Horseradish peroxidase hrp conjugated secondary antibodies

Total protein was obtained by RIPA buffer (New Cell & Molecular Biotech, Suzhou, China) and nuclear protein was extracted using the Nucleoprotein Extraction Kit (Sangon Biotech, Shanghai, China). Blots were incubated overnight at 4 °C with respective primary antibodies listed in Supplementary Table 2, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000; Affinity Biosciences, OH, USA) for 1 h at room temperature, then developed with high-sensitivity ECL (Vazyme, Nanjing, China). The protein bands were visualized using a Tanon 5200 Chemiluminescent Imaging System (Tanon, Shanghai, China). ACTB or histone H3 were used as loading controls. The validation information of antibodies is provided in the Reporting Summary. ELISA assays of human TGFβ1 and mouse IL1β/IL6/TNFα/IL17A (Multi Sciences, Hangzhou, China) were performed as per the manufacturer’s protocol using a Varioskan Multimode Microplate Reader (Thermo Fisher Scientific, Waltham, MA, USA).

After different treatments, proteins were harvested and their contents were determined by the BCA protein assay kit (Beyotime, China). Thereafter, 10%–12% SDS-PAGE was applied in separating 30 μg proteins, followed by transfer onto the PVDF membranes (Millipore, USA). The membranes were later immersed in 5% BSA for a 2 hours period, followed by overnight incubation under 4°C using the primary antibodies as follows: NF-κB, p-NF-κB, IκB-α, p-IκB-α, JNK, p-JNK, p38, p-p38, ERK, p-ERK, TNF-α, IL-6, Cox-2 (1 : 1000), and GAPDH (1 : 2000). Then, horseradish peroxidase (HRP)-conjugated secondary antibodies (Affinity Biosciences Co. Ltd., China) were added to further incubate membranes. WB assay results were analyzed with the ECL system and ImageJ software.

We used the RIPA Lysis Buffer (Fudebio, Hangzhou, China) containing phosphatase/protease inhibitors (Fudebio, Hangzhou, China) to extract total protein from the cultured cells following the manufacturer’s method. SDS‐polyacrylamide gel electrophoresis (10%; SDS‐PAGE) was performed to separate the protein samples (20 µg), which were then transferred to Millipore polyvinylidene difluoride (PVDF) membranes. These PVDF membranes were kept in overnight incubation at 4°C with anti‐CD206, anti‐ODC, anti‐IL‐33, anti‐iNOS, anti‐CD68, anti‐ARG1 and anti‐GADPH primary antibodies (Affinity, USA), followed by incubation at room temperature for 60 minutes with horseradish peroxidase (HRP)–conjugated secondary antibodies (Affinity, USA) that were diluted to 1:5000 in 5% skim milk. After washing with TBST, the membranes were treated for 1 minutes with SuperSignal™ West Dura Extended Duration Substrate (Fudebio, Hangzhou, China) and were visualized through chemiluminescence.