Ab180630

Tissues or cells were lysed using RIPA Lysis Reagent (Boster, China) for 20 min, and then centrifuged at 10 000×g for 10 min on ice. Proteins in supernatant were harvested and quantified by BCA protein assay kit (Thermo Fisher, USA). Subsequently, 20 μg total proteins were subjected to each lane and separated by 12% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Thermo Fisher, USA). After that, the membrane was blocked with 5% non-fat dry milk for 1 h at 37°C and probed with specific primary antibodies overnight at 4°C, including: rabbit anti-MON1B (Novus, NBP1-92131, 1: 2000), anti-MMP2 (Abcam, ab92536, 1: 1000), anti-MMP9 (Abcam, ab38898, 1: 1000), anti-TIMP-2 (Abcam, ab180630, 1: 1000), anti-MTA-1 (Abcam, ab, 1: 1000), anti-NF-κB (Abcam, ab16502, 1: 1000), anti-IκB (Abcam, ab32518, 1: 1000), anti-CXCR-4 (Abcam, ab181020, 1: 1000), and anti-β-actin (Abcam, ab8227, 1: 2000). Subsequently, the membranes were incubated with Goat Anti-Rabbit IgG H&L (HRP) secondary antibody (Abcam, ab6721, 1: 5000) for 1 h at 37°C. The immunoblots were visualized by enhanced chemiluminescence (ECL) detection reagents (Thermo Fisher, USA). Digital images of immunoreactive bands were analyzed by Bio-Rad ChemiDoc™ XRS+ System with Image Lab™ Software #1708265 (Bio-Rad, USA).

Protein was obtained from tissues using a chilled lysis buffer containing protease inhibitor and phosphatase inhibitor. Lysates were centrifuged at 12,000 g for 10 min at 4°C. Equal amounts of sample proteins (40 μg–80 μg) mixed with loading buffer were separated in 10% (wt/vol) SDS/PAGE gels. Afterward, proteins were transferred from SDS/PAGE gels to 0.45 μm polyvinylidene fluoride membranes. Unspecific antibody binding was prevented by blocking for 1 h at room temperature with 5% nonfat milk powder in triethanolamine-buffered saline/Tween 20 (TBST). Blots were incubated with primary antibodies overnight at 4°C. The primary antibodies used in this study were rabbit anti-IGFBP7 (abs136651, Absin, 1:1,000), rabbit anti-TIMP2 (ab180630, Abcam, 1:1,000), and mouse anti-β-actin (100166-MM10, Sino Biological, 1:5,000). After three times of washing in TBST, the blots were incubated with HRP-conjugated goat anti-rabbit antibody (A0208, Beyotime) or HRP-conjugated goat anti-mouse antibody (A0216, Beyotime) diluted 1:5,000 in TBST for 1 h. Chemiluminescent signal was detected using chemiluminescence substrate (Tanon, Shanghai). After chemiluminescent detection, β-actin was performed on the same blots as housekeeping protein. The density of the bands was measured using ImageJ software and normalized to healthy controls.