Anti vlcad

Western blotting of whole cell lysates from patient and control fibroblasts was performed as previously described [20 ]. Twenty five micrograms of protein were loaded onto a 4–15% gradient precast SDS-PAGE gel (Bio-Rad Laboratories, Hercules, CA). Following electrophoresis separated proteins were transferred onto a nitrocellulose membrane. Primary antibodies included anti-ITCH (1;1000, BD Transduction Laboratories, San Jose, CA, #611198), Anti-VLCAD (1:1000, antigen produced by the Vockley Lab, UPMC Children's Hospital of Pittsburgh and antibody generated by Cocalico Biologics, Stevens, PA), Anti-TFP α/β (1:2000, produced by the Vockley Lab), anti-Ubiquitin (1:1000, Abcam, Cambridge, MA, #ab19247), and anti-TXNIP (1:500, Abcam, #ab188865). Blots were incubated with HRP conjugated secondary antibodies. Primary purified mouse anti-GAPDH monoclonal antibody (1,25,000, Abcam, #ab8245) was used as a loading control (Supplemental Table S1). All western blot studies were performed more than once from different celluar lysates (technical replicates).