Journal: Journal of Biological Chemistry

Article Title: Phosphatidylethanolamines Modified by γ-Ketoaldehyde (γKA) Induce Endoplasmic Reticulum Stress and Endothelial Activation

doi: 10.1074/jbc.m110.213470

Figure Lengend Snippet: FIGURE 8. KA-PE induces elements of the ER stress response. HUVEC were treated with 3 M PE or KA-PE for 10–60 min, lysates generated, and induction of three representative elements of the ER stress response measured by immunoblotting as described under “Experimental Procedures.” A, immunoblots for BiP/grp78, phospho-p38, CHOP, and actin from a representative experiment. Actin was used as an internal loading control for each sample. B, quantitation of three replicate CHOP immunoblotting experiments. For each replicate, sample was normalized to the 10-min PE treatment to determine fold increase in expression.C,quantitationofthreereplicateBiPimmunoblottingexperiments.D,quantitationofthreereplicatephospho-p38experiments.E,immunoblotfor phospho-p38 and total p38 after 60 min of incubation with 3 M PE or KA-PE in additional experiments to determine whether increased phospho-p38 expression resulted from increased total p38 expression or increased phosphorylation. Samples were in nonadjacent wells on same membrane. F, quantitation of duplicate phospho-p38 and total p38 immunoblotting experiments.

Article Snippet: Each strip was incubated with appropriate primary antibody for CHOP (L63F7, Cell Signaling Technology), BiP/grp78 (C50B12, Cell Signaling Technology), or phospho-p38 (9211S, Cell Signaling Technology) and then visualized using fluorescent anti-mouse or anti-rabbit IgG antibody and scanned by the Odyssey Imaging System (LI-COR Biosciences).

Techniques: Generated, Western Blot, Control, Quantitation Assay, Expressing, Incubation, Phospho-proteomics, Membrane