Signaling pathways western immunoblotting analysis

To investigate the molecular mechanism of purple corn anthocyanins and PCA on AGEs induced MAPK and NF-κB activation, the levels of phosphorylated signaling proteins were analyzed by Western blotting. The concentration of 2.5 × 10⁵ per well of HACs at passage 4 was cultured in 6-well plates. The 80% confluent of HACs was cultured without serum overnight and then pretreated with anthocyanins (C3G, Pg 3-glc and P3G) and PCA (6.25–50 µg/ml) for 2 h followed by stimulation with 10 μg/ml of AGEs for another 10 min. The cytoplasmic proteins of HACs were extracted using RIPA buffer and were separated using 12% (w/v) of SDS-PAGE. Proteins were transferred onto nitrocellulose membranes which were later blocked with 5% (w/v) non-fat dried-milk proteins in PBST. After blocking, various probes, including p65, p-p65, IκB-α, p38, p-p38, JNK, p-JNK, ERK, p-ERK or β-actin antibody (Cell Signaling Technology, Inc., Beverly, MA, USA) were added and incubated overnight. The membranes were then washed with PBST and HRP-conjugated secondary antibodies were added. Incubation was performed for 1 h and the bands were visualized using enhanced chemiluminescence (ECL) reagent (GE Healthcare, Chicago, IL, USA). The quantification of band intensity was performed by TotalLab TL120 v2006 (for Windows, TotalLab software, Newcastle upon Tyne, UK, www.nonlinear.com).