Ab171738

Recombinant human TNF-α was purchased from Peprotech and 2′,7′-dichlorofluorescein diacetate (DCF-DA) was purchased from Sigma–Aldrich. The following antibodies were used: anti-SOD3 for western blotting (ab83108) and anti-SOD3 for immunohistochemistry (IHC; ab171738) from Abcam; anti-glyceraldehyde dehydrogenase (GAPDH; ab83108) from AbClone; anti-phospho-NF-κB inhibitor protein ɑ (IκBɑ), anti-phospho-NF-κB p65, anti-phospho-c-jun N-terminal kinase (JNK), anti-JNK, anti-phospho-p38 mitogen-activated protein kinase (MAPK) (Cell Signaling Technology), anti-NF-κB p65, anti-p38 MAPK, anti-phospho-extracellular signal-regulated kinase (ERK), and anti-ERK2 from Santa Cruz Biotechnology; and anti-collagen type I cleavage site from ImmunoGlobe. Anti-MMP-1 and procollagen α1(I) N-propeptide (pN-ColIα1) antibodies have been previously described [21 (link)]. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were purchased from KOMA Biotech (Seoul, Korea). Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) and rhodamine Red-X-conjugated goat anti-mouse IgG were purchased from Thermo Fisher Scientific (Waltham, MA, USA). SPlink HRP Detection Bulk Kit for Mouse and Rabbit Antibodies and Liquid AEC Substrate Kit (20×) for IHC were purchased from GBI labs (Bothell, WA, USA).

Following the LSS experiment, EA.hy926 cells were harvested for Western blot analysis. Briefly, the proteins were extracted using RIPA buffer (89900, Thermo Fisher Scientific, Waltham, UK). According to the manufacturer’s protocol, protein concentrations were determined by the BCA protein assay kit (P0012S, Beyotime Biotech, Shanghai, China), and equal amounts of proteins were resolved on the 10% SDS-PAGE gel followed by transferring to the PVDF membranes. After blocked with 5% skim milk for 90 min at room temperature, the blots were incubated at 4 °C overnight with primary antibodies against NRF2 (16396-1-AP, Proteintech Group, Inc., Chicago, IN, USA), HMOX1 (10701-1-AP, Proteintech Group, Inc.), eNOS (27120-1-AP, Proteintech Group, Inc.), SOD2 (ab171738, Abcam, Cambridge, MA, USA), and Beta Actin (ab8226, Abcam, Cambridge, MA, USA) as needed. After that, the membranes were further probed by the HRP-conjugated secondary antibodies. Blotted protein bands were visualized with enhanced chemiluminescence detection reagents (Thermo Fisher Scientific). Relative changes in protein expression were estimated from the mean pixel density using Image J, normalized to Beta Actin.