To generate the expression plasmids for the GST-fused JMJD6 protein, we integrated the JMJD6 cDNA sequence between 1075 and 1834 into the EcoRI/XhoI site of pGEX-4T-1 vector (Cytiva, Marlborough, MA, USA), as previously described [17 (link)].
Moreover, E. coli BL-21 cells transformed with the pGEX-4T-1 clone were cultured in 200 mL of Luria–Bertani broth and treated with 0.1 mM IPTG for 3 h. The cells were then collected and lysed by sonication in BugBuster Master Mix (Novagen, San Diego, CA, USA), followed by centrifugation at 13,000× g and 4 °C for 10 min. GST-tagged JMJD6 proteins were purified through Glutathione-Sepharose column chromatography (GE Healthcare Life Sciences, Chicago, IL, USA) and dialyzed as previously described [17 (link),18 (link),19 (link)].
Moreover, E. coli BL-21 cells transformed with the pGEX-4T-1 clone were cultured in 200 mL of Luria–Bertani broth and treated with 0.1 mM IPTG for 3 h. The cells were then collected and lysed by sonication in BugBuster Master Mix (Novagen, San Diego, CA, USA), followed by centrifugation at 13,000× g and 4 °C for 10 min. GST-tagged JMJD6 proteins were purified through Glutathione-Sepharose column chromatography (GE Healthcare Life Sciences, Chicago, IL, USA) and dialyzed as previously described [17 (link),18 (link),19 (link)].
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