Original ContributionNeuroprotective effects of PEP-1-carbonyl reductase 1 against oxidative-stress-induced ischemic neuronal cell damage
Section snippets
Materials
Ni2+–nitrilotriacetic acid Sepharose Superflow was purchased from Qiagen (Valencia, CA, USA). The indicated primary antibodies and actin were obtained from Cell Signaling Technology (Beverly, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA). 4-Hydroxy-2-nonenal (4-HNE) and 8-hydroxy-2-deoxyguanosine (8-OHdG) antibodies were purchased from Santa Cruz Biotechnology. CBR1 inhibitor (hydroxyl-PP-Me) was obtained from Sigma–Aldrich (St. Louis, MO, USA). The polymerase chain reaction (PCR)
Purification and transduction of PEP-1–CBR1 protein into HT-22 cell lines
Supplementary Fig. 1 shows the PEP-1–CBR1 protein expression vector that is based on a pET-15b vector that has a PEP-1 sequence, six histidines, and a CBR1 gene. PEP-1–CBR1 proteins were overexpressed by adding IPTG and purified from the overexpressed whole extract of E. coli using a Ni2+–nitrilotriacetic acid Sepharose affinity column and PD-10 column chromatography. As shown in Figs. 1A and B, purified PEP-1–CBR1 protein was confirmed through SDS–PAGE and Western blotting using a
Discussion
Human CBR1 is known to be a ubiquitous NADPH-dependent enzyme belonging to the short-chain dehydrogenase/reductase superfamily, which catalyzes the reduction of carbonyl compounds. So far, the known kinds of carbonyl reductases are CBR1, CBR3, and CBR4 [43]. These enzymes are known to have a crucial role in detoxification in cancer, diabetes, and other neuronal diseases [10], [11], [12], [13], [14], [15], [43], [44], [45], [46], [47], [48]. CBR1 plays a critical role in biological processes as
Acknowledgments
This work was supported by a Priority Research Centers Program grant (NRF-2009-0093812) and a Mid-career Researcher Program grant (NRF-2012R1A2A2A06043084) through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning in the Republic Korea.
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These authors contributed equally to this work.