Anti fak antibody

The protein samples were harvested using a total protein lysates kit (cat no. KGP250; Nanjing KeyGen Biotech Co. Ltd., Nanjing, China). The protein concentrations were determined using the BCA Protein Assay kit (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) according to the manufacturer’s instructions. A total of 25 µg protein from each sample was subjected to SDS-polyacrylamide gel electrophoresis (gel, 10%; Bio-Rad Laboratories, Inc., Hercules, CA, USA) and transferred to a PVDF membrane (Immobilon; EMD Millipore, Bedford, MA, USA). Immunoprobing was performed by incubating the membrane with anti-GAPDH antibody (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-CTSB antibody (1:1,000; cat. no. ab230401, Abcam), anti-FAK antibody (1:1,000; cat. no. WL01696; Wanleibio Co., Ltd., Shanghai, China) and anti-MMP-9 antibody (1:1,000; cat. no. 10375-2-AP; Proteintech Group, Inc., Chicago, IL, USA) overnight at 4°C, followed by binding with a HRP-conjugated secondary antibody (1:2,000; cat. no. 7074; Cell Signaling Technology, Inc.) for 1 h at room temperature. The Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Inc.) was used for detection.

The RIPA protein lysis buffer (Beyotime, Shanghai, China) was used to split the proteins in tissues or cells, and the protein concentration was measured using a bicinchoninic acid disodium kit (Beyotime). An equivalent amount of cell lysate was separated on a 10% SDS-PAGE gel and transferred onto polyvinylidene difluoride membranes. The membranes were subsequently sealed, and anti-TRIM59 antibody (1: 5000) was added. GAPDH (1: 10 000; Multisciences, Hangzhou, China) as an internal reference. The membranes were incubated with goat anti-rabbit IgG secondary antibody (1: 5000; Bioworld Technology, lnc., St. Louis Park, MN, USA). The intensity of protein bands was analyzed with Image J software (National Institutes of Health, Bethesda, MD, USA). Other primary antibodies used in this study included anti-FAK antibody, anti-phospho-FAK, anti-AKT, anti-phospho-AKT, anti-MMP2 antibody, and anti-MMP9 antibody, which were all purchased from Abcam or Santa Cruz Biotechnology (Dallas TX, USA).