Odyssey infrared imaging system v3

Total protein extraction from macrophages plated in 6-well plates was performed using a lysis buffer containing 40 mM Tris pH 7.5, 150 mM KCl, 1 mM EDTA, 1% Triton-X-100, PIC (Protease Inhibitor Cocktail, Roche), PIB 25x (Phosphatase Inhibitor Buffer, 25 mM Na₃VO₄, 250 mM PNPP, 250 mM β-glycerophosphate, 125 mM NaF). Cell lysate was recovered and centrifuged for 5 min at 15,700 g and 4 °C to pellet cell debris. The supernatant was collected and stored at −70 °C before western blotting. 20 µg of proteins were separated on 10% SDS-PAGE gels and transferred onto a low fluorescence background PVDF blotting membrane (Millipore). Quantitative LI-COR technology was used for western blot analyses (Odyssey Infrared Imaging System v3.0.16, LI-COR, Biosciences). Membranes were blocked with Odyssey blocking buffer diluted 1:2 in PBS for 1 h at RT. Primary antibodies diluted in Odyssey Blocking buffer-Tween 0.1% were incubated overnight at 4 °C, then membranes were washed with PBS-Tween 0.1%, and finally incubated with secondary antibodies diluted 10,000 x for 1 h at RT. Membranes were washed with PBS-Tween 0.1%, and then with PBS, and finally dried before scanning. Loading control was assessed with α-tubulin or β-actin according to the molecular weight of the protein of interest. Antibodies used are listed in Supplementary Table S2.

Nuclear, cytoplasmic and total proteins were subjected to electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gels and then transferred to PVDF membranes (Thermo Fisher Scientific, Millipore, MA). After using 5% fat-free milk to block non-specific binding, we incubated the membranes with primary monoclonal antibodies against Nrf2 (1:1000), HO-1 (1:1000), p-p65 (1:1000), P65 (1:1000), p-IκBα (1:1000), IκBα (1:1000), lamin B (1:1000) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:1000) for 12 h at 4 °C. The membranes were then incubated with fluorescence-labeled anti-rabbit immunoglobulin G (1:10000) for 1.5 h at room temperature. Images were captured using an Odyssey Infra-red Imaging System v3.0.16 (LI-COR Biosciences, Nebraska, NE) and quantified by densitometry scanning using ImageJ Analysis software (National Institutes of Health, Bethesda, MD). Experiments were repeated three times (n = 3).

The protein expression of VEGF, TGF-β1, PDGF, MMP-9, Collagen IV, EGF, and β-actin was measured by western blotting. Proteins were extracted by grinding the tissues using a mortar and pestle in liquid nitrogen. Proteins were separated by electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gels and then transferred to polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific, Millipore, MA, USA). The membranes were then incubated with rabbit polyclonal antibodies against VEGF (1 : 1000), TGF-β1 (1 : 1000), PDGF, Collagen IV (1 : 4000), EGF (1 : 1000), and MMP-9 (1 : 1000) for 12 h at 4°C on the shaker. After washing, membranes were incubated with fluorescence-labelled anti-rabbit immunoglobulin G (1 : 10000) for 1.5 h on the shaker at room temperature. The blots were subsequently scanned using an Odyssey Infrared Imaging System v3.0.16 (LI-COR Biosciences, NE), and the band intensity was quantified by ImageJ software (NIH, MD, USA). The reagents and kit numbers are provided as a supplementary table (Table S1).