Pmal c2

MBP-tagged Tctp22 (link) and GST-tagged Brm fragments (GST::Brm 1-311, 304-747, 748-1638, Δ304-500, Δ574-648, Δ694-747, ΔHSA, ΔBRK, and ΔHSA, ΔBRK) were cloned into pMAL-c2 (NEB) and pGEX5X-1 (GE healthcare), respectively. In-Fusion system (Clontech) was used for cloning. Primer sets used for cloning were listed in Supplementary Table 2. GST-tagged and MBP-tagged proteins were expressed in E.coli Rosetta2 (Novagen, Germany). IPTG (0.1 mM) induction was performed in 14 °C shaking incubator at 250 r.p.m. for 4 h. The extracted proteins were dialysed in TBS buffer (10 mM Tris-HCl (pH8.0), 150 mM NaCl) containing 20% glycerol and 1 mM DTT and stored at -20 °C before use.
Five micrograms of MBP fusion proteins were used as prey and the same amount of GST fusion proteins as baits. Protein complexes were formed in PDB (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, 10% glycerol, 0.1% Triton X-100, 1 mM DTT, proteinase inhibitor cocktail (Roche)) containing 0.2% bovine serum albumin for 2–3 h at 4 °C. After washing three times in PDB at RT for 15 min, the samples were boiled in protein loading buffer at 94 °C for 5 min and loaded for western blotting. Primary antibodies were as follows: Ms anti-GST (1:4,000, SantaCruz sc-138) and Rb anti-MBP (1:10,000, NEB E8030S).

Binding between TbPex3 and TbPex19 was examined essentially as described (Knoblach et al, 2013 (link)). GST fusion to TbPex19 was constructed in pGEX4T-1 (GE Healthcare). MBP fusions to TbPex3 and to mutants TbPex3-F102A and TbPex3-L105A were constructed in pMAL-c2 (New England Biolabs). Recombinant proteins were expressed in the Escherichia coli strain BL21 (Invitrogen). GST alone or GST-TbPex19 was immobilized on glutathione-sepharose beads and incubated with E. coli lysates containing MBP-TbPex3 or MBP-TbPex3-F102A or MBP-TbPex3-L105A in binding buffer (20 mM Tris–HCl, pH 7.5, 100 mM KCl, 5 mM MgCl₂, and 0.5% (vol/vol) Triton X-100). Unbound proteins were removed by washing five times in binding buffer. Immobilized proteins were eluted in sample buffer (50 mM Tris–HCl, pH 6.8, 2% SDS, 5% [vol/vol] glycerol, 0.002% bromophenol blue, 100 mM 2-mercaptoethanol) and subjected to SDS–PAGE and immunoblotting.

The plasmids pMAL-C2-AtYchF1 and pGex-4T-1-AtGAP1 and their corresponding empty-vector control plasmids (pMAL-C2: New England Biolabs Inc., Beverly, MA, United States; pGex-4T-1: GE28-9545-49, GE Healthcare, Chalfont St Giles, England) were transformed into the E. coli BL21 (DE3) strain. Protein expression was induced by 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) in the growth medium (LB) with 100 mg L⁻¹ ampicillin with shaking at 20°C overnight. SpinClean^TM MBP Excellose^® spin kit (Cat# 23020, Mbiotech, Haman, Korea) and MagneGST™ Protein Purification System (Cat# V8600, Promega, CA, United States) were then used to purify the expressed MBP- and GST-tagged proteins according to the user manuals, respectively.

The ORF of GhHOX3 and GhHD1, in frame, was fused to the maltose-binding protein tag of the expression vector pMAL-C2 (New England Biolabs), and the recombinant proteins were affinity purified following the manufacturer’s manual. The ORF of GhSLR1 and the yellow fluorescence protein gene were in frame fused to the glutathione S-transferase tag of the expression vector pGEX-4T-1 (GE Healthcare), respectively, and the recombinant proteins were affinity-purified using Glutathione Sepharose 4B (GE Healthcare) following the manufacturer’s manual. The 6 × promoter segments of GhRDL1 and GhEXPA1, containing the intact or mutated L1-box cis-element, were labelled with Cy5 on both ends. The assay was performed by incubation of the DNA fragment with the purified protein at 25 °C for 30 min, separated with 5% native PAGE in 0.5 × TBE (Tris/Borate/EDTA) buffer (10 V cm⁻¹, 4 °C). Fluorescence was observed with an image scanner (FLA-9000, FUJIFILM).

The whole HsMar1 excision cassette (Fig. 1a) and the HSMAR-RA ORF ([19 (link)]) were synthetized by Eurofins and cloned in pBluescript KS+. The plasmid containing the HsMar1 cassette is named pHsMar1. The mPB plasmid, expressing PiggyBac transposase, was obtained from the Welcome trust Sanger institute ([39 (link)]).
HSMAR-RA ORF was cloned in fusion with Maltose Binding Protein (MBP) in pMalc2 (New England Biolabs). The resulting fusion MBP-HSMAR-RA and HSMAR-RA were cloned in pCS2 plasmid. After PCR amplification, CRE ORF was cloned in pCS2.
All constructs were controlled by sequencing (Eurofins MWG Biotech). DNA preparations to be transfected were performed with Nucleobond Xtra EF kit (Macherey-Nagel).
Genomic DNAs needed for analyses were extracted and purified with Nucleospin Tissue kit (Macherey-Nagel).
PCR primers used in the study are listed in Additional file 1: Table S1 and were purchased by Eurofins.