Anti rabbit or anti mouse secondary antibodies

Cells were hydrolyzed in RIPA buffer with a mixture of protease inhibitors and phosphatase inhibitors. Protein products were isolated by SDS–PAGE and transferred onto nitrocellulose membranes. We cut the membranes according to the molecular weight of the target proteins. The membranes were then blocked in TBST buffer with 3.0% BSA for 1 hour and incubated at 4 °C with primary antibodies overnight. The membranes were rinsed three times with TBST buffer and incubated at room temperature for 2 hours with the corresponding secondary antibody. Protein detection was performed using an enhanced chemiluminescence system. The expression of proteins of interest was normalized to that of β-actin. The following primary antibodies used in WB analysis: anti-mouse snail, anti-mouse Vimentin, anti-mouse E-cadherin, and anti-mouse N-cadherin (Cell Signaling Technology, Danvers, MA, USA); anti-rabbit CST1, anti-mouse GAPDH, and anti-mouse β-actin (ABclonal, Biotechnology Co. Ltd. Wuhan, China); and anti-rabbit or anti-mouse secondary antibodies (Beyotime Biotechnology Co. Ltd. Shanghai, China). Relative protein expression was normalized to that of β-actin. The same set of lysate samples were run in parallel (sister) gels to test different proteins (same for all figures).

Protein isolation and Western blotting were performed as previously described [35 (link)]. Total cellular protein was extracted in RIPA lysis buffer (Beyotime, China) supplemented with 1% PSMF (Beyotime, China) and 1% phosphatase inhibitor (Beyotime, China). Protein was quantified using the bichoninic acid assay (BCA; Beyotime, China) according to the manufacturer’s instructions. Equal amounts of protein (30 μg) were separated through a 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a PVDF membrane. Membranes were blocked in 5% milk-Tris-buffered saline with Tween 20 (TBST), followed by overnight incubation with primary antibodies for phosphor (p)-ERK, ERK, VEGF, p-Akt, Akt, eNOS, CXCR4, KLF-8, and GAPDH (dilution 1:1000, Abcam, USA) at 4 °C. The membranes were washed with TBST and then incubated with anti-rabbit or anti-mouse secondary antibodies (dilution 1:1000, Beyotime, China). All signals were detected by the Molecular Imager ChemiDocTM XRS+ System (BIO-RAD, USA), and data were normalized by GAPDH levels (p-ERK and p-Akt were normalized by total ERK and total Akt, respectively).

Total cellular protein was extracted in RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with 1% phenylmethanesulfonyl fluoride (PMSF) (Beyotime, Shanghai, China) and 1% phosphatase inhibitor (Beyotime, Shanghai, China). Equal amounts of protein (30 μg) were separated through a 10% or 12% SDS-PAGE and transferred to a PVDF membrane. Membranes were first probed with primary antibodies for p65, LC3B, galectin-3, adenosine monophosphate kinase (AMPK), phosphate-AMPK (Thr172), mammalian target of rapamycin (mTOR), phosphate-mTOR (Ser2448), and GAPDH (all the above 8 antibodies: 1 : 1000; from Cell Signaling Technology, USA) and then incubated with anti-rabbit or anti-mouse secondary antibodies (1 : 1000; Beyotime, Shanghai, China). All signals were detected by the Molecular Imager ChemiDoc™ XRS+ System (Bio-Rad, Hercules, CA, USA). p65, LC3B, and galectin-3 were normalized by GAPDH levels. Phospho-AMPK and phosphate-mTOR were normalized by total AMPK and total mTOR.