Escherichia coli bl21

The GST pulldown assay was carried out as previously described^{58 (link)}. The GST alone and GST-fusion proteins were expressed in Escherichia coli BL21 (Takara) and purified using Pierce GST Spin Purification Kit (Thermo scientific) as per the manufacturer’s instruction. The purified proteins were subjected to immobilized on the Pierce Spin Column and then preyed ERα from MCF-7 cells lysate.

The open reading frames (ORF) of the five glycoside hydrolases and the three SBP, excluding the signal peptide sequences predicted by the PSORT server (https://psort.hgc.jp/), were amplified by PCR and cloned into pCold I DNA (TaKaRa Bio Inc., Shiga, Japan), using restriction enzymes (NdeI and XhoI) or an In-fusion HD cloning kit (TaKaRa Bio Inc.). The primers used in this study are listed in Table 1. Each plasmid was transformed into Escherichia coli BL21 (TaKaRa Bio), and the transformants were cultured to express the recombinant N-terminal His-tagged proteins. The culture conditions were according to the manufacturer’s standard protocol, except for E. coli BL21 harboring the BpXyl43A-expressing plasmid, which was cultured at 15°C after the addition of 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside) and 3% ethanol. The bacterial cells were lysed using 4 ml/g of lysis buffer (B-PER bacterial cell lysis reagent; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 100 μg/ml lysozyme and 10 U/ml DNase I, followed by centrifugation at 4°C (15,000 × g, 10 min) to obtain the protein fraction. Recombinant proteins were further purified using an Ni-nitrilotriacetic acid spin column (Qiagen) and concentrated by use of a Vivaspin ultrafiltration membrane (GE Healthcare UK Ltd., Little Chalfont, UK). The recombinant proteins prepared were verified using SDS-PAGE (Fig. S4).

GST alone, GST-KLF12 and GST-p53 were expressed in Escherichia coli BL21 (Takara, Dalian, China) and purified using Pierce GST Spin Purification Kit (Thermo Scientific, MA, USA) as per the manufacturer’s instruction. The purified GST and GST-KLF12 were seperately immobilized on a different Pierce Spin Column and used to pull down endogenous p53 from MCF-7 cell lysate. The purified GST and GST-p53 were seperately immobilized on a different Pierce Spin Column and used to pull down exogenous p300 from HEK293T cell lysate. An immunofluorescence assay was conducted as described previously [63 (link)].