Bradford assay kit

The exosomes isolated by Exoquick™ precipitation or colon cancer cells were lysed with RIPA buffer (Sigma, USA). The protein concentration of lysates using the Bradford Assay Kit (Abcam, USA) with Thermo Scientific™ NanoDrop™ One (USA). Samples were subjected to SDS-PAGE on 12% tris–glycine gels and blotted onto nitrocellulose membranes. Membranes were probed with specific primary antibodies over-night at 4 °C followed secondary antibody for 1 h at room temperature and visualized by the ECL detection system.

HaCaT cells were plated in 100mm culture dishes. Twenty-four hours later, the cells were treated with 0.25, 0.5, 1, 2 mg/mL of ACTPER for 72 h. After treatment, these same cells were washed with cold PBS and lysed using a phosphosafe extraction buffer (Novagen, Madison, WI, USA). Total protein contents in the cell lysates were determined by a Bradford assay kit (Abcam, Cambridge, UK) according to the manufacturer’s protocol. After reconstituting in the sample buffer, 10 micrograms of protein samples were subjected to SDS-PAGE on Bolt™ 10% Bis-Tris Plus Gels (ThermoFisher, Waltham, MA, USA), and electrophoretically transferred to PVDF membranes (Millipore, Burlington, MA, USA). The membranes were reacted with primary antibodies against filament aggregating protein (Filaggrin) (sc-80609, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and β-actin (A5441, 1:5000, Sigma). Membranes were then incubated with HRP-conjugated anti-mouse or anti-rabbit IgG (1:100,000, Sigma) and visualized in films using ECL solution (Millipore, Billerica, MA, USA).