Western Blot Analysis of Ox-LDL and GXHP Effects

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.