Ne per nuclear extraction reagent kit

Nuclear fractions of hyperoxia-exposed PAECs were isolated using NE-PER Nuclear Extraction Reagent Kit (Thermo Scientific, Rockford, IL) according to manufacturer’s protocol. Lysates were subjected to Western blot using AIF antibody.

Two sets of complementary 22-mer oligonucleotides with biotin end labelling (Supplementary Table S4) were generated by Zoonbio Biotechnology Co., Ltd. (Nanjing, China), which were centred on rs508419 (T/C) with T or C alleles. We annealed each set to create double-stranded oligonucleotides; the sequences of which are specified in Supplementary Table S6. Nuclear protein lysates were extracted from C2C12 cell pellets with the NE-PER Nuclear Extraction Reagent Kit (Thermo Scientific), and protein concentrations were determined with the Pierce BCA Protein Assay Kit (Thermo Scientific). EMSAs were performed with the LightShift Chemiluminescent EMSA Kit (Thermo Scientific), according to the manufacturer’s instructions.
The JASPAR program (http://jaspar.genereg.net/) was used to computationally predict transcription factors that might differentially bind at rs508419 by contrasting the predictions and/or scores generated for each SNP allele35 (link)36 (link). Transcription factor ChIP-seq data for the region overlapping rs508419 were obtained from the ENCODE project17 (link).