Chemiluminescent detection reagent

Cells and exosomes were lysed in ice-cold cell lysis buffer containing 50 mM Tris/HCl (PH7.5), 2 mM EDTA, 150 mM NaCl, 1 % (v/v) Triton X-100, complete™ proteinase and phosphatase inhibitors (Roche), as described previously [5 (link)]. Lysates were loaded and subjected to SDS–polyacrylamide (4–20 %) electrophoresis and electroblotted onto PVDF membranes. After blocking in 5 % non-fat dry milk, membranes were incubated with mouse anti-polyglutamine (1C2, Cat. No. MAB1574, Millipore), mouse anti-total huntingtin (4C8, Cat. No. MAB2166, Millipore) and mouse anti-mutant huntingtin (EM48; 1:1000) antibodies. Exosomes were identified with the antibodies raised against CD9 (1:1000, Cat. No. EXOAB-CD9A-1, Systems Biosciences), CD63 (1:1000, Cat. No. EXOAB-CD63A-1, Systems Biosciences), CD81 (1:1000, Cat. No. EXOAB-CD81A-1, Systems Biosciences) and HSP70 (1:1000, Cat. No. EXOAB-Hsp70A-1, Systems Biosciences). Membranes were incubated with the appropriate secondary antibody (goat anti-mouse or anti-rabbit) according to the manufacturer’s instructions, followed by the addition of the chemiluminescent detection reagent (Millipore). Bands were visualized using a ChemiDoc™ XRS + system (Bio-Rad) and quantified using the image lab software (Bio-Rad).

The tumor was lysed with RIPA buffer (Beyotime Institute of Biotechnology) on ice for 30 min and followed by centrifugation at 12,000 g for 20 min at 4°C. Next, we perform the quantitation by the BCA method (Thermo Fisher Scientific). The proteins were fractionated by western blot (WB) analysis, and finally detected with chemiluminescent detection reagent (Millipore).

Cells had been separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, lysed in RIPA buffer, and then transferred to a PVDF membrane. After being blocked for one to two hours at room temperature within 10% nonfat milk, the membranes had been incubated at 4°C overnight with antibodies particular to p-β-catenin (1:1,000, Cell Signaling Technology), p-ERK (1:1,000, Cell Signaling Technology), runt-related transcription factor 2 (RUNX2; 1:1,000, Cell Signaling Technology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:1,500, Cell Signaling Technology, Danvers, MA, America). After washing within Tris-buffered saline including Tween 20 (TBST) three times (10 minutes each), the membranes had been incubated by using horseradish-peroxidase-conjugated goat anti-rabbit IgG (1: 5,000, Cell Signaling Technology,) as a secondary antibody at normal temperature for an hour. After being washed with TBST for three times (ten minutes each), the immunoreactive bands had been noticed adopting a developed chemiluminescent detection reagent (Millipore) and further visualized through the explosion of the blot to X-ray film (Bio-Rad) for 0.2-2 minutes. The expression of proteins had been quantified through the determination of the ratio of the proteins' absorbance to that of the inner GAPDH control.