Mlb00c

To detect secreted IL-1β in conditioned media of cultured fibroblasts, the media was concentrated using Amicon Ultra-15 Centrifugal Filter Units with Ultracel-10 membrane (Merck Millipore, UFC901024) and ELISA was performed using Mouse IL-1 beta/IL-1F2 Quantikine ELISA Kit, according to manufacturer’s instructions (R&D Systems, MLB00C).

The concentrations of inflammatory factors IFN-γ, IL-1β, and IL-6 in nasal mucosal tissues were assessed using ELISA assays. A total of 1 mL 0.9% NaCl was added to each 0.1 g sample, and all samples were homogenized at 1000 rpm for 5 min and centrifuged at 1500 × g for 10 min at 4°C to collect the supernatant. Concentrations of inflammatory factors IFN-γ (DIF50C, R&D Systems, Minneapolis, MN, USA), IL-1β (DLB50, R&D Systems), IL-6 (D6050, R&D Systems), IL-4 (PI618, Beyotime), and IL-13 (ab47353, Abcam, Cambridge, MA, USA) in nasal mucosal tissues of patients with CRSsNP and controls were measured using ELISA kits.
The concentrations of inflammatory factors IFN-γ (MIF00, R&D Systems), IL-1β (MLB00C, R&D Systems), IL-6 (M6000B, R&D Systems), IL-4 (PI612, Beyotime), and IL-13 (ab219634, Abcam) in nasal mucosal tissues of mice in the CRSsNP and control groups were assessed using ELISA kits as well. All operations were performed strictly in accordance with the ELISA kit instructions.

Lipopolysaccharide (LPS) and SP600125 were purchased from Sigma-Aldrich (St Louis, MO, U.S.A.). The cell culture Medium (DMEM, MEM, and RPMI 1640), fetal bovine serum (FBS), penicillin–streptomycin (10,000 U/ml) solution, and phosphate-buffered saline (PBS) were purchased from Thermo Fisher Scientific (Waltham, MA, U.S.A.).
Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α (MTA00B), IL-6 (M6000B), and IL-1β (MLB00C) were obtained from R&D Systems (Minneapolis, MN, U.S.A.). The primary antibodies for Ufm1 (ab109305), LZAP (CDK5RAP3) (ab157203), NF-κB p65 (ab16502), p-NF-κB p65 (ab86299), ATF2 (ab47476), p-ATF2 (ab32019), c-Jun (ab32137), p-c-Jun (ab32385), anti-GFP antibody (ab290), and β-actin (ab8226) were from Abcam (Cambridge, U.K.). Anti-Flag antibody (F7425) was form Sigma. Anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (ab97040) was also from Abcam.

The levels of several factors such as the tumor necrosis factor-α (TNF-α, MTA00B, R&D Systems Inc., Minneapolis, MN, USA), IL-1β (MLB00C, R&D Systems), interleukin (IL)-6 (M6000B, R&D Systems), and DA (Shanghai Yueyan Biological Technology, Shanghai, China) in the BALF and hippocampal tissue homogenate were detected using the commercially available ELISA kits [24 (link)].