Typhoon scanner

EMSA was carried out as described with minor modifications (33 (link)). Briefly, the chemically synthesized EspR cognate sequences (FL, ΔS1, ΔS2) (IDT/Eurofins, Germany) were annealed and radiolabeled with γP³² using T4 PNK (NEB, England). The binding assays of recombinant EspR to different probes were performed in NEB 2.1 buffer supplemented with 50 ng/ml pdIdC, 10% glycerol, and in the presence or absence of unlabeled molar excess of cognate DNA (cold/homologous competition) for 30 min at ambient temperature (25-30°C). The DNA-protein complexes were resolved on a 5% native acrylamide gel (39: 1) at 200 V in 0.5 X TBE. Subsequently, the gels were dried and exposed to a phosphor imaging screen overnight (16-20 h) and imaged using a Typhoon scanner (BIO-RAD, USA) with Quant One software.

DNA oligonucleotides labelled with biotin at the 5′-end were annealed to produce double-stranded probes. After the overexpression of POU-M2 in BmE cells, the nuclear proteins were extracted from the cells using the nuclear and cytoplasmic protein extraction kit (Beyotime). EMSA was performed using an EMSA/Gel-Shift kit (Beyotime) [39 (link)]. DNA-binding assay was performed in 10 µl reaction system containing 2 µl nucleoprotein, 1 µl labelled probe and 2 µl binding buffer (Beyotime) at 25°C for 20 min. For the competition assay, a 10- to 100-fold molar excess of unlabelled (cold) probe or mutant probe was incubated with nucleoprotein for 10 min and then incubated with the labelled probe for 20 min. For antibody-based EMSA analysis, the nucleoprotein, labelled probe and anti-POU-M2 (1 µl) were co-incubated at 25°C for 20 min. The reaction mixture was separated on 5% SDS-PAGE in 0.5 × TBE buffer (45 mM Tris-borate, 1 mM EDTA, pH 8.3) by electrophoresis. Finally, the gel was photographed by a Bio-Rad Typhoon scanner (CA, USA). The primers for the probes are listed in the electronic supplementary material, table S1.