Nf κb

NF-κB and COX-2 expression levels were measured using the immunofluorescent method. NF-κB (ThermoFisher Scientific, PA5-16545, Waltham, MA, USA), COX-2 (Invitrogen, PA1-9032, Carlsbad, CA, USA), FITC-conjugated IgG (Jackson Immunoresearch, 315-095-003, West Grove, PA, USA), Alexa Fluor 555-conjugated IgG (ThermoFisher Scientific, A-21127), and DAPI (ThermoFisher Scientific, 62249) were used. A K1-Fluo Confocal Microscope (Nanoscope System, Daejeon, Korea) was used for image acquisition and for analyzing the fluorescent intensity.

Total protein was extracted from the kidney tissues using Pro-Prep Protein Extraction Solution (iNtRON Biotechnology, Gyeonggi-Do, Republic of Korea) according to the manufacturer’s instructions. For NF-κB expression, nuclear proteins were prepared using the NE-PER nuclear and cytoplasmic extraction kit (Thermo Fisher Scientific, Rockford, IL, USA). Western blot analysis was performed using the following antibodies: ATX (Proteintech Group Inc., Rosemont, IL, USA), LPAR1 (Santa Cruz Biotechnology, Dallas, TX, USA), LPAR3 (Gene Tex, Inc., Irvine, CA, USA), PI3K (Abcam, Cambridge, UK), Akt (Cell Signaling Technology Inc., Danvers, MA, USA), phospho-Akt (Sre473) (Cell Signaling Technology Inc., Danvers, MA, USA), NF-κB (Santa Cruz Biotechnology, Dallas, TX, USA), TGF-β (R&D Systems, Minneapolis, MN, USA), TNF-α (Proteintech Group Inc., Rosemont, IL, USA), IL-1β (Cell Signaling Technology Inc., Danvers, MA, USA), IL-6 (Proteintech Group Inc., Rosemont, IL, USA), GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA), and Lamin-B1 (Cell Signaling Technology Inc., Danvers, MA, USA).

Following treatment, coverslips were gently washed with PBS before fixation with 4% PFA. Following fixation, cells were permeabilized using 5% goat serum (Thermo Fisher Scientific, MA, USA) and 0.01% Tween20 (Sigma-Aldrich, MA, USA). Cells were stained using 4’6-diamidino-2-phenylindole (DAPI) to identify nuclei, as well as the indicated primary antibodies, including MBP (1:500; Abcam, Cambridge, UK), Olig2 (1:500; Abcam, Cambridge, UK), and NFκB (1:200; Thermo Fisher Scientific, MA, USA). Conjugated secondary antisera directed against the species of the primary were used according to manufacturer instructions (1:1,000, Abcam, Cambridge, UK). Coverslips were then affixed to slides (Denville Ultraclear, MA, USA) with fluromount-G (Invitrogen, MA, USA) and imaged (Olympus 1X71, CellSens Software; Olympus, MA, USA). Five fields of view at 20× magnification using identical image capture settings were assessed by an experimenter blinded to treatments. To analyze the amount of OPC differentiation, Olig2+ cells and MBP+ cells were counted and the percent MBP+ cells was calculated. Olig2+ was also used to distinguish astrocytes in the cultures to refine the cell counts to only MBP+/Olig2+ OL-lineage cells. Data are presented as the percentage of MBP+/Olig2+ cells relative to the control, differentiation media only, condition set as 100%.

The lung tissue from the piglets was extracted using a buffer similar to that used in our previous research. Extracted lung proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bionovas Inc., Toronto, Ontario, Canada) and blotted onto a nitrocellulose membrane. The membrane was treated with SOD2, 3-NT, TNF-α, NF-κB, caspase-3, and β-actin antibodies (Thermo Fisher Scientific Inc.). Secondary antibodies and chemiluminescent substrates (GE Healthcare Life Sciences, USA, Barrington, IL, USA) were also used to induce chemiluminescence that was detected by an LAS-4000 (GE Inc. Healthcare Life Sciences). The optical density of the Western blot was evaluated in ImageJ (version 1.48 t, NIH, Washington, DC, USA).

The inflammatory markers in the ipsilateral cortex homogenates were measured in experimental mice 7 days post-rmTBI. The levels of NF-κB (Cat #: KHO037; Thermo Fisher Scientific, Massachusetts, USA) and TNF-α (Cat #: DY410; R&D Systems, Minneapolis, MN, USA) were analyzed using commercially available kits.

Examination of protein expression has been described previously [62 (link)]. The investigation has been conducted using primary antibodies directed against β-actin, β-catenin, E-cadherin, N-cadherin, Snail, vimentin (Cell Signaling Technology, Danvers, MA, USA), α-smooth muscle actin, fibronectin, occludin, NFκB, TGFβ, ZEB1, and ZEB2 (ThermoFisher Scientific, Waltham, MA, USA). The amount of protein was densitometrically determined using ImageJ software.