Anti igf2bp2

Tumor tissues were fixed with 4% paraformaldehyde for 24 h and then embedded in paraffin. Paraffin-embedded slices (4 μm) were dried at 62 °C for 60 min and dewaxed twice in fresh xylene for 30 min, followed by hydration using gradient alcohol. After antigen retrieval in citrate buffer at 100 °C for 10 min, the slices were blocked in 5% bovine serum albumin for 1 h and incubated at 4 °C for 12 h in primary antibodies, including anti-GLI1 (Santa Cruz Biotechnology, 1:200), anti-METTL3 (BOSTER Biological Technology, 1:500), anti-METTL14 (BOSTER Biological Technology, 1:500), and anti-IGF2BP2 (Proteintech1:500). Next, the slices were incubated with biotin-labeled secondary antibodies (BOSTER Biological Technology) for 60 min and incubated with horseradish enzyme-labeled chain avidin solution (BOSTER Biological Technology) for 30 min at room temperature. Positive staining was visualized with brown 3,3’-diaminobenzidine tetrahydrochloride (DAB, ZSGB, Beijing, China) and counterstained for 3 min using hematoxylin. Finally, the slices were dehydrated by increasing concentrations of ethanol and xylene and sealed with neutral balsam. Images were taken by a microscope (Olympus, Japan) with ×200 magnification.

Western blot analysis was performed as described previously [91 (link)] using anti-METTL3 (Cat. no. 15073-1-AP; 1:1,000), anti-IGF2BP1 (Cat. no. 22803-1-AP; 1:1,000), anti-IGF2BP2 (Cat. no. 11601-1-AP; 1:1,000), anti-HNRNPC (Cat. no. 11760-1-AP; 1:5,000), and anti-HNRNPA2B1 (Cat. no. 14813-1-AP; 1:2,000) antibodies, all of which were purchased from Proteintech. Anti-GADPH antibody (Affinity; Cat. no. #T0004; 1:3,000) was used to determine GAPDH protein levels. The target protein bands were quantified using the ImageJ v1.53a software (NIH, Bethesda, Maryland, USA). Differential expression was determined by calculating the ratios of target protein values for tumor tissues relative to the corresponding paired adjacent noncancerous lung tissues followed by log₂FoldChange for each patient.