Amersham western blot detection reagent

Total protein was extracted with RIPA lyse buffer containing 1 mmol/L of PMSF, 1 mmol/L of NaF, and 1 mmol/L of Na₃VO₄. The cell lysates were separated using polyacrylamide gel electrophoresis (PAGE) and subsequently transferred to a PVDF membrane by electrotransfer. Afterward, the membrane was blocked for 1 h and probed with primary antibodies against HSF1 (1:1,000), β-catenin (1:500), and β-actin (1:300) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) followed by a horseradish peroxidase (HRP)-conjugated secondary antibody (Abcam). Blots were visualized using Amersham Western blot detection reagent (GE Healthcare, Piscataway, NJ, USA). Blot density of each band was quantified by Gel imaging system and Quantity One 4.62 software (Bio-Rad, Hercules, CA, USA).

Total proteins were extracted with RIPA (Beyotime, Jiangsu, China) agents from AS plaques and macrophages and the amount of proteins were determined using the bicinchoninic acid (BCA) method. Afterwards, the cell lysates were subjected to electrophoresis in sodium dodecyl sulfate (SDS)-PAGE and subsequently transferred to a PVDF membrane (Millipore, USA). Then, after the blockage with goat serum, the membrane was incubated with primary antibodies against STAT6, pSTAT6 and β-catenin (Abcam, Cambridge, UK) overnight at 4°C. After 3 washes with TBST, the membrane was probed with horseradish peroxidase-conjugated secondary antibody for an additional 2 h at room temperature. Blots were visualized using Amersham Western blot detection reagent (GE Healthcare, Piscataway, NJ, USA).