Supersep ace 5 20 gel

Western blotting was performed as previously described [22 (link)] with slight modifications using specific antibodies. Briefly, lysates of SH-SY5Y cells were separated by SDS-PAGE using a SuperSep Ace 5–20% gel (Wako), and the resulting proteins were transferred to a polyvinylidene difluoride membrane (Merck Millipore, Darmstadt, Germany). The membrane was blocked with 5% nonfat milk for 1 h at room temperature and then reacted with primary antibodies (all antibodies used at 1:1000 dilution) for 18 h at 4 °C, followed by reaction with the corresponding secondary horseradish peroxidase-conjugated antibody (all antibodies used at 1:1000 dilution) for 1 h at room temperature. Signals were detected by Western Lightning Plus-ECL (PerkinElmer, MA, USA). Chemiluminescence was captured using a cooled CCD Light-Capture camera system and analyzed using CS Analyzer software version 2.0 (ATTO, Tokyo, Japan).
The caspase pathway was analyzed by detecting changes in proteins cleaved upon activation (caspase-3, PARP, and AIF), translocation of cytochrome c out of mitochondria, and regulators that promote (Bax) or suppress (Bcl-2) apoptosis by western blotting.

Samples dissolved in Laemmli SB were separated by Novex 3–8% Tris-Acetate Gel (binding assay and tau oligomerization assay; Invitrogen) or SuperSep Ace 5–20% gel (other assays; WAKO Pure Chemical Industries) and transferred onto membranes with semi-dry transfer systems (Bio-Rad Laboratory). The membrane was blocked with 5% milk in PBS-T for 1 h at room temperature. They were probed with antibodies overnight at 4 °C. After washing the membrane with PBS-T, blots were incubated with horseradish peroxidase-linked second antibodies and then examined by enhanced chemiluminescence detection on Las3000 or Las4000 (GE Healthcare).