Luminata forte western hrp substrate system

Cells were harvested for SDS-PAGE and immunoblotting by scraping into SDS-PAGE sample buffer containing 2% SDS, 100 mM dithiothreitol, 10% glycerol, 0.1% bromophenol blue and protease inhibitors (Complete Roche) in 50 mM Tris–HCl pH 6.8 and heating to 100 °C for 5 min. Other samples were prepared by addition of SDS-PAGE sample buffer and heating to 100 °C for 5 min. Samples were separated on 8–15% (w/v) acrylamide gels and transferred to Protran nitrocellulose membranes (Schleicher and Schuell) using a Mini-PROTEAN 3 gel electrophoresis system and Transblot system (BioRad). The immunoblots were then blocked by incubation in 5% (w/v) dried milk/0.1% (w/v) Tween-20 in Tris buffered saline (TBS) pH 7.5 for 1 h and then probed with primary antibodies diluted in blocking solution for 16 h at 4 °C. Following washing in blocking solution, they were then incubated with horseradish peroxidase-conjugated goat anti-mouse, anti-rabbit or anti-rat Igs (GE Healthcare). Immunoblots were developed using an enhanced chemiluminescence Luminata Forte Western HRP substrate system according to the manufacturer’s instructions (Millipore). Signals on immunoblots were quantified using ImageJ after scanning with an Epson Precision V700 Photo scanner essentially as described by us in previous studies [78 (link)].

Cells were harvested for sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) by washing with calcium-free phosphate buffered saline (PBS) pre-warmed at 37 °C and scraping into SDS-PAGE sample buffer containing protease inhibitors (Complete Roche), 1 mM Na₃VO₄ and 5 mM NaF. Samples were then heated for 10 min at 100 °C, sonicated and centrifuged at 10000 g (av) for 10 min. Total, cytosolic and synaptoneurosome proteins were prepared from rat brains essentially as described [35 (link), 47 (link)]. Protein concentrations were determined using a commercial BCA assay (Pierce). Samples were prepared for SDS-PAGE by addition of sample buffer and then resolved on 10 or 15% SDS-PAGE gels and transferred to nitrocellulose membranes (Schleicher & Schuell Bioscence) by wet electroblotting (BioRad). Membranes were blocked with Tris-HCl-buffered saline (TBS) containing 5% dried milk and 0.1% Tween-20 for 1 h at 20 °C, and then incubated with primary antibodies in blocking buffer for 16 h at 4 °C. Following washing in TBS containing 0.1% Tween-20, the blots were incubated with horseradish peroxidase conjugated secondary antibodies and developed using chemiluminescence with a Luminata Forte Western HRP substrate system according to the manufacturer’s instructions (Millipore). Chemiluminescence signals were detected using a BioRad ChemiDoc MP Imaging system.

Cultured cells were processed and analysed by SDS‐PAGE and immunoblotting as described previously (Gomez‐Suaga et al., 2017 (link)). After probing with primary antibodies, the blots were incubated with horseradish peroxidase conjugated secondary antibodies and developed using chemiluminescence with a Luminata Forte Western HRP substrate system according to the manufacturer's instructions (Millipore). Signals were detected using a BioRad ChemiDoc MP Imaging system. Mouse brains were prepared for SDS‐PAGE as described previously (Stoica et al., 2016 (link)), and samples were analysed as above for cultured cells.