Gtx114209

Cultured cells were fixed with 4% formaldehyde, permeabilized with PBS containing 0.1% Triton X-100, blocked with 5 mg/mL BSA, and incubated overnight with appropriate primary antibodies followed by FITC or TRITC-secondary antibody (ZSBIO, dilution 1:200) before mounting with Hoechst. The antibodies include anti-HIF1a (1:100, GeneTex, GTX127309), anti-ALKBH5 (1:100, Millipore, ABE547), anti-SFPQ (1:100, GeneTex, GTX114209), and anti-NONO (1:100, Abclonal, A3800).

To obtain whole-cell protein extracts, cells were harvested and lyzed as previously described (22) . Cell culture medium and serum from the allografted mice were prepared to measure IL8 levels. The corresponding primary antibodies used for immunoblotting included anti-ALKBH5 (1:1,000, Proteintech, 16837-1-AP), anti-HIF1a (1:1,000, GeneTex, GTX127309), anti-FTO (1:1,000, Santa Cruz Biotechnology, sc-271713), anti-METTL3 (1:1,000, Abclonal, A19079), anti-b-actin (1:10,000, Abclonal, AC026), anti-b-tubulin (1:10,000, Abclonal, AC021), anti-GAPDH (1:20,000, Abclonal, AC002), anti-IL8 (1:1,000, GeneTex, GTX115959), anti-albumin (1:1,000, Abclonal, A0353), anti-SFPQ (1:1,000, GeneTex, GTX114209), and anti-NONO (1:1,000, Abclonal, A3800). And Direct-load Color Prestained Protein Marker (M221, GenStar) was used in all Western blot assays.