Anti ilk

Protein extracts (20 μg) from the different groups of BMSCs were loaded onto 6–12% SDS-PAGE gel (Beyotime), then subjected to electrophoresis for 90 min at 120 V and transferred to PVDF membranes. The membranes were blocked and incubated with primary antibodies overnight at 4°C, followed by washing and incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL, Beyotime) and exposed to x-ray film. Relative protein expression was determined by image analysis using an Alpha Innotech gel analysis system.
The primary antibodies used were anti-Itgb1 (Abcam, USA, 1:500), anti-FAK (Abcam, USA, 1:1000), anti-ILK (Abcam, USA 1:5000), anti-VEGF (Abcam, USA 1:1000), anti-Bax (Cell Signaling Technology, USA 1:1000), anti-caspase 3 (Cell Signaling Technology, USA 1:1000), anti-Bcl-2 (Cell Signaling Technology, USA 1:1000), anti-β-actin (ZSGB BIO). The secondary antibodies were HRP-conjugated goat anti rabbit IgG (BIOSS, China 1:5000).

Proteins were extracted from cultured HESCs using RIPA lysis buffer, and the concentration of protein was detected by the BCA Protein Assay Kit (Beyotime). Samples were then separated by 10% SDS-PAGE gels (Beyotime). The primary antibodies were applied according to the provided recommendations: anti-ILK (1:5000, Abcam), anti-TGFβ1 (1:200, Boster), anti-SMAD2 (1:1000, Affinity), anti-VEGF (1:200, Boster), anti-COX-2 (1:700, Proteintech Group), anti-MMP-9 (1:800, Abcam) and anti-GAPDH (1:1000, Abcam). Finally, positive bands were detected using the chemiluminescent ECL Plus reagent (Beyotime) according to the manufacturer’s protocol. The densitometry of bands was quantified using Quantity One version 4.6.0 software (Bio-Rad). Expressions of proteins were normalized to GAPDH protein.

Cells were lysed in lysis buffer (50 mM Tris-HCl, pH 6.8, 1% SDS) and applied for protein quantification (Pierce BCA protein assay kit, Fisher). Lysates were diluted in Laemmli buffer containing 62.5 mM Tris-HCl, pH 7.4, 2% SDS, 5% 2-mercaptoethanol, and 10% glycerol to the same concentration and subjected to SDS-PAGE. Proteins were then transferred to PVDF and probed with primary antibodies (anti-GFP, Cell Signaling; anti-ILK, Abcam; anti-GAPDH, Cell Signaling; anti-PINCH, BD Bioscience; anti-Parvin, Cell Signaling; anti-actin, Cell Signaling), which was followed by HRP-conjugated secondary antibody (anti-mouse HRP, Cell Signaling; anti-rabbit HRP, Cell Signaling). All antibodies and their dilution in the study are listed in Supplementary Table 1, uncropped western blots are provided in Supplementary Figures 9–14.