Ni charged magbeads

The full-length coding sequence of MeSAUR1 was amplified with V2 primer pair (Table 1). The PCR product was ligated into the pCold Pros2 vector (TaKaRa, JPN) at the site of Nde I/Sal I, generating pCold Pros2-MeSAUR1. pCold Pros2-MeSAUR1 was introduced into Escherichia coli strain BL21 (DE3) for protein expression. E. coli cells containing pCold Pros2-MeSAUR1 were cultured in LB medium supplied with 100 mg/L Ampicillin at 37°C. When the OD₆₀₀ of the culture reaches 0.4–0.8, quickly cool the culture to 15°C in ice water, and then 1.0 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added and the cultures were incubated at 15°C, 120 rpm for 24 h. The cells were collected and resuspended in the BugBuster^® Protein Extraction Reagent (Novagen, GER) and incubated at 30°C for 1 h. The supernatant was collected and purified with Ni-Charged MagBeads (GenScript, United States). pCold Pros2 was expressed and purified as a control in accordance with the above methods. The purified protein was verified by SDS-PAGE and Western Blotting (Sambrook and Russell, 2001 ). Tag Anti-ProS2 (Takara, JPN) and Goat Anti-Mouse IgG/HRP (Boster, United States) were the primary and secondary antibodies used in Western Blot, respectively.

Escherichia coli Rosetta (DE3) was used for protein prokaryotic expression. GST‐ and His‐tagged proteins were purified by Glutathione MagBeads and Ni‐Charged MagBeads (GenScript, Nanjing, China), respectively. Purified His‐tagged protein (10 µg) and/or GST‐tagged protein (10 µg) were mixed and incubated with 30 µL Glutathione MagBeads in phosphate‐buffered saline (PBS) for 3 h. MagBeads were separated using a magnetic frame and washed five times with 1 × PBS containing 0.5% NP‐40. The bound proteins were analysed by Western blotting.

To get a large amount of MePHD1 protein, the full-length coding sequence of MePHD1 was amplified with P1 primer pairs and then fused to the Nde I/Sal I sites in the pCold Pros2 vector (TaKaRa, Tokyo, Japan), which generated the pCold Pros2-MePHD1. pCold Pros2-MePHD1 was introduced into Escherichia coli strain BL21 (DE3) competent cells for protein expression. The transformed strains were cultured in LB medium supplied with 100 mg/L Ampicillin, at 37 °C, and 250 rpm until the OD600 of the culture reached 0.4–0.8, and the bacterial cell fluid temperature cooled down to 15 °C. The cultures were incubated at 15 °C and 120 rpm, for 24 h, after adding 1.0 mM isopropyl β-d-1-thiogalactopyranoside. The soluble proteins of the cultured cells were extracted and purified by BugBuster^® Protein Extraction Reagent (Novagen, Darmstadt, Germany) and Ni-charged MagBeads (GenScript, Jiangsu, China), respectively. pCold Pros2 was expressed and purified as control. The purified protein was verified by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. When performing Western blotting, Tag Anti-ProS2 (TaKaRa, Tokyo, Japan) and Goat Anti-Mouse IgG/HRP (Boster, Wuhan, China) were used as primary and secondary antibodies, respectively.