Journal: PLoS ONE

Article Title: Electroacupuncture-Like Stimulation at the Baihui (GV20) and Dazhui (GV14) Acupoints Protects Rats against Subacute-Phase Cerebral Ischemia-Reperfusion Injuries by Reducing S100B-Mediated Neurotoxicity

doi: 10.1371/journal.pone.0091426

Figure Lengend Snippet: Representative western blot images show the expression of (A) mitochondrial cytochrome c and (C) cytosolic cytochrome c, TRADD, FADD, cleaved caspase-8 and cleaved caspase-3 in the ischemic cortical penumbra in the Sham-7 d, Model-7 d, EA-7 d, and Non-acup-7 d groups 7 d after reperfusion. COX IV and actin were used as internal controls for the mitochondrial and cytosolic fractions, respectively. The relative mitochondrial expression of (B) cytochrome c, and the relative cytosolic expression of (D) cytochrome c, (E) TRADD, (F) FADD, (G) cleaved caspase-8, and (H) cleaved caspase-3 were evaluated in the ischemic cortical penumbra in the Sham-7 d, Model-7 d, EA-7 d, and Non-acup-7 d groups (n = 4). Data are presented as mean ± SD. * P <0.05 compared with the Sham-7 d group; # P <0.05 compared with the Model-7 d group.

Article Snippet: They were then incubated with a mouse anti-GFAP (1∶1000 dilution, #3670 Cell Signaling Technology), rabbit anti-phospho-SAPK/JNK (p-JNK (Thr183/Tyr185); 1∶1000 dilution, #9251S Cell Signaling Technology), rabbit anti-phospho-p44/42 mitogen-activated protein kinase (MAPK (p-ERK); 1∶1000 dilution, #9101 Cell Signaling Technology), rabbit anti-phospho-p38 MAP kinase (p-p38 MAP kinase (Thr180/Tyr182); 1∶1000 dilution, #9212 Cell Signaling Technology), rabbit anti-cytochrome c (1∶1000 dilution, #4272 Cell Signaling Technology), rabbit anti-tumor necrosis factor receptor type 1-associated death domain (TRADD) (1∶1000 dilution, #3694 Cell Signaling Technology), rabbit anti-Fas-associated death domain (FADD) (1∶1000 dilution, #341282 Calbiochem), rabbit anti-cleaved caspase-8 (1∶1000 dilution, 3259-100 BioVision), or rabbit anti-cleaved caspase-3 (1∶1000 dilution, #9661S Cell Signaling Technology) antibody overnight at 4°C.

Techniques: Western Blot, Expressing

Journal: British Journal of Pharmacology

Article Title: 14-Deoxyandrographolide desensitizes hepatocytes to tumour necrosis factor-alpha-induced apoptosis through calcium-dependent tumour necrosis factor receptor superfamily member 1A release via the NO/cGMP pathway

doi: 10.1111/j.1476-5381.2010.00836.x

Figure Lengend Snippet: 14-DAG inhibited TNF-α-mediated DISC formation. (A) Hepatocytes were pretreated with 14-DAG (5, 10 and 15 nM) for 1 h and then exposed to TNF-α/ActD for 12 h. Control cells were treated with the vehicle, DMSO. Immunoreactive bands of the active caspase-3 fragment were analysed by Western blot. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. Caspase-3 activity was determined by using the fluorometric substrates DEVD–AFC. The data are shown as mean ± SD of three independent experiments. *P < 0.02, **P < 0.01, versus TNF-α/ActD-treated cells. (B) Caspase-8 activity was also determined by using the fluorometric substrates IETD–AFC in different treatment groups similar to caspase-3. The data are shown as mean ± SD of three independent experiments. *P < 0.02, **P < 0.01, versus TNF-α/ActD-treated cells. (C) Hepatocytes were pretreated with 14-DAG for 1 h at 37°C followed by treatment with TNF-α (10 ng·mL−1) and ActD (200 ng·mL−1) for 1 h at 37°C. TNFRSF1A was immunoprecipitated from the cell lysate, and the immunoprecipitates (IPs) were electrophoresed and immunoblotted with anti-FADD, anti-TRADD and anti-caspase-8 (p43) antibodies. Densitometric analyses of IPs were performed. The data are shown as mean ± SD of three independent experiments. (D) Hepatocytes were pretreated with 14-DAG for 1 h followed by incubation in PBS (pH 7.4) containing biotinylated TNF-α (10 ng·mL−1). TNFRSF1A internalization in hepatocytes was viewed under laser scanning confocal microscope using streptavidin–FITC. The magnification of the photomicrograph is 100×. (E) Evaluation of TNFRSF1A internalization in the presence of 5, 10 and 15 nM 14-DAG (experimental conditions were same as D) was quantified by FACS analysis using CELL Quest software. Results are representative of seven independent experiments with similar results.

Article Snippet: Fluorescent dyes were from Molecular Probes (Invitrogen Corporation); Ru360 was from Calbiochem (EMD Bioscience, Gibbstown, NJ, USA); 1,3,4,6-tetrachloro-3a, 6a di phenyl glycoluril (chloroglycoluril) (PIERCE, Rockford, IL, USA); antibodies of TRADD, FADD were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); antibody of TNFRSF1A was from Sigma.

Techniques: Control, Western Blot, Activity Assay, Immunoprecipitation, Incubation, Microscopy, Software