In fusion system

The coding region of OsNAC14 amplified from the cDNA was cloned into the pHBT vector carrying GFP-myc using the In-fusion system (Clonetech). The final construct (35S::OsNAC14-GFP) and the control vectors (35S::GFP) were transfected into protoplasts (Oryza sativa cv. Ilmi) using PEG-mediated protoplast transformation system. 35S::NF-YA7-GFP was used as control for nuclear localization (Lee et al., 2015 (link)). GFP and mCherry signals were observed 12 h after transfection using SP8 STED confocal fluorescence microscope (Leica).

The coding region of transcript Pif1A-RI cDNA (GH05455) was amplified by PCR with forward primer 5′-ACTATACAAAGAATTCGCTGCCATGGGC-3′ and reverse primer 5′-GGTACTAGTGGATCCGTAGTATTTCTTAG-3′ (synthesized by IDT, Coralville, IA, USA). The Clone Tech InFusion System (Mountain View, CA, USA) was used to clone the PCR product into the CsprHS83 vector directly upstream and in frame with a sequence encoding GFP. Downstream of the inserted gene was a fly myosin VI 3′-UTR. Final vectors were confirmed by sequencing on premises (Biology Department, Washington University, St. Louis, MO, USA).

We created minigenes using the procedure described by Ueyama et al. [20 (link)]. Minigenes contained a full-length human OPN1LW cDNA with the full-length introns 2 and 3 inserted on either side of exon 3. Site-directed mutagenesis was performed using the QuickChange kit (Agilent) or the In-Fusion system (Clonetech) to create 128 different exon 3 haplotypes which were cloned into the minigene using the HindIII and AflII sites flanking exon 3. The 128 minigenes generated were sequence-verified, and the only differences among them were the sequences of exon 3. Exon 3 haplotypes varied at eight nucleotide positions, described here using SNP ID numbers: rs949930 was A or G, rs713 was A or C, rs731614 was C or G, rs5986963 was A or G, rs5986964 was T or G, rs149897670 was C or T, rs145009674 was A or G, and rs155715655 was G or T. Minigenes represented all combinations of these SNPs except rs5986964. Typically, in OPN1LW genes, rs5986963 and rs5986964 are A and T, or G and G, so these are the only combinations represented in the 128 minigenes. Exons 2 and 4 of the human OPN1LW gene are also polymorphic. The minigenes all had the following residues at the variable positions: c. 194C (rs782815809) c.300A (rs1065420), c.331A (rs1065421), c.347C (rs1065422), c.689T (rs148583295), c.697G (rs781936473), c.698C (rs1292153674), c.699T (rs1226772901), and c.706A (rs1065426).

To generate the Luc and the Luc-Cre vectors, the pS/MARt-GFP DNA vector was first digested with the restriction enzymes NheI and BglII to linearize the vector and eliminate the transgene GFP. The InFusion system provided by Clonetech was used to introduce the luciferase gene alone or in combination with the Cre recombinase gene to generate the vector pS/MARt-Luc or the vector pS/MARt-Luc-P2A-Cre, respectively.

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).

pMX-OCT4, pMX-SOX2, pMX-KLF4 and pMX-cMYC, were obtained from Addgene (plasmids 17217, 17218, 17219 and 17220, respectively). The shRNA p53 construct was previously described and validated49 (link). Constructs for the expression of mutant oncogenes were obtained from Addgene (RasV12 Neo (w108-1) #22259; EGFR D770-N771 insNPG #11016; EGFR (del3) #11015; src Y527F #13660). Coding sequence for the mutant oncogenes were removed by restriction enzymes and subcloned into a lentiviral backbone with the In-Fusion system according to manufacturer's instructions (Takara/Clontech).