Quantifying Neuronal Protein Levels

As described in Trostnikov et al. (2019) (link), for evaluation of the protein amount in motor neurons and the brains, approximately 30 thoraxes and 30 heads, respectively, of 3–5 days old adults of each genotype were dissected and homogenized in 8 M urea solution. Equal amounts of samples from the supernatants were preincubated with sample buffer (deionized water, 0.5 M Tris-HCl, glycerol, 10% SDS, 0.5% bromphenol blue, DTT) for 5 min at 95°C and separated in a 4–12% (w/v) acrylamide Bis/Tris SDS-PAGE gel using the vertical electrophoretic chamber Mini-Protean Tetra (Bio-Rad). Proteins were transferred from the gel to the PVDF membrane (Immobilon-P Membrane) using electroblotting (Mini Trans-Blot Modul, Bio-Rad), blocked in BlockPro blocking buffer (Visual Protein) and incubated with anti–GSK3 beta primary antibodies (1:300; ab18893, Abcam) for 1 h. Bound antibodies were detected with goat anti–rabbit secondary antibodies conjugated with alkaline phosphatase (1:20000; A3687, Sigma). Prior to visualization, the membranes were incubated in the alkaline CDP buffer for 5 min and then in the Immun-Star AP- Substrate (Bio-Rad) for 7 min. After scanning, the relative intensity quantification of each band was evaluated with Image Lab software (Bio-Rad). Three to four independent protein extractions (biological replicates) per sex per genotype were made.