Immun star ap substrate

Proteins were extracted using Complete Lysis-M (Roche, Indianapolis, IN, USA). Extracts were subjected to SDS-PAGE (Super Sep Ace 7.5, 10 or 15%, Wako Pure Chemical Industries, Osaka, Japan) and transferred onto a membrane (Amersham Hybond-P PVDF Membrane, GE Healthcare, Buckinghamshire, UK). Primary and secondary antibodies are listed above in the Reagents subsection. Antibody-protein complexes were detected using Immun-Star™ AP substrate (Bio-Rad Laboratories), and protein bands were visualized using an ImageQuant™ LAS 4000 image analyzer (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).

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.

Approximately 50 heads of 3- to 5-day-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,Millipore, Burlington, MA, USA) using electroblotting (Mini Trans-Blot Modul, Bio-Rad), blocked in BlockPro blocking buffer (Visual Protein Biotechnology Corporation, Taiwan), and incubated with anti–GSK3 beta primary antibodies (1:300; ab18893, Abcam, Cambridge, Great Britain) for one hour. 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 independent protein extractions (biological replicates) per genotype were made.