Anti fto

Hypoxia-treated cells were rapidly lysed with 1% SDS and boiled in SDS loading buffer and analyzed by Western blot assays. The following antibodies were used: anti-ALKBH5 (1:1000, Proteintech, 16837–1-AP), anti-HIF1a (1:1000, GeneTex, GTX127309), anti-FTO (1:1000, Abclonal A3861), anti-METTL3 (1:1000, Abclonal, A19079), anti-b-Tubulin (1:10,000, Abclonal, AC021), anti-SFPQ (1:1000, Abclonal, A3494), anti-NONO (1:1000, Abclonal, A3800), anti-PSPC1 (1:1000, Proteintech, 116714-1-AP), and anti-GFP (1:1000, Proteintech, 50430-2-AP). Direct-load Color Prestained Protein Marker (GenStar, M221) was used in all assays.

Cells were harvested and lysed with RIPA Lysis Buffer (beyotime, China) containing 1% protease inhibitor  PMSF (beyotime). The cell lysates were separated by SDS-PAGE and transferred to PVDF membranes (Thermo Fisher Scientific, USA). After blocking with 5% (M/V) BSA, the membrane was incubated with primary antibodies including anti-FTO (1:1000, ABclonal, China), anti-phospho STAT3 (Tyr705) (p-STAT3, 1:500, affinity, China), anti-STAT3 (1:500, affinity), anti-cleaved poly (ADP-ribose) polymerase (PARP) (1:500, CST, USA), anti-cleaved caspase-3 (1:500, affinity) and anti-β-actin (1:2000, proteintech, China) at 4°C overnight, followed by incubation with HRP-conjugated Goat anti-Rabbit or Goat anti-Mouse IgG secondary antibodies (1:10,000, proteintech) at 37°C for 40 min. The protein bands were quantified using the Gel-Pro-Analyzer software. β-actin was served as an endogenous control.

Western blotting analysis was performed as described in the previous report (Zhao et al., 2021 (link)). The cells were lysed in lysis buffer. After centrifugation, the supernatant was collected and the protein concentration was quantified by BCA assay kit. The samples were boiled at 95°C for 5 min and immediately frozen in a –80°C refrigerator. The primary antibodies were used as follows: anti-CD36 (ABclonal, Wuhan, China, A5792, 1:1,000), anti-GAPDH (ABclonal, Wuhan, China, AC033, 1:1,000), anti-FTO (ABclonal, Wuhan, China, A1438, 1:1,000), anti-P65-S536 (ABclonal, Wuhan, China, AP0475, 1:1,000), anti-IKB-α (Proteintech, Wuhan, China, 10268-1-AP, 1: 1,000), and anti-β-actin (ABclonal, Wuhan, China, AC026, 1: 1,000).