Tnt t7 sp6 coupled reticulocyte lysate system

Radiolabeled substrates were generated by in vitro transcription/translation using the plasmid pcDNA3.1-Linc013026-MYC, the SP6/T7-coupled TNT reticulocyte lysate system (Promega, Madison, WI, USA), and [³⁵S]methionine (370 kBq/μL, >37 TBq/mmol, Hartmann Analytic, Braunschweig, Germany) according to the manufacturer’s instructions.

Radiolabeled substrates were generated by in vitro transcription/translation using the plasmids pSNAP-23his6 as a positive control and pcDNA3.1-C20orf204-189AA-MYC, the SP6/T7-coupled TNT reticulocyte lysate system (Promega), and [³⁵S]methionine (370 kBq/μl, >37 TBq/mmol, Hartmann Analytic, Braunschweig, Germany) according to the manufacturer’s instructions.

EHF protein was obtained when 1 μg of pcDNA3.1(-)A-EHF or empty vector as DNA template was used according to the protocol of the TNT T7/SP6 Coupled Reticulocyte Lysate System (Promega Corp., Madison, WI, USA). EMSA was performed by using LightShift Chemiluminescent EMSA Kit (Pierce Biotechnology, Rockford, IL, USA) following the manufacturer's protocol. The binging reaction mixtures contained 2 μl of the translation products, 100 fmol of biotin-labeled oligonucleotide probes (Sangon, Shanghai, China), 2.5% glycerol, 5 mM MgCl2, 50 ng/μl Poly (dI-dC) and 1% NP-40 were incubated in binding buffer at room temperature for 20 min. Unlabeled wild type or mutant oligonucleotides (10 pmol) were incubated with the translation products at room temperature for 15 min prior to the addition of biotin-labeled probes. The mixtures containing loading buffer were separated on a 6% non-denaturing polyacrylamide gel in 0.5 × TBE buffer at 100 V, and oligonucleotides were electrophoretic transferred to a nylon membrane (Roche Diagnostics, Mannheim, Germany). After cross-linking with HL-2000 HybriLinker Hybridization Oven (UVP, Upland, CA, USA), the membrane was detected using a LightShift Chemiluminescent EMSA Kit. The sequences of the double-stranded oligonucleotides used to detect the DNA-binding activity of EHF were presented in Supplementary Table 7.