Western bright ecl western blotting detection kit

Total protein was collected from the cells using RIPA buffer (MilliporeSigma) containing protease inhibitors and the protein concentrations were determined by the BCA method. Equal amounts of protein (20 µg/lane) were loaded and separated on SDS-polyacrylamide gels (8-10%) for electrophoresis. Thereafter, the protein bands on the gels were transferred to nitrocellulose membranes (MilliporeSigma). The membranes were then blocked with 5% bovine serum albumin (diluted in Tris-Cl-buffered saline with 0.1% Tween-20) for 2 h at room temperature and incubated with primary antibodies at 1:3,000 dilution overnight at 4˚C. Subsequently, the membrane was incubated with secondary antibodies (1:3,000 dilution; anti-mouse IgG or anti-rabbit IgG; cat. nos. ab205719 and ab205718; Abcam). The blot was then detected using the Western Bright ECL western blotting detection kit (Bio-Rad Laboratories, Inc.). Equal sample loading was verified by detection of GAPDH. The primary antibodies were as follows: Mouse monoclonal anti-GAPDH (cat. no. sc-32233; Santa Cruz Biotechnology, Inc.), rabbit monoclonal anti-MCU (cat. no. ab272488; Abcam), rabbit polyclonal anti-Aβ (cat. no. 51-2700; Invitrogen; Thermo Fisher Scientific, Inc.), mouse monoclonal anti-tau (cat. no. sc-390476; Santa Cruz Biotechnology, Inc.) and rabbit monoclonal anti-tau (phospho Ser214; cat. no. ab170892; Abcam).