Western ecl substrate kit

EPCs were washed three times with PBS and then covered with 100-μl RIPA lysis buffer (Boster, China, AR0102) containing 1% proteinase inhibitor cocktail for 40 min on ice. Then cell lysis was collected using cell scrapers. The concentration of protein samples was determined by BCA assay kit (Boster, China, AR0146). Then protein samples were separated on 10% of SDS-PAGE (40 μg per lane) and transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking with 5% skim milk at room temperature for 1 h, the membranes were incubated overnight with the corresponding primary antibodies at 4 °C. In the second day, the membranes were incubated in 1:5000 diluted horseradish peroxidase-conjugated goat anti-rabbit IgG and goat anti-mouse IgG secondary antibody for 1 h (Boster, China, BA1056). The protein bands were visualized with Western ECL Substrate Kit (Thermo Pierce, USA). Images were captured by Bio-Rad scanner (Hercules, CA), and the density was determined by ImageJ version 1.48.

Cells were washed with PBS three times and lysed with RIPA containing 1 mM protease inhibitor cocktail and 1 mM phosphatase inhibitor cocktail (Boster). Then 30 μg protein samples were separated by SDS-polyacrylamide gels and transferred to PVDF membranes. The membranes were then blocked with 5% bone serum albumin for 1 h and incubated with appropriate antibodies at 4 °C overnight. Subsequently, blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. The bands were detected by the Western ECL Substrate Kit (Thermo Pierce, USA). Protein expressions were determined by mormalizing to GAPDH, and representative bands are shown. Compared to the Control group, the EMF group was treated with EMF for 7 days before culturing in inductive medium.

Briefly, chondrocytes were homogenized on ice and lysed with RIPA lysis buffer (Boster, Wuhan, China). After 30 minutes, the lysates were collected and centrifugated at 12 800 g for 20 minutes at 4℃. 25 μg proteins from each sample were separated by 10% SDS‐PAGE gel and then transferred to polyvinylidine difluoride PVDF membranes (pore size 0.45 μmol/L, Millipore, Billerica, MA), which were subsequently incubated with targeted primary antibodies anti MMP3 (17873‐1‐AP, proteintech, USA), MMP13 (ab39012, Abcam, USA), FIS1 (10956‐1‐AP, proteintech, USA), DRP1 (#8570, CST, USA), MFF (#84580, CST, USA), iron regulatory protein 1 (IRP1, ab126595, Abcam, USA), iron regulatory protein 2 (IRP2, ab110321, Abcam, USA), transferrin receptor (TfR1, ab84036, Abcam, USA), Ferroportin (FPN, ab78066, Abcam, USA), β‐actin (#BM0627, Boster, Wuhan) at 4°C overnight. After being washed for three times with TBST, membranes were incubated with respective secondary antibodies at room temperature for 1 hour. Bands and band density were detected using Western ECL Substrate Kit (Thermo Pierce, USA) and analyzed with the built‐in software of Bio‐Rad scanner (Bio‐Rad, Hercules, CA). Images were acquired with Bio‐Rad scanner (Hercules, CA) and densitometry was quantified by digital image analysis software (Quantity One, Bio‐Rad, Hercules, CA).