Glutathione sepharose bead

GST-fusion proteins were prepared by growing transformed BL21 E. coli and inducing recombinant protein expression by adding IPTG (0.5 mM final concentration) for 2 hr. Bacteria were pelleted, and the GST-fusion protein was purified employing standard procedures using glutathione Sepharose beads (Thermo Scientific).
Hippocampi and cortices dissected from adult rat brains were pooled together, lysed in RIPA buffer by homogenisation in a tephlon-glass homogeniser, rotated for 1 hr at 4°C and then centrifuged at 10,000 × g for 30 min at 4°C. Supernatants were incubated with glutathione Sepharose beads for 3 hr at 4°C and then washed and resuspended in 3× sample buffer and analysed by SDS-PAGE followed by western blotting.

GST-fusion proteins were prepared by growing transformed BL21 E. coli and inducing recombinant protein expression by adding IPTG (0.5mM final concentration) for 2 h. Bacteria were pelleted, resuspended in lysis buffer (8 M urea, 50 mM Tris-HCl, 1 mM EDTA, 1 mM DTT, pH 7.5) and rotated for 1 h at 4°C. Lysed bacteria were inserted into a dialysis membrane (cutoff 13kDa) and dialyzed for 1 h at 4°C in dialysis buffer I (4 M urea, 1 mM Tris-HCl, 1 mM DTT, pH 5). The membrane was then moved to dialysis buffer II (50 mM Tris-HCl, 50 mM NaCl, 2 mM MgCl2, 200 μM DTT, pH7.5) overnight at 4°C. Membranes were dialyzed with new dialysis buffer II for 3 h at 4°C. Lysates were then collected and centrifuged at 10,000 g for 30 min at 4°C. The supernatant was incubated with glutathione Sepharose beads (Thermo Scientific) overnight at 4°C and then washed with dialysis buffer II.
Hippocampi and cortices dissected from adult rat brains were pooled together, lysed in RIPA buffer by homogenization in a teflon-glass potter, rotated for 1 h at 4°C and then centrifuged at 10,000 g for 30 min at 4 °C. Supernatants were incubated with glutathione Sepharose beads for 3 h at 4°C and then washed and resuspended in 3X sample buffer and analyzed by SDS-PAGE followed by western blotting.

The calli expressing 35S::Myc and 35S::MdbHLH3-Myc were subjected to ChIP-qPCR analysis. We used an anti-Myc antibody (Beyotime)^{6 (link)}. The immunoprecipitated DNA samples were used as templates for qPCR assays.
EMSA was conducted as described by Hu et al.^{6 (link)}. MdbHLH3-His recombinant protein was expressed in Escherichia coli strain BL21 and purified using glutathione sepharose beads (Thermo Scientific, San Jose, CA, USA). The EMSA probe biotin labeling kit (Beyotime) was used to label an oligonucleotide sequence corresponding to the MdDEP1 promoter, which was subsequently used for the binding assays with a LightShift chemiluminescent EMSA kit (Thermo).