Secondary antibody conjugated with horseradish peroxidase

Cells were lysed in lysis buffer and protein concentrations of the lysates were determined using the Bradford protein assay system (Bio‐Rad, Hercules, CA, USA). Equal amounts (30μg protein each lane) of total cellular protein were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The membrane was blocked and then incubated with primary antibody overnight at 4°C. Primary antibodies used included antibodies for ING5 and SMAD3 (Proteintech Group, Inc., Rosemont, IL, USA); IL‐6 (Bioworld Technology Co., Ltd., Nanjing, China); pAKT (Ser473/Thr308) and STAT3 (Cell Signaling
Technology, Danvers, MA, USA); p‐STAT3 (Y705), AKT, β‐catenin, p‐β‐catenin (Ser33/S37), E‐cadherin, N‐cadherin, Snail, Slug, Twist, EGFR, and CEACAM6 (Abcam, Cambridge, MA, USA); and β‐actin (Actin) (Sigma‐Aldrich, St. Louis, MO, USA). The membrane was incubated with corresponding secondary antibody conjugated with horseradish peroxidase (1:5000, Sigma‐Aldrich) at room temperature for one hour. The blots were developed using an enhanced chemiluminescence Western blot detection system (Amersham Bioscience, Buckinghamshire, UK).

After treatment of the cells with 24s-OHC and Aβ for 24 hours, they were washed with phosphate buffered saline, scrapped with lysis buffer (RIPA plus protease inhibitor), and mechanically homogenized. Protein concentration was determined by Lowry protein assay and then samples were separated on 10% SDS-polyacrylamide gel and transferred to PVDF membranes. Membranes were incubated overnight with anti HMG-CoA reductase (1:5000; Abcam), ABCA1 (1:2000), and anti GAPDH (1:10000) at 4°C. After incubation time, the membranes were washed three times with tris-buffered saline/Tween (TBST) and then incubated with secondary antibody conjugated with horseradish peroxidase (1:4000; Sigma) and detection was performed using enhanced chemiluminescence substrates. The ImageJ software was used to quantify the western blot data.