Sc 8008

Immunofluorescence was realized according to the previously described methodology of Guerra et al. (2016) (link) and Ribeiro et al. (2017) (link). Sections of the oral mucosa from the hamsters, three per group, were dewaxed using xylol and, posteriorly, washed in concentrations different of ethanol and PBS. In process of antigen retrieval, was utilized a 10 mM sodium citrate solution containing 0.05% Tween 20 for a period 40 min at 95°C. Posteriorly, the samples were incubated in Sudan-Black 0.1% diluted in 70% ethanol, for 40 min, at room temperature to decrease the tissue autofluorescence. The slides again were incubated at 4°C overnight with mouse monoclonal anti-NFκB (1:200; sc-8008, Santa Cruz Biotechnology) or rat polyclonal anti-iNOS (1:200, sc-8332, Santa Cruz Biotechnology) primary antibodies and were washed three times for 5 min in PBS containing 0.2% Triton X-100. Subsequently, the samples were incubated with an Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (1:500 in 1% BSA blocking solution; Dako, Brazil, and United States). Finally, coverslips were applied using VECTASHIELD^® mounting medium with DAPI (4′,6-diamidino-2′-phenylindole). Immunofluorescence images were obtained using a Carl Zeiss Laser Scanning microscope (LSM 710, 20× objective, Carl Zeiss, Jena, Germany) (Araujo Junior et al., 2016 (link); de Assis et al., 2016 (link)).

Interferon-gamma (IFN-γ, 300-02) and tumor necrosis factor alpha (TNF-α, 300-01A) were purchased from Peprotech. AMPK agonists Metformin hydrochloride (PHR1084), AICAR (A9978), Resveratrol (R5010); SIRT1 agonist SRT1720 (SRT1720) and NRF-2/HO-1 agonist Protoporphyrin IX cobalt chloride (C1900) were purchased from Sigma-Aldrich. Inhibitors of NF-κB Emodin (E7881) and JSH23 (J4455); IKKβ inhibitor IKK16 (SML1138); STAT1 inhibitor Fludarabine (F2773) and NOS2 inhibitor 1400W (W4262) were purchased from Sigma-Aldrich. Inhibitors of MEK1 and MEK2 PD98059 (ab120234), JNK SP600125 (ab120065) and MAPK SB202190 (ab120638) were purchased from Abcam. Reagents were dissolved in DMSO or water (Metformin and AICAR) at 100x stock concentrations and maximum DMSO concentration used in cells was 1%. Monoclonal antibody against 3-nitrotyrosine (ab61392) was purchased from Abcam. Monoclonal antibody against NF-κB p65 (sc-8008) was purchased from Santa Cruz Biotechnology.

Protein lysates were boiled for 5 minutes at 95°C in SDS protein buffer (5x laemmli sample buffer, Thermo Fisher Scientific) and separated by SDS-PAGE following transfer to a polyvinylidene fluoride (PVDF) membrane. Primary antibodies were anti-alpha-Amylase (rabbit polyclonal, CST-4017, Cell signaling, Boston, USA), anti-Pancreatic-Lipase-A3 (mouse monoclonal, SC-374612, Santa-Cruz, Texas, USA), anti-Glyoxalase-I (Glo-I, mouse monoclonal, SC-133214, Santa-Cruz), anti-NF-ĸB (p65 subunit, mouse monoclonal SC-8008, Santa-Cruz) and anti-Vinculin (mouse monoclonal, SC-73614, Santa-Cruz). Secondary antibodies were anti-mouse (goat anti-mouse, 1858413, Pierce / Thermo Fisher Scientific) and anti-rabbit (goat anti-rabbit, 1858415, Pierce). Western Blot signals were quantified using imager (G-Box Chemie XX9, Syngene, Cambridge, UK). Signals were normalized to their respective loading controls using ImageJ-Software (v. 1.48, http://imagej.nih.gov) and: GeneTools (Syngene).

Western blot analyses were performed as described previously (Zheng et al., 2021 (link)). Total proteins were extracted from hepatocytes or liver samples using RIPA lysis buffer (P0013B, Beyotime, China), separated by SDS-PAGE gel electrophoresis, and transferred to polyvinylidene fluoride membranes (ISEQ00010, Millipore, United States). The membranes were blocked with 5% milk (232100, BD Biosciences, United States), and subsequently hybridized overnight with primary antibodies at 4°C. Finally, the secondary antibody (1:10000, 14708, CST, United States) was used to combine chemiluminescent horseradish-peroxidase substrate (WBKLS0500, Millipore, United States), and the membranes were imaged by Bio-Rad ChemiDoc XRS System (Hercules, United States).
Primary antibodies against p-TBK1 (1:2000, 5483), p-NF-κB (1:2000, 3033) and beta-actin (1:2000, 4970) were obtained from Cell Signaling Technology (Beverly, United States), against TBK1 (1:1000, sc-398366) and NF-κB (1:1000, sc-8008) were purchased from Santa Cruz Biotechnology (California, United States), and against CCR2 (1:1000, abs136993) was purchased from Absin (Shanghai, China).

The protein expression in RAW264.7 cells were detected by Western blot. Cells (1.6 × 10⁵ cells/well) were plated overnight and then treated with the indicated concentrations of bioactive compounds. After 1h, 1μg/ml LPS were added. Then, the supernatant and precipitation of cells were collected 24h later. Immunoblotting was performed using antibodies against the target proteins, including AKT1 (1:2,500; ab89402; Abcam, Cambridge, MA, United States), p-AKT1 (1:5,000; ab81283; Abcam, Cambridge, MA, United States), PI3K (1:1,000; 4,249; Cell Signaling Technology, Inc., Danvers, MA, United States), p-PI3K (1:1,000; bs-3332R; Bioss Antibodies, Biotechnology, Inc., Beijing, China), NFκB-p65 (1:1,000; sc-8008; Santa Cruz Biotechnology, Inc.,Dallas, TX, United States), p-NFκB-p65 (1:1,000; YP0191; Immuno Way, Biotechnology, Inc., Plano, TX, United States). The blots were developed with an enhanced chemiluminescence kit (ECL, Amersham Biosciences, Buckinghamshire, United Kingdom) and measured by using a luminescent image analyzer (LAS-3000, Fuji Photo Film Co. Ltd., Japan).