Bca method

Whole lysates of MSCs were prepared using the RIPA Lysis Buffer (Beyotime, China, Cat# P0013B). Proteins were extracted and the protein concentration was quantified using the BCA method (Beyotime, China, Cat# P0010). Equal amounts of protein samples were loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA, Cat# ISEQ00010) which were blocked with 5% bovine serum albumin (BSA) (Gemini Bio, USA, Cat# 700-100P) in Tris buffered saline (TBS) for 2 h at room temperature. Then, the membranes were incubated overnight at 4°C with the following primary antibodies: anti-YAP1 (Abcam, UK, Cat# ab56701; diluted at 1:1000) and anti-GAPDH (CWBio, China, Cat# CW0100; diluted at 1:1000). After washing with TBS containing 0.1% Tween-20, membranes were incubated with a horseradish peroxidase (HRP)-conjugated Goat Anti-Mouse secondary antibody (Signalway, China, Cat# L3032) for 1 h at room temperature. The protein bands were visualized using an enhanced chemiluminescence kit (Amersham Biosciences, USA) and detected by a gel imaging system (Tanon, China) 72 (link), 73 (link). Quantification of WB results was performed using the ImageJ 1.47 software (NIH, USA).

Cells were harvested and homogenized in the RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM MgCl₂, 1.5% NP-40, 0.1% SDS, and 0.5% sodium deoxycholate) supplemented with protease cocktail inhibitor, 10 μM MG-132, 10 μg ml⁻¹ leupeptin and 1 mM phenylmethylsulfonyl fluoride. The protein concentrations of lysates were determined using BCA method (Beyotime). Samples were mixed with the membrane protein solubilization buffer (62.5 mM Tris-HCl, pH 6.8, 15% SDS, 8 M urea, 10% glycerol, and 100 mM DTT) plus the 4× loading buffer (150 mM Tris-HCl, pH 6.8, 12% SDS, 30% glycerol, 6% 2-mercaptoethanol, and 0.02% bromophenol blue) and incubated for 30 min at 37 °C. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare). Immnoblots were blocked with 5% non-fat milk in TBS containing 0.075% Tween-20 (TBST) and probed with indicated antibodies overnight at 4 °C. After washing in TBST for five times, blots were incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. Immunoreactivity was developed with Clarity Western ECL chemiluminescent substrates (170–5061, Bio-Rad). Western blot images were captured by Amersham Imager 600 (GE Healthcare), and integrated optical intensities of each band were quantified by Image-Pro Plus 6 software (Media Cybernetics). Uncropped blots are shown in Supplementary Figs. 9–13.

The cells were dissociated, centrifuged, and collected. Cell lysis buffer (Beyotime company, Jiangsu, China) was added, and the cells were incubated in an ice bath for 15–20 min. The cells were centrifuged at 4°C at 20000 rpm for 10 min. The supernatant was collected for future use. Proteins were quantified using the BCA method (Beyotime), and the concentrations of samples in all groups were adjusted to the same level. Loading buffer was added to the samples; after boiling in water for 5 min, the samples were stored at -20°C for subsequent use. A 12% SDS-polyacrylamide gel was prepared, and the samples were subjected to electrophoresis. The samples were then transferred onto a 0.2 μm polyvinylidene fluoride (PVDF) membrane using the wet transfer method at 90 V. After membrane transfer was complete, the PVDF membrane was removed and blocked for 1 h at room temperature. Primary antibodies were added and incubated at 4°C overnight. After the membrane was washed with phosphate-buffered saline/tween (PBST), a horseradish peroxidase-labeled secondary antibody was added, and the membrane was incubated at room temperature for 1 h. After the membrane was washed with PBST, the proteins were detected using the enhanced chemiluminescence (ECL) method (Byotime). β-actin was used as a control and obtained from Beyotime Biotechnology. All of the primary antibodies and dilutions contained the following: goat anti-LGMN antibody (1:500; R&D, Shanghai, China), mouse anti-MMP2 antibody (1:1000; ZSGN-BIO, Beijing, China), and mouse anti-MMP9 antibody (1:1000; ZSGN-BIO). Secondary antibodies coupled to horseradish peroxidase (HRP) were purchased from Beyotime.