Ab137332

The proteins of each group were collected, and the precipitate was separated by lysis, shock, and centrifugation. The precipitate was separated by electrophoresis in 10% SDS-PAGE gel then transferred to PVDF membrane. Following 5% non-fat milk or BSA blocking, the membranes were incubated with related primary antibodies at 4°C overnight. Then, the membranes were incubated with secondary antibodies for 1 h and imaged by ECL chemiluminescence kit (Bio-Rad). The antibodies utilized as follows: MMP1 (1 : 1000; ab137332; Abcam), Vimentin (1 : 2000, #5741, CST), Snail (1 : 1000, #3879, CST), E-cadherin (1 : 2000, #3195, CST), N-cadherin (1 : 1000, #13116, CST), and GAPDH (1 : 2000 dilution, #2118, CST).

The harvested skin tissues were homogenized in Pro-prep solution (iNtRON Biotechnology; Seoul, Korea), and their lysates were centrifuged at 12,000× g for 30 min. The proteins were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, MA, USA). The membranes were blocked with 5% nonfat milk for 2 h and washed with Tris-Buffered Saline containing 0.05% Tween-20 (TBST) buffer. The membranes were incubated with primary antibodies of Matrix metalloproteinase (MMP)-1 (ab137332, 1:1000, abcam, Cambridge, UK) -2, -9, and TGF-β1 (sc-13595, sc-393859, sc-130348, 1:1000, Santa Cruz, CA, USA), at 4 °C overnight. The blots were incubated with a horseradish peroxidase-conjugated secondary antibody (1:1000, Thermo Scientific, IL, USA) for 1 h. Immunoreactive bands were visualized with the Pierce ECL Western blotting substrate (Thermo Scientific, IL, USA), using ChemiDoc (BioRad Laboratories, CA, USA).

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.

Mouse dorsal back skin was isolated after the mice were sacrificed, and IHC analyses were performed with slight modifications according to previously reported methods [20 (link), 21 (link)]. Briefly, the tissue was fixed in a 10% formaldehyde solution in PBS for 24 h and then embedded in a paraffin block. Each section was cut into 5 μm thick slices and treated with xylene for deparaffinization. Then, the sections were treated with 3% hydrogen peroxide in a methanol solution to prevent endogenous peroxidase activity, and the epitopes were retrieved. The slides were treated with 10% normal goat serum for 1 h and then incubated overnight at 4°C with antibodies to silent mating type information regulation 2 homolog 1 (SIRT1; ab166821; Abcam, Cambridge, MA), interleukin- (IL-) 1β (ab9722; Abcam), and matrix metalloproteinase- (MMP-) 1 (ab137332; Abcam). Finally, the sections were stained with hematoxylin and eosin (H&E) and Masson's trichrome stain, as previously reported [22 ].