Imagestudio ver 3

Amniotic fluid or spinal cord samples were resolved on 8% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred onto 0.22 µM nitrocellulose membranes (Li-Cor Biosciences, Lincoln, NE, USA). After transfer, blots were blocked for non-specific binding with 5% blotting-grade blocker (Bio-Rad Laboratories, Hercules, CA, USA) in TBS and incubated with one of the following antibodies: 1F6 neurocan mouse monoclonal antibody (1:500; Developmental Studies Hybridoma Bank—DSHB, Iowa City, IA, USA), 650.24 neurocan mouse monoclonal antibody (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), or 3F8 phosphacan mouse monoclonal antibody (1:250; DSHB, Iowa City, IA, USA). The sizes of the detected proteins were estimated using Precision Plus Protein™ all-blue prestained protein standards (Bio-Rad Laboratories, Hercules, CA, USA). Blots were incubated with goat anti-mouse antibody conjugated to IRDye^® 680RD dye (LI-COR Biosciences, Lincoln, NE, USA), and signals were detected using Odyssey CLx Imaging System (LI-COR Biosciences, Lincoln, NE, USA). Image Studio Ver. 3.1. (LI-COR Biosciences, Lincoln, NE, USA) was used to quantify the signal intensity and presented as arbitrary fluorescence units (AFU).

Immunoblotting was performed to detect protein expression using the Quick Western Kit–IRDye® 680RD (LI-COR Biosciences) following standard protocol. Primary antibodies used were: anti-SETD7 (a gift from Dr. Susanne Gräslund at SGC), anti-H3K4me1, anti-Histone H3, anti-NFE2L2, Anti-PPARGC1A (Abcam), anti-DDK (Origene), anti-SOD2 (Abcam). Membrane transfer was performed on iBlot 2 Gel Transfer Device with iBlot 2 Transfer stacks (PVDF) (Life Technologies). Imaging analysis was performed on the Odyssey CLx Infrared Imaging System using Image Studio Ver 3.1 (LI-COR Biosciences).