Smart bicinchoninic acid protein assay kit

Pro-Prep Protein Extraction Solution (Intron Biotechnology Inc., Seongnam, Korea) was used to obtain total proteins from NHDF cells and skin tissues, on the basis of the manufacturer’s protocol. After determination of the protein concentration using a SMARTTM Bicinchoninic Acid Protein Assay Kit (Thermo Fisher Scientific Inc., Wilmington, DE, USA), we separated total proteins by 4–20% SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) for 2 h, followed by transfer to nitrocellulose membranes at 40 V for 2 h. They were independently incubated overnight at 4 °C with the specific primary antibodies (Supplementary Table S1). These membranes were then incubated with 1:2000 diluted horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Invitrogen) at room temperature for 1 h. Enzyme on blots were developed using the Amersham ECL Select Western Blotting detection reagent (GE Healthcare, Little Chalfont, UK). Finally, chemiluminescence signals for each band were detected under FluorChemi^®FC2 (Alpha Innotech Co., San Leandro, CA, USA).

Liver proteins were extracted using Pro-Prep Protein Extraction Solution (Intron Biotechnology Inc., Seongnam, Republic of Korea) following the manufacturer’s instructions. The protein concentration was determined with the SMARTTM Bicinchoninic Acid Protein assay kit (Thermo Fisher Scientific Inc., Wilmington, MA, USA). Next, 30 μg of protein was separated on a 4–20% SDS-PAGE gel, transferred onto nitrocellulose membranes, and blocked with 5% nonfat milk in Tris-buffered saline with Tween 20 (TBST). The membranes were then incubated overnight at 4 °C with specific primary antibodies. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using a chemiluminescence kit, and images were captured using a digital camera equipped with the FluorChem^® FC2 Imaging system. Protein densities were analyzed using the AlphaView Program, version 3.2.2.

The total proteins were obtained from NHDF cells and skin tissues using Pro-Prep Protein Extraction Solution (Intron Biotechnology Inc., Seongnam, Korea) based on the manufacturer’s protocol. After centrifugation at 13,000 rpm for 5 min, the protein concentrations were determined using a SMARTTM Bicinchoninic Acid Protein Assay Kit (Thermo Fisher Scientific Inc.). Proteins were separated by 4–20% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) for 2 h, and then transferred to nitrocellulose membranes at 40 V for 2 h. Membranes were then incubated at 4 °C with the following primary antibodies overnight; anti-MMP-1/8 (H-300) (Cat. No. SC-30069, Santa Cruz Biotechnology) or anti-β-actin antibody (Cat. No. A5316, Sigma-Aldrich Co.); washed with washing buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 0.05% Tween 20); and incubated with 1:1,000 diluted horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cat. No. G21234, Invitrogen) at room temperature for 1 h. Blots were developed using Amersham ECL Select Western Blotting detection reagent (Cat. No. RPN2235, GE Healthcare, Little Chalfont, UK). Chemiluminescence signals from specific bands were detected using FluorChemi^®FC2 (Alpha Innotech Co., San Leandro, CA, USA).