Anti mmp9

For preparation of protein extracts, 12 pairs of cancer and adjacent normal tissues were crushed with a mortar under ice cold conditions and lysed with RIPA lysis buffer together with protease inhibitors. Cells were collected and lysed with RIPA lysis buffer together with protease inhibitors. After centrifugation at 12,000 rpm at 4°C for 20 min, supernatants were collected and protein concentration was determined using the Pierce™ BCA protein assay (Thermo, Waltham, MA, USA). Proteins were separated by electrophoresis on a 10% SDS-polyacrylamide gel, electroblotted onto a PVDF membrane, and blocked by 5% nonfat dry milk for 1 h. Membranes were then washed in TBST three times for 5 min and then incubated with anti-MMP1 (Abcam, Cambridge, MA, USA), anti-MMP2 (Abcam), anti-MMP3 (Abcam, USA), anti-MMP7 (Abcam, USA), anti-MMP8 (Abcam, USA), anti-MMP9 (Invitrogen, Waltham, MA, USA), anti-MMP11 (Bioss, Beijing, China), anti-MMP12 (Abcam, USA), anti-MMP14 (Abcam, USA), anti-MMP17 (Abcam, USA), anti-MMP19 (Bioss, China), anti-MMP28 (Abcam, USA), anti-Collagen (Abcam), anti-TIMP2 (Bioss, China), or anti-GAPDH (Abcam). Subsequently, the membranes were washed with PBST and incubated for 1 h with goat anti-rabbit IgG (Abcam). Finally, membranes were washed three times and immunoreactivity was determined by using a Chemi DOC™ XRS+ system (BioRad Laboratories, Hercules, CA, USA).

Liver tissues and cell lines were lysed in a RIPA lysis buffer containing 1 mmol·L⁻¹ phenylmethanesulfonyl fluoride (Beyotime Institute of Biotechnology, Beijing, China) at 4 °C for 30 min. The lysates were then centrifuged at 15,000× g for 10 min at 4 °C and their protein concentrations were determined using the BCA Protein Assay (Beyotime Institute of Biotechnology, Beijing, China). Samples were mixed with 5× SDS loading buffer, boiled for 5 min, and 50 μg of total protein was subjected to SDS-PAGE in 10% gels and transferred to nitrocellulose membranes. After blocking, the membranes were incubated overnight at 4 °C with the following primary antibodies: anti-MMP-9 (1:1000 dilution; Invitrogen, MA5-15886, Carlsbad, CA, USA), anti-p-AMPK (1:1000 dilution; CST, #2535, Danvers, MA, USA), anti-AMPK (1:1000 dilution; CST, #2532, Danvers, MA, USA), and anti-GAPDH (1:5000; CST, #2118, Danvers, MA, USA). The membranes were washed with Tris-buffered saline/0.1% Tween 20 (TBST) and incubated with secondary antibodies for 1 h at 25 °C. Signals were detected using Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA). Protein levels were quantified by calculating the grayscale value of each band using ImageJ (version 1.43, National Institutes of Health, Bethesda, MD, USA) software.

The cells were prepared using RIPA lysis buffer as well as protease and phosphatase inhibitor (Bimake). 30mg of protein loaded per well and immune blotted overnight at 4 °C with antibody. The primary antibodies included anti-MUC15 (Abcam, ab224468), anti-Livin (Abcam, ab97350), anti-MMP2 (Invitrogen, 35-1300Z), anti-MMP9 (Invitrogen, MA5-15886), anti-E-cadherin (GeneTex, GTX124198), anti-Vimentin (Abcam, ab92547), anti-b-Catenin (CST, #9562) and anti-c-Myc (CST, #5605). The secondary antibodies included goat anti-mouse IgG HRP and goat anti-rabbit IgG HRP (Invitrogen).