Pvdf membrane

Total protein was extracted using a protein extraction kit (Shanghai Yeasen Biotechnology Co., Ltd.) according to the manufacturer's protocol, and the protein concentration was determined using an ultraviolet spectrophotometer (Onedrop1000; Shanghai Genechem Co., Ltd.). A total of 10 µg protein/lane was loaded on a 12% polyacrylamide gel, resolved using SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Sangon Biotech Co., Ltd.). The PVDF membrane was blocked with 5% non-fat milk for 2 h at room temperature. Subsequently, the PVDF membrane was incubated with the corresponding primary antibody against the target protein overnight at 4°C followed by incubation with the secondary antibody at room temperature for 1 h. The protein band was detected using a Beyo ECL Star kit (Beyotime Institute of Biotechnology). The antibodies used were as follows: Anti-PI3K (ab70912; 1:100), anti-MEK1 (ab32091; 1:1,500), anti-ERK1 (ab32537; 1:600), anti-cdc25 (ab111830; 1:2,000) (all Abcam), anti-C-fos (554C1a; 1:500; Santa Cruz Biotechnology, Inc.), anti-ALOX12B (PA5-23608; 1:800; Invitrogen; Thermo Fisher Scientific, Inc.), anti-GAPDH (ab181602; 1:10,000; Abcam), goat anti-mouse IgG antibody (ab97035; 1:2,000) and goat anti-rabbit IgG antibody (ab7090; 1:5,000) (both Abcam).

Whey was isolated as above and then boiled in SDS loading buffer for 10 min, after which samples were electrophoretically separated using 12% polyacrylamide Tris-glycine gels. Afterwards, these gels were stained using Coomassie Brilliant Blue G-250, and the sample purity and concentrations were determined using Tanon Gis software (Bio-Tanon). For western blotting, separated proteins were then transferred onto PVDF membranes (F019531; Sangon). The membranes were blocked using 5% BSA/TBST overnight at 4°C, and then probed using a polyclonal rabbit-anti-LF antibody (1:2,000, 10% FBS/TBST; Sangon) at 37°C for 1.5 h. next, an hRP-conjugated secondary goat-anti-rabbit IgG (1:1,000, 10% FBS/TBST; Sangon) was used to probe blots at 37°C for 1 h. The blots were then washed three times in TBST (20 mM Tris-base, 137 mM naCl, 0.05% Tween-20), and protein bands were detected with an ECL substrate solution (Millipore Corporation) based on provided directions.

Liver or intestinal tissues were lysed using M-PER mammalian protein extraction reagent supplemented with HALT proteinase and phosphatase inhibitor cocktails (Thermo-fisher Scientific, USA), Protein concentrations were determined by Bradford protein assay with BSA standards (Bio-Rad). Total protein was extracted and equal quantities of proteins (10 µg) were electrophoresed on 10% SDS-PAGE, and then transferred onto PVDF membranes (Sangon Biotech, China). The membrane was incubated with primary antibodies and then with horseradish peroxidase (HRP)-conjugated secondary antibody. ECL Detection System (CliNX, Shanghai, China) was used to detect antibody reactivity. Primary antibodies included rabbit monoclonal antibody β-actin (Abcam, USA) and rabbit polyclonal antibody Lcn2 (Abcam, USA), which were all diluted at 1:1000. Appropriate second antibodies (Cell Signaling Technology, USA) were all diluted at 1:2000.