Bca protein assay

MG63 and U2OS cells were treated with si-CD44 or si-NC for 48 h, and total proteins were extracted using RIPA buffer containing protease inhibitor cocktail. Protein concentrations were determined using the BCA Protein Assay (Multi sciences). Proteins (30 µg/lane) were separated by 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk for 2 h at room temperature (RT). Next, the membranes were incubated with anti-CD44 (1:2000, ABclonal, China), cathepsin S (1:2000, Affinity, China), anti-MMP-9 (1:2000, ABclonal, China) at 4 °C overnight. Subsequently, the appropriate horseradish peroxidase (HRP)-linked secondary antibodies (1:5000, Sera care) were used to visualise the immunoreactivity. GAPDH was used as an internal control. The intensity of each band was measured with ImageJ.

Cells were divided into the following five groups for western blotting: control group (untreated RAW 264.7 cells), model group (RAW 264.7 cells treated with ox-LDL), and three treatment groups (RAW 264.7 cells treated with ox-LDL and 0.2, 0.6, and 1.8 g/L of GXHP, respectively). Cells were collected, and a protein extraction kit (Gene pool, Beijing, China) was used to extract proteins according to the manufacturer’s protocol. The protein concentration was determined using a BCA protein assay (Multi Sciences, Hangzhou, China). Protein separation using 12% SDS-PAGE gel was then performed, and the protein samples were transferred to a PVDF membrane. After blocking, the PVDF membrane was incubated with specific primary antibodies (Cell Signaling Technology, Danvers, MA, USA), such as PI3 kinase p85 antibody (dilution ratio was 1:500), phospho-PI3 kinase p85 antibody (dilution ratio was 1:1,000), AKT1 antibody (dilution ratio was 1:1500), and phospho-AKT1 antibody (dilution ratio was 1:500), overnight at 4°C. This was followed by 1 hour incubation at room temperature with horseradish peroxidase conjugated goat anti-rabbit IgG (Abcam, Cambridge, MA, USA)(dilution ratio was 1:5000).Antigen–antibody binding was detected using enhanced chemiluminescence reagents (ThermoFisher Scientific, Waltham, MA, USA). Quantification of each protein was determined using Quantity One v.4.6.2.

Total protein extraction was performed using RIPA lysis buffer containing PMSF (Solarbio, Beijing, China). Protein concentration was determined using the BCA Protein Assay (Multi Sciences, Hangzhou, China). Equal amounts of protein samples were electrophoresed on 10% sodium dodecyl sulfate–polyacrylamide electrophoresis gel and then transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA), followed by blocking with 5% skim milk for an hour. Afterwards, the membranes were probed with specific primary antibodies overnight at 4 °C and then with secondary antibody (KPL, Milford, MA, USA) for an hour at room temperature. The primary antibodies used were listed in Supplementary Table 6. Finally, the signals were visualized using an enhanced chemiluminescence reagent (Multi Sciences, Hangzhou, China).