Protein samples were prepared from the cultured HaCaT cells with a protein extraction buffer (Cell Lytic™ M; Sigma, St. Louis, MO, USA), in accordance with the instruction manual. The protein concentration of the samples was measured with a Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA) using a spectrophotometer with optimal density at 595 nm. The protein samples were separated on precast gradient polyacrylamide gels (Bolt™ 4–12% Bis-Tris Plus Gels; Thermo Fisher Scientific, Waltham, MA, USA) and transferred to nitrocellulose membranes (GE Healthcare, Little Chalfont, Buckinghamshire, UK) by using the Bolt™ Mini Blot Module and Mini Gel Tank (Thermo Fisher Scientific, Waltham, MA, USA), in accordance with the manufacturer’s recommendations. The membrane was blocked in 5% bovine serum albumin (BSA; Sigma, St. Louis, MO, USA). The blocked membrane was probed with a primary antibody and a horseradish peroxidase-conjugated secondary antibody. Following a repeat of the washing step, the membrane was kept in enhanced chemiluminescence detection reagents (Thermo Fisher Scientific, Waltham, MA, USA) for 1 min. Signal intensity was measured with an image analyzer (ChemiDoc™ XRS+; Bio-Rad Laboratories, Hercules, CA, USA).
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