Mouse anti il 4

Total protein was extracted from BMDMs by incubating the cells for 10 min at 4 ℃ in RIPA lysis buffer (Fdbio Science, Guangzhou, China) with additional protease inhibitor. After BCA protein determination (Fdbio Science), 5 × loading buffer (Fdbio Science) was added in protein lysates. Equal quantities of lysates were isolated by SDS-PAGE and transferred onto 0.22 um PVDF microporous membranes (Beyotime Institute of Biotechnology, Jiangsu, China). The membranes were blocked with 5% whole milk and then incubated with primary antibodies for 16 h at 4 °C. Afterwards, the membranes were incubated at room temperature for 60 min with the secondary antibodies. Target protein bands were visualized by FDbio-Dura ECL (Fdbio Science). Antibodies applied for western blot in this study were: rabbit anti-IGF2BP3 (Proteintech, Rosemont, USA, 1:1,000, 14642-1-AP), rabbit anti-iNOS (Proteintech, 1:1,000, 22226-1-AP), rabbit anti-CD206 (Proteintech, 1:1,000, 18704-1-AP), rabbit anti-IL-1β (Proteintech, 1:1,000, 16806-1-AP), mouse anti-IL-4 (Proteintech, 1:1,000, 66142-1-Ig), species-matched HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA).

The lymph nodes and maxilla were lysed by RIPA (Invitrogen) with phosphatase and protease inhibitors to extract protein as stated by the manufacturer’s protocol. The concentration of total protein was measured and then subjected to electrophoresis in SDS-PAGE gel. Protein in the gel was transferred to PVDF membranes through the semi-dry electrophoretic transfer unit. Afterward, skimmed milk powder was used to block the non-specific binding sites. The membranes were then incubated overnight with primary antibodies including rabbit anti-β-actin (1:20000, Bioss), mouse anti-GATA-3 (1:2000, Proteintech), mouse anti-IL-4 (1:1000, Proteintech), rabbit anti-T-BET (1:500, Proteintech), rabbit anti-phospho-p38 MAPK (1:1000, Bioss), rabbit anti-p38 MAPK (1:1000, Bioss), mouse anti-ERK1 (1:1000, Bioss), mouse anti-phospho-ERK1 (1:1000, Bioss). Finally, secondary antibodies were incubated with anti-rabbit (1:10000, Bioss) or anti-mouse (1:10000, Bioss) before detecting the HRP signal using ECL.

BMDMs were seeded onto coverslips in a 12-well plate. After transfected with IGF2BP3 overexpressing plasmids/IGF2BP3 siRNA for 60 h, BMDMs were fixed with 4% paraformaldehyde for 15 min and administered with 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 15 min at room temperature. Afterwards, BMDMs were blocked with 10% bovine serum and incubated with primary antibodies at 4 °C for 16 h. After rinsing three times in PBS, coverslips were incubated with fluorescent dye for 1 h at room temperature. Nuclei were stained with DAPI mounting medium. Antibodies used for cell immunofluorescence staining include: rabbit anti-iNOS (Proteintech, 1:150, 22226-1-AP), rabbit anti-CD206 (Proteintech, 1: 150, 18704-1-AP), rabbit anti-IL-1β (Proteintech, 1: 150, 16806-1-AP), mouse anti-IL-4 (Proteintech, 1: 150, 66142-1-Ig), species-matched Alexa-488 or -594-labeled secondary antibody (Life Technologies, Carlsbad, CA, USA).