2.8. Wes Simple Western System

Protein quantification was done with the Wes Simple Western system, as per manufacturer’s specified protocols. Wes Simple Western uses an automated process of capillary gel electrophoresis. In brief, proteins were extracted from tissue with a lysis buffer comprising 50-mM Tris-HCl (pH 7.5), 150-mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, and 0.1% SDS; lysates were quantified using a micro BCA assay reagent kit (Pierce, Rockford, IL, USA). Proteins were loaded onto Wes 25-well plates with Simple Western Sample Buffer and ProteinSimple MasterMix, containing dithiothreitol and fluorescent standards. Primary antibodies, horseradish-peroxidase-conjugated secondary antibodies, separation gel matrix, stacking gel matrix, and luminol-peroxide mixture were placed into the appropriate wells of the Simple Western plate. Chemiluminescent signal was quantified as peak area in the electropherogram (see Supplementary Fig. 2A) and also represented by the “gel view” function of the ProteinSimple software (Supplementary Fig. 2B).