Protein Expression Analysis of EAHY Cells

Generally 1.5 × 10⁶ EAHY were inoculated onto 10-cm culture dishes and exposed to various concentrations of LPC for 24h. After removal of medium and washed with PBS, cell lysates were prepared by dissolving cells in lysis buffer (10 mM Tris-HCl, pH 7; 140 mM sodium chloride; 3 mM magnesium chloride; 0.5% NP-40; 2 mM phenylmethylsulfonyl fluoride; 1% aprotinin; and 5 mM dithiothreitol). Then the equal amounts of proteins (20–50 μg/ml) were loaded to run 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein separation and transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were first blotted with primary antibodies of cdc2, cyclin B1, IL-8 and GAPDH for 2 hr as described before [35 (link), 36 (link)]. The membranes were then incubated in respective horseradish peroxidase-link secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 hr. After washing the membrane with buffer, ECL reagents (Amersham) were added and the chemiluminescence of protein bands was determined by exposure of membranes to Fuji films for 30 sec to 10 min. The intensity of GAPDH bands was used as control.