Peroxidase conjugated secondary antibody

After cells were incubated with the inhibitors, respectively, for 12 h, Caveolin-1 enriched membrane fractions were isolated as described above and the final samples were detected by western blotting. The protein samples were separated by SDS-PAGE gel and then electrotransferred to PVDF membranes. Subsequently, blots were subjected to immunostaining with antibodies against Caveolin-1 (Cell Signaling Technology, 1 : 800), Cavin-1 (ANBO, 1 : 500), and lectin-like oxLDL receptor (Lox-1, WuXi AppTec, 1 : 1000). After incubation for 1 h with a peroxidase-conjugated secondary antibody (Abbkine, 1 : 10000), bands were visualized by an ECL western blotting detection system (NDR, Israel). The band intensities were quantified using ImageJ software.

Cells were lysed in IP lysis buffer containing protease inhibitors and then measured protein concentration by BCA assay. An equal amount of protein from cells loaded into the 8% SDS-PAGE, transferred to polyvinylidene difluoride membranes (PVDF), then incubated with primary antibody against VWF (1:500, Cell Signaling Technology, Shanghai, China) and SPP1 (1:2,000, Proteintech, Wuhan, China) at 4°C overnight, followed by incubation with peroxidase-conjugated secondary antibody (1:5,000, Abbkine, Wuhan, China) at room temperature for 1 h, finally visualized by enhanced chemiluminescence detection. The β-actin (1:5,000, Abbkine, Wuhan, China) was used as internal references in this study.

After cells were incubated with the inhibitors, respectively, for 12 h, Caveolin-1 enriched membrane fractions were isolated as described above and the final samples were detected by western blotting. The protein samples were separated by SDS-PAGE gel and then electrotransferred to PVDF membranes. Subsequently, blots were subjected to immunostaining with antibodies against Caveolin-1 (Cell Signaling Technology, 1 : 800), Cavin-1 (ANBO, 1 : 500), and lectin-like oxLDL receptor (Lox-1, WuXi AppTec, 1 : 1000). After incubation for 1 h with a peroxidase-conjugated secondary antibody (Abbkine, 1 : 10000), bands were visualized by an ECL western blotting detection system (NDR, Israel). The band intensities were quantified using ImageJ software.