Sc 8784

We mixed 30-µg protein extracts with a sample buffer, boiled them for 10 min, and performed electrophoresis using 8–15% sodium dodecyl sulfate-polyacrylamide gels. We transferred the proteins in the gels to a polyvinylidene difluoride membrane and incubated the blots with primary antibodies against COL1α1 (sc-8784, Santa Cruz, CA, USA), PPARγ (16643-1-AP, PROTEINTECH, IL), TFAM (sc-166965, Santa Cruz, CA, USA), CD36/SR-B3 (NB400-144, Novus Biologicals, CO, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (60004-1-lg, PROTEINTECH, IL, USA) for protein control. After washing the blots with tris-buffered saline and incubating them with horseradish peroxidase-coupled anti-rabbit immunoglobulin-G antibodies (dilution, 1:5000, NEF812001EA, PerkinElmer, MA, USA), HRP anti-mouse immunoglobulin-G antibodies (dilution, 1:10,000, NEF822001, PerkinElmer, MA, USA),), and HRP anti-goat immunoglobulin-G antibodies (dilution, 1:10,000, sc-2354, Santa Cruz, CA, USA) at room temperature for 1 h, we developed them with enhanced chemiluminescence detection (GE Healthcare Biosciences AB, Uppsala, Sweden), exposed them to film, and quantified the signals by using densitometry.

Dorsal skin tissue samples were collected at various time points following the final UVA irradiation; 1, and 6 h post-irradiation for Nrf2 and its target proteins, respectively; 1 h post-irradiation for oxidative DNA damage; 24 h post-irradiation for MMP-1 and collagen. Tissue sections were washed with PBS for 5 min/time (3 times) and blocked with phosphate-buffered saline (PBS) containing 2% BSA for 30 min. After removing excess blocking buffer, the slides were incubated with Nrf2 Ab (ab31163; Abcam, Cambridge, MA, United States), GST Ab (sc-459; Santa Cruz Biotechnology, Santa Cruz, CA), NQO1 Ab (ab34173; Abcam, Cambridge, MA, United States) (1:50), 8-OHdG [N45.1] Ab (ab48508; Abcam, Cambridge, MA, United States) (1:50), MMP-1 Ab (ab137332; Abcam, Cambridge, MA, United States) (1:50), collagen I (C-18) Ab (sc-8784; Santa Cruz Biotechnology, Santa Cruz, CA) (1:50) for 1 h. The slides were then washed for 5 min/time (3 times) with a PBS solution and incubated for 1 h at room temperature with FITC-conjugated the secondary Ab (green) and with DAPI (blue) to counterstain the nuclei for detection of nuclear Nrf2, the secondary Ab Alexa Fluor 488 goat anti-rabbit (Abcam) for detection of MMP-1 and collagen levels. An inverted fluorescent microscope equipped with a Nikon Intensilight was used for the imaging of IF stainings (20X) which were quantified using ImageJ software.

After treatment with TGFβ, LX2 and Huh7 cells were scraped in RIPA buffer (50 mM Tris HCl, pH 7.4, 1% Triton X-100, 0.2% SDS, 1 mM EDTA, 1 mM PMSF and 5 μg/ml leupeptin) to obtain total protein extracts. Also, protein content from LX2 and Huh7 culture supernatants were concentrated using ultrafiltration units (Vivaspin Turbo 4 Ultrafiltration Unit.VS04T21, Sartorius, Thermo Fisher Scientific Inc.). Protein extracts and concentrated supernatants were boiled in Laemmli sample buffer prior to electrophoresis in 10% SDS-PAGE. Proteins were then transferred to an immunoblot nitrocellulose membrane (Bio-Rad) that was blocked with 5% non-fat dry milk and exposed to primary BMP8A antibody (ab154373 Abcam plc, Cambridge, UK), αSMA (A-2547, Merck Life Science, Darmstadt, Germany) and COL1A1 (SC-8784, Santa Cruz Biotechnology Inc., Heidelberg, Germany) antibodies overnight at 4 °C. After the incubation with the corresponding secondary antibody (Santa Cruz Biotechnology Inc.), immunoreactive bands were visualized using the ECL Western blotting protocol (Bio-Rad). Densitometric analysis of the bands was performed using Image J software. Ponceau staining was used as loading control.