Anti α tubulin

Cells were lysed in RIPA lysis buffer and the protein concentration of the cell lysates determined by BCA Protein Assay (Thermo Scientific, 23227). Equal amounts of protein were loaded onto SDS-PAGE gel and transferred to PVDF membranes. Western blotting was performed using primary antibodies and secondary antibodies conjugated with HRP. For immunoblotting, the following antibodies were used: anti-SENP2 (Abcam, ab58418, 1:1000), anti-HIF-1α (Cell Signaling Technology, 79233, 1:1000), anti-phospho-threonine (Cell Signaling Technology, 9386, 1:1000), anti-Ubc9 (Cell Signaling Technology, 4786, 1:1000), anti-BrdU (Cell Signaling Technology, 5292, 1:1000), anti-ubiquitin (Cell Signaling Technology, 3936, 1:1000), anti-cleaved-caspase 3 (Cell Signaling Technology, 9661, 1:1000), anti-SUMO1 (Cell Signaling Technology, 4930, 1:1000), anti-SUMO2/3 (Cell Signaling Technology, 4971, 1:1000), anti-SENP1 (Cell Signaling Technology, 11929, 1:1000), anti-SENP3 (Cell Signaling Technology, 5591, 1:1000), anti-HA-Tag (Cell Signaling Technology, 3724, 1:2000), anti-Flag-Tag (Cell Signaling Technology, 14793, 1:2000), anti-hexokinase 1 (Proteintech, 19662-1-AP, 1:1000), anti-hexokinase 2 (Proteintech, 22029-1-AP, 1:1000), anti-VDAC1 (Proteintech, 10866-1-AP, 1:1000), anti-alpha tubulin (Proteintech, 66031-1-Ig, 1:5000), anti-GAPDH (Proteintech, 10494-1-AP, 1:5000).

Western blotting was performed as previously described [29 (link)]. Protein samples were separated by SDS–PAGE electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes. Then, the PVDF membranes with proteins were incubated with 5% nonfat milk, specific primary antibodies at 4 °C overnight, and secondary antibodies (1:5000, BA1050, BA1054, Boster, Wuhan, China) in sequence and detected using ECL reagent. The primary antibodies were as follows: anti-Annexin A1 (1:1000, 32,934, Cell Signaling Technology), anti-E-cadherin (1:1000, ab231303; Abcam, Cambridge, U.K.), anti-vimentin (1:1000, ab20346; Abcam), anti-MMP9 (1:1000, ab76003; Abcam), anti-EGFR (1:1000, 66,455–1-Ig; Proteintech), anti-phospho-EGFR (1:1000, 3777, Cell Signaling Technology), anti-AKT(1:1000, 4685, Cell Signaling Technology), anti-phospho-AKT (1:1000, 4060, Cell Signaling Technology), anti-ERK (1:1000, 4659, Cell Signaling Technology), anti-phospho-ERK (1:1000, 4370, Cell Signaling Technology), anti-STAT3, anti-phospho-STAT3, anti-GAPDH (1:10,000, 60004–1-Ig; Proteintech), and anti-alpha tubulin(1:10,000, 66031-1-Ig; Proteintech) antibodies.

Huh7, LM3 cells were seeded and cultured until the confluence reached 70%. Western Blotting (WB) was performed by the protocols of a previous research. Using a 10% gel, amounts of protein (25 µg) were subjected to Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The primary antibodies applied for the WB experiment were bought from Proteintech Group (Rosemont, United States): anti-METTL18 (Catalog number: 25553-1-AP; 1:1000), anti-GAPDH (Catalog number: 10494-1-AP; 1:5000) antibodies and antialpha tubulin (Catalog number: 11224-1-AP; 1:5,000) antibodies.

The human ovarian cancer cell lines A2780 and SKOV3 were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Both cell lines were cultured in RPMI-1640 medium (Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) at 37 °C in 5% CO2. RIPA was purchased from Sigma–Aldrich (St Louis, MO, USA). Transfections were performed using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). The following antibodies were used: anti-HDAC9, anti-vimentin (Abcam, Cambridge, MA, USA); anti-TGF-β, anti-SMAD2/3, anti-P-SMAD2(S465 + S467)/P-SMAD3(S423 + S425) (Wanleibio, China); anti-E-cadherin, anti-N-cadherin, anti-snail, anti-FOXO1, anti-β-catenin, anti-beta actin, anti-lamin A/C, anti-alpha-tubulin (Proteintech, Chicago, IL, USA); anti-Acetyl-β-catenin (Lys49), anti-slug (Cell Signaling Technology, Beverly, MA, USA).