Anti inos

Longitudinal stomach tissue strips from H. felis-infected or uninfected mice were fixed with 10% formaldehyde, embedded in paraffin, and cut into 4-μm-thick sections. Hematoxylin and eosin staining was performed as per standard protocols, and the presence of lymphoid follicles and lymphoepithelial lesions (LELs) were recorded. Sections of murine stomach or human MALT lymphoma tissues were subjected to heat-induced epitope retrieval and then incubated overnight with anti-TCRγδ (Abcam, Cat. no. ab118864), anti-IL-17 (Abcam, Cat. no. ab79056), anti-IL-1β (Abcam, Cat. no. ab9722), anti-IL-23 (Santa Cruz, Cat. no. sc-50303), anti-Arg-1 (Proteintech, Cat. no. 16001-1-AP), anti-iNOS (Proteintech, Cat. no. 18985-1-AP), anti-Gr-1 (R&D systems, Cat. no. MAB1037-100), anti-CD11b (Abcam, Cat. no. ab133357), anti-CD33 (Abcam, Cat. no. ab11032), and anti-CD11b (Abcam, Cat. no. ab133357) primary antibodies at 4°C. The following day, the sections were probed with biotin–streptavidin horseradish peroxidase-conjugated secondary antibody, and stained using 3,3′-diaminobenzidine reagent. For immunofluorescence, fluorochrome-conjugated secondary antibodies were used. Positively stained cells were counted in five random non-overlapping fields (400× magnification; Nikon, Ni-U) using ImageJ software. The histoscore of each biomarker was evaluated according to Table 2.

Limonin (purity ≥ 98%), DMSO, Safranin O, collagenase type-II, Fast Green FCF, and hematoxylin were provided by Solarbio (Beijing, China). Recombinant rat IL-1β was purchased from Multi Sciences Biotech, Co., Ltd. (Hangzhou, China). Anti-Collagen II, Aggrecan, ADAMTS5, and MMP13 were purchased from Abcam (Cambridge, UK). Anti-p65, IκBα, and Lamin-B1 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-INOS, COX-2, HO-1, NQO1, SOD1, Nrf2, and GAPDH were purchased from Proteintech (Wuhan, China). Reagents for cell culturing were purchased from Gibco (Grand Island, USA).

The whole cell protein was extracted with RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China, Cat#S711-001S), and the protein concentration was measured with BCA protein analysis kit (Beyotime Biotechnology, Cat#P0012). Equal amounts of total protein were loaded on 6%–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then the separated proteins were transferred to PVDF membrane (Merck Millipore, Darmstadt, Germany, Cat#IPVH00010). After blocking with 5% skim milk, membranes were incubated with primary antibodies at 4 °C overnight, followed by incubation with the IRDye conjugated secondary antibody for 1 h at room temperature. The antibodies used in this study were as follows: Anti-β-Actin (1:1000, Abcam, Cambridge, MA, USA, Cat#ab97626), Anti-GOLPH3 (1:1000, Abcam, Cat#ab91492), Anti-COX-2 (1:1000, Proteintech, WuHan, China, Cat#27308-1-AP), Anti-iNOS (1:1000, Proteintech, Cat#18985-1-AP) and IRDye-conjugated (680RD Goat anti-Mouse/800CW Goat anti-Rabbit) IgG secondary antibodies (1:10000, Li-COR Biosciences, Lincoln, NE, USA, Cat#926-68070/Cat#926-32211). Membranes were imaged with an Odyssey CLX Infrared Imaging System (Li-COR Biosciences).

The expression of α-smooth muscle actin (α-SMA) and inducible nitric oxide synthase (iNOS) in carotid artery tissues was determined by immunohistochemical staining. In brief, 5-μm paraffin sections were immersed in antigen retrieval solution and heated for 10 min in a microwave oven. Then, the sections were then incubated with 3% H₂O₂ for 15 min at room temperature. After blocking with goat serum (Solarbio, Beijing, China) for 15 min at room temperature, the sections were incubated with primary antibodies separately at 4°C overnight (anti-α-SMA, 1:200, BOSTER, Wuhan, China; or anti-iNOS, 1:200, Proteintech, Wuhan, China). Biotinylated Goat anti-Mouse or Goat anti-Rabbit IgG (1:200, Beyotime, Haimen, China) were then added to the sections for 30 min at 37°C. After development with DAB and counterstaining with hematoxylin, the sections were photographed by a light microscopy.

The mouse recombinant LIGHT protein (rLIGHT, cat#HY-P73837), PI3Kγ specific inhibitor (Eganelisib, IPI549, cat# HY-100716), and SGK1 inhibitor (GSK650394, cat#HY-15192) were purchased from MedChemExpress (MCE, New Jersey, USA). The primary antibodies anti-phospho-Akt (Ser473) (cat#4060) and anti-Akt (cat#4691)used for western blotting were purchased from Cell Signalling Technology (Danvers, Essex County, MA, USA); anti-PIK3CG (cat#A0266), anti-SGK1 (cat#A3936), and anti-phospho-Smad2/Smad3 (cat#AP1343) were purchased from ABclonal (Wuhan, China); anti-Smad2 (cat#ET1604-2), anti-Smad3 (cat#ET1607-4) and anti-PI3K p85α (cat# ET1608-70)were purchased from HUABIO (Hangzhou, China); and anti-F4/80 (cat#28463-1-AP), anti-CD3 (cat#17617-1-AP), anti-CD163 (cat#16646-1-AP), anti-ARG1 (cat#66129-1-Ig), anti-CD206(cat#60143-1-Ig), anti-iNOS (cat#18985-1-AP), anti-MCP1 (cat#66272-1-Ig), anti-TGFβ1 (cat#21898-1-AP), anti-collagen I (cat#14695-1-AP), anti-collagen III (cat#68320-1-Ig), anti-α-SMA (cat#14395-1-AP), anti-β actin (cat#20536-1-AP), and anti-GAPDH(cat#10494-1-AP) were purchased from Proteintech (Wuhan, China).