Anti twist1

Total proteins were extracted from fresh tissues and cells using RIPA Protein Lysis solution (Pierce, IL, USA) and quantified by the Bradford method. Prepared samples were electrophoresed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotted onto a polyvinylidene fluoride membrane (Millipore, MA, USA) using the Tetra Handcast system (Bio-Rad, USA). The membrane was blocked for 1 h at room temperature or overnight at 4 °C in Tris-buffered saline with 0.05% Tween (TBST) containing 5% non-fat milk. Then it was incubated overnight at 4 °C with the appropriate primary antibody, and followed by the secondary antibody for 70 min at room temperature. The protein bands were detected and quantified using the Gene Gnome Syngene Bio Imaging System (Syngene, UK) with an electrochemiluminescence kit (Pierce, IL, USA).
The primary anti-SOX3 antibody was purchased from Abcam (MA, USA), and the anti-β actin antibody was purchased from Santa Cruz Biotechnology Inc. (CA, USA). Other primary antibodies (anti-E-cadherin, anti-CK-18, anti-N-cadherin, anti-vimentin, anti-Snail1, anti-Twist1, anti-Slug, anti-ZEB1, anti- ZEB2, anti-FLAG) were purchased from Cell Signaling Technology (MA, USA). Anti-mouse or anti-rabbit HRP-conjugated secondary antibodies were purchase from Santa Cruz Biotechnology Inc. (CA, USA).

Total proteins were prepared, and western blot analysis was performed as previously described [19 (link)]. We used the following primary antibodies: anti-CD81 (Abcam, Cat# ab79559), anti-TSG101 (Abcam, Cat# ab125011), anti-E-cadherin (Cell Signaling Technology, Danvers, MA, USA, Cat# 14472S), anti-Vimentin (Abcam, Cat# ab92547), anti-N-cadherin (Cell Signaling Technology, Cat# 13116S), anti-MMP-2 (Cell Signaling Technology, Cat# 40994S), anti-MMP-9 (Cell Signaling Technology, Cat# 13667T), anti-ZEB1 (Cell Signaling Technology, Cat# 70512S), anti-Snail (Cell Signaling Technology, Cat# 3879S), anti-Slug (Cell Signaling Technology, Cat# 9585S) anti-Twist1 (Cell Signaling Technology, Cat# 46702S), anti-EGFR (Cell Signaling Technology, Cat# 4267S), anti-ERK antibody (Cell Signaling Technology, Cat# 4696S) and anti-p-ERK antibody (Cell Signaling Technology, Cat# 4370T), anti-androgen receptor (Abcam, Cat# ab74272), anti-β-actin (Cell Signaling Technology, Cat# 3700S) and anti-GAPDH (Cell Signaling Technology, Cat# 5174S).

Assays were performed as previously described, with modification [40 (link), 69 (link)]. The antibodies were obtained from the following companies: anti-HA(Y-11), anti-c-Src, and anti-Myc (9E10) were from Santa Cruz; anti-V5 was from Invitrogen; anti-TrkB and anti-SOCS3 were from Abcam; anti-phospho-c-Src, anti-Twist-1, anti-STAT3, anti-phospho-STAT3, anti-JAK2, and anti-phospho-JAK2 were from Cell Signaling Technology; and anti-E-cadherin, anti-fibronectin, anti-N-cadherin, anti-α-catenin, and anti-β-catenin were from BD Transduction.

The chemical reagents and working concentrations used were as follows: BHB (for cells: 0, 15, and 25 mM; for mice: 100 mg/kg; Sigma-Aldrich, cat. 52,017) and p300 inhibitor A485 (for cells: 2.5 µM; for mice: 30 mg/kg; Sigma-Aldrich, cat. SML2192). The antibodies used were as follows: anti-SLUG (Abcam, cat. ab27568), anti-TWIST1 (Cell Signaling Technology, cat. #90,445), anti-E-cadherin (Abcam, cat. ab231303), anti-vimentin (Sigma-Aldrich, cat. V6630), anti-snail (Sigma-Aldrich, cat. SAB5700806), anti-ZEB1 (Abcam, cat. ab276129), anti-ZEB2 (Abcam, cat. ab191364), anti-GAPDH (Abcam, cat. ab8245), anti-SOX2 (Cell Signaling Technology, cat. #14,962), anti-BMI1 (Abcam, cat. ab269678), anti-CD133 (Abcam, cat. ab284389), anti-KLF4 (Abcam, cat. ab129473), anti-β actin (Cell Signaling Technology, cat. #3700), anti-INMT (Abcam, cat. ab181854), anti-METTL3 (Abcam, cat. ab195352), anti-METTL14 (Abcam, ab220030), anti-ALKBH5 (Abcam, cat. ab195376), anti-FTO (Abcam, cat. ab280081), anti-m6A (Invitrogen, cat. MA5-33030), anti-Ki67 (Abcam, cat. ab15580), and anti-pan BHB-lysine (BHB-K) (PTM BioLabs, China, cat. #PTM-1201RM).