Anti flot1

Whole cell lysates were collected according to the manufacturer's instruction (Beyotime Biotechnology) and then centrifuged at 10,000 g for 10 min at 4°C. Then 1 mL supernatant was incubated with 1 μg anti-SDC1, anti-FLOT1, anti-Flag antibody (1:100, Proteintech Group), or anti-IgG antibody (1:100, Cell signaling Technology) for 16 h followed by addition of 20 μL protein A/G plus agarose beads (Santa Cruz Biotechnology) and incubated overnight at 4 °C. Protein samples were spun down, washed four times with immunoprecipitation buffer and heated for 10 min at 100 °C prior to loading on an SDS-PAGE gel for Western blot assay. Finally, the protein bands were visualized by ChemiDoc XRS system (Bio-Rad Laboratories Inc.). Antibodies are listed in supplementary Table S2.

Western blotting was carried out as previously described with a minor modification. The antibodies used in the study are as follows: rabbit polyclonal anti-MMP2, anti-MMP7, anti-MMP9, anti-Bcl-xl, anti-Bcl-2, anti-Bax, anti-E-cadherin, anti-PI3K, anti-p53, anti-GSK3β, anti-Flot-1, anti-Akt3 (Proteintech, Wuhan, China), anti-phospho-Akt3 (S472) (Abgent, Suzhou, China), anti-phospho-p65 (S536), p50, anti-IκB, anti-Foxo1, anti-phospho-Foxo1 (S256), anti-p38, anti-p27, anti-p21, anti-CCNA1, anti-CCNE2 (Sangon Antibody R&D Center, Shanghai, China), anti-phospho-mTOR (S2448) (ImmunoWay, Newark, DE, USA), mouse monoclonal anti-Flot-2, anti-α-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-β-actin (Sigma, USA). Akt Inhibitor VIII, a specific Akt inhibitor, was purchased from Merck Millipore (Merck KGaA, Darmstadt, Germany). Quantification of signal intensity (IOD, integral optical density) was performed with Gel-Pro Analyzer software(Version 4.0). Expression change was indicated by IOD ratio of targeted protein before and after treatments. And the intensity was normalized by β-Actin signal. All detections were repeated for three independent times.

Protein samples mixed with 5Xloading buffer were boiled at 100°C for 5 min. Then, cooled down the samples and subjected them into sodium dodecyl sulfate–polyacrylamide gel, electrophoresed and transferred the sample to PVDF membrane. Then, blocked the membrane with 5% skimmed milk for 1 h, and then incubated the membrane with primary antibodies, anti‐Flot1 (1:1000, Proteintech, Rosemont, IL, USA), anti‐p‐ERK1/2 (Thr202/Tyr204) (1:1000, Proteintech) at 4°C overnight. Washed the membrane, incubated it into secondary antibodies, and washed it again 3 times. At last, detected the signals with ChemiDoc (Bio‐Rad).