et al., 1998 (link)), the plasma-membrane PIP aquaporins (Heinen et al., 2014 (link)), the mitochondrial dihydrolipoyl dehydrogenase (DLD; de Castro Silva Filho et al., 1996 (link)), or against NtPDR3. The anti-NtPDR3 antibody used in the first figure has been described previously (Ducos et al., 2005 (link)), while that used in other figures was obtained as follows. The 120-bp DNA fragment encoding Pro-795 to Arg-834 of NtPDR3 was amplified by PCR using primers, one of which allowed the insertion of a six-histidine tag at the C-terminal end, and the PCR reaction product was inserted into the Escherichia coli expression vector pGEX-KG (Guan and Dixon, 1991 (link)). The glutathione S-transferase/NtPDR3 fusion protein was produced in E. coli, purified on a nickel–nitrilotriacetic acid agarose resin (Qiagen, Valencia, CA), and used to immunize rabbits.

The purification of PhoB and TctD was performed as previously described (27 (link)). Briefly, E. coli BL21(DE3) cells carrying either pET21::phoB or pET21::tctD were grown in LB supplemented with 100 mg/ml ampicillin broth at 20°C. Expression was induced with 0.1 mM isopropyl-1-thio-b-Dgalactopyranoside (IPTG) at an OD600 of 0.5 to 0.7. After overnight incubation, bacteria were harvested and bacterial pellets were resuspended in cold lysis buffer (50 mM NaH₂PO₄, pH 8.0, 300 mM NaCl, 10 mM imidazole) containing 1 mM DTT, 1 mg/ml lysozyme, protease inhibitors (Complete mini, EDTA free, Roche) and Benzonase Nuclease (Novagen). Cells were then lysed by sonification and the cell-free supernatant was incubated with nickel-nitrilotriacetic acid agarose resin (Qiagen) for 1 h at 4°C. The resins were washed with lysis buffer and proteins were eluted with 50 mM NaH₂PO₄, pH 8.0, 300 mM NaCl and 250 mM imidazole. After SDS-PAGE analysis, fractions containing pure protein were pooled and dialyzed for 16 h at 4°C in 50 mM NaH₂PO₄, pH 8.0, 300 mM NaCl. The protein concentration was determined by using the Bradford-based Roti^®-Quant solution following manufacture's instructions (Roth).