Journal: Oxidative Medicine and Cellular Longevity
Article Title: Modulation of Glutathione Hemostasis by Inhibition of 12/15-Lipoxygenase Prevents ROS-Mediated Cell Death after Hepatic Ischemia and Reperfusion
Figure Lengend Snippet: Western blot assays of liver tissue (right anterior segment) exposed to ischemia-reperfusion injury for detection of activity levels of proapoptotic proteins JNK (stress-activated protein kinase/Jun-amino-terminal kinase), ERK (mitogen-activated protein kinase p44/42), caspase-3, and PARP (poly-ADP-ribose-polymerase). (a) Representative images of Western blots demonstrating the differences in enzyme activity levels for each experimental group. Inactive proteins are shown on top for ERK (total ERK, 42 + 44 kDa), PARP (uncleaved, 116 kDa), caspase-3 (loading control GAPDH, 37 kDa), and JNK (total JNK, 46 + 54 kDa). Activated protein forms are shown in the middle for ERK (phosphorylated ERK, 42 + 44 kDa), PARP (cleaved, 89 kDa), caspase-3 (cleaved, 20 kDa), and JNK (phosphorylated JNK, 46 + 54 kDa). Loading controls are shown below in each group for glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 37 kDa). (b) Quantitative photometric analyses of Western blots prove the mostly significant downregulation of proapoptotic enzyme activity levels after Baicalein pretreatment. Activated enzyme ratio is calculated as a quotient of active/inactive protein form. Changes in protein level activation after sole vehicle administration (DMSO) could also be observed. Data are presented as the mean ± SEM. ∗ p
Article Snippet: SDS-gel electrophoresis and protein transfer onto nitrocellulose membranes and followed by overnight incubation with antibodies at 4°C were performed for activity detection of stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK, rabbit, 46/54 kDa, Cell Signaling Technology, USA), mitogen-activated protein kinase p44/42 (ERK1/2, rabbit, 42/44 kDa, Cell Signaling Technology, USA), caspase-3 (rabbit, 17/19/35 kDa, Cell Signaling Technology, USA), and poly-ADP-ribose-polymerase (PARP, rabbit, 89/116 kDa, Cell Signaling Technology, USA).
Techniques: Western Blot, Activity Assay, Activation Assay