Enhanced chemiluminescence plus kit

Total protein from cells and tissues was lysed in Whole Cell Lysis Assay buffer (cat. KGP250, KeyGen BioTECH, China) containing protease inhibitors (cat. KGP603, KeyGen BioTECH, China) and phosphatase inhibitors (cat. KGP602, KeyGen BioTECH, China). Then, 30 µg of protein per sample was resolved on 10% SDS-PAGE gels and transferred onto PVDF membranes. The membranes were first incubated overnight at 4°C in TBS containing BSA, 0.05% Tween 20, and primary antibodies against GAPDH (1:4000; cat. M20006S, Abmart, USA), β-hCG (1:1000; cat. AP13036b, Abgent, USA), luteinizing hormone/chorionic gonadotropin receptor (LHCGR) (1:1000, cat. 19968-1-AP, Proteintech, USA), ERK1/2 (1:1000; cat. 4695, Cell Signaling Technology, USA), P-ERK1/2 (1:1000; cat. 4370, Cell Signaling Technology, USA) and MMP-2 (1:1000; cat. 10373-2-AP, Proteintech, USA), followed by incubation with secondary antibodies (cat. KGAA3d5, KeyGen BioTECH, China) conjugated with horseradish peroxidase at room temperature for 1 h. The protein bands were detected using an enhanced chemiluminescence plus kit (Millipore) according to the manufacturer’s recommendations.

Western blotting was performed as previously described (31 (link)). In brief, total protein was extracted from tissues and cell lines using RIPA lysis buffer (EMD Millipore). Total protein concentration was measured using a BCA Protein assay kit (Nanjing KeyGen Biotech Co., Ltd.), according to the manufacturer's protocol. Equal amounts of protein (~30 µg) were separated via SDS-PAGE on a 12% gel, and subsequently transferred onto PVDF membranes (EMD Millipore). After blocking with 5% skimmed milk at room temperature for 2 h, the membranes were incubated overnight at 4°C with the following primary antibodies: BZW1 (cat. no. ab85090; 1:800; Abcam) and GAPDH (cat. no. ab8245; 1:8,000; Abcam). Following primary incubation, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG H&L (cat. no. ab205718; 1:2,000; Abcam) and HRP-conjugated goat anti-mouse IgG H&L (cat. no. ab205719; 1:2,000; Abcam) at room temperature for 40 min. Protein bands were visualized using the Enhanced Chemiluminescence Plus kit (EMD Millipore) and semi-quantified by densitometric analysis of protein signals using ImageJ version 1.49 (National Institutes of Health).

Total protein was isolated from tissues and cell lines using RIPA lysis buffer (EMD Millipore). The concentration of total protein was measured using a BCA Protein assay kit (Nanjing KeyGen Biotech Co., Ltd.). Protein (30 μg) was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (EMD Millipore). The membranes were then blocked with 5% skim milk at room temperature for 2 h. Subsequently, the membranes were incubated with the following primary antibodies: E-cadherin (1:1,000; cat. no. 14472), N-cadherin (1:1,000, cat. no. 13116), Vimentin (1:1,000, cat. no. 5741), Slug (1:1,000, cat. no. 9585), GAPDH (1:1,000, cat. no. 5174) (all from Cell Signaling Technology, Inc.) overnight at 4°C. Following incubation with the primary antibodies, the membranes were incubated with a the goat anti-rabbit IgG H&L (HRP) (1:2,000, cat. no. ab205718, Abcam) and goat anti-mouse IgG H&L (HRP) (1:2,000, cat. no. ab205719, Abcam) secondary antibodies for 40 min at room temperature. Finally, protein bands were visualized using the Enhanced Chemiluminescence Plus kit (EMD Millipore) and quantified by densitometric analysis of protein signals using ImageJ version 1.49 (National Institutes of Health).