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: International Journal of Molecular Sciences

Article Title: TNF/IFN-γ Co-Signaling Induces Differential Cellular Activation in COVID-19 Patients: Implications for Patient Outcomes

doi: 10.3390/ijms26031139

Figure Lengend Snippet: TNF H IFNγ H patients have more molecules associated with TLR signaling and cell death mediated by the TNF/TNFR1 pathway than the TNF N-L IFNγ N-L group. The relative quantification (RQ) of MYD88 ( A ), NFKB1 ( B ), IRF7 ( C ), TRAF2 ( D ), FADD ( E ), and TRADD ( F ) genes was measured at the transcriptional level. The RQ of the HD group was normalized to 1 (dotted red line). Data are represented as median and IQR (25–75) values, and each dot represents a patient. The Kruskal–Wallis test was performed for statistical comparisons; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Gene expression was assessed using TaqMan probes for the target genes, Fas-associated via death domain ( FADD , Hs00538709_m1), Tradd-TNFRSF1A associated via death domain ( TRADD , Hs00601065_g1), Traf2-TNF receptor associated factor 2 ( TRAF2 , Hs00184192_m1), Myd88-myeloid differentiation primary response 88 (MyD88, Hs01573837_g1), Nfkb1-nuclear factor kappa B subunit 1 ( NFKB1 , Hs00765730_m1), and Irf7-interferon regulatory factor 7 (IRF7, Hs01014809_g1).

Techniques: Quantitative Proteomics

Journal: Cellular and Molecular Life Sciences

Article Title: TNF-induced necroptosis and PARP-1-mediated necrosis represent distinct routes to programmed necrotic cell death

doi: 10.1007/s00018-013-1381-6

Figure Lengend Snippet: PARP-1 is activated with distinct kinetics by TNF and MNNG. In ( a – f ), L929Ts cells were treated with 100 ng/ml TNF for the indicated times. a Cells were analyzed for PARP-1 by immunoblotting with antibody 9542. The same lysates were additionally analyzed on separate gels for TRADD, FADD, caspase-8, and RIP1. Arrows indicate the positions of the full-length proteins, which were additionally verified for TRADD, FADD, caspase-8, and RIP1 by parallel separation of lysates from 293T cells overexpressing the respective protein (293T+). Untransfected 293T cells (293T−) were included for control. Shifted signals for TRADD and RIP1 in these lanes represent the cross-reactive endogenous human proteins that differ in size from the endogenous murine proteins of L929Ts cells. b Western blot with antibody 9542 that detects all cleavage fragments of PARP-1. Arrows indicate the position of the full-length protein and the 89-kDa caspase-3 cleavage product present only in apoptotic control lysates (Co, extracts from cells treated with 100 ng/ml TNF and 2 μg/ml of the protein biosynthesis inhibitor CHX for 1 h). A longer exposure is shown for the lower part of the blot to increase the visibility of the reactive bands. c Cells were additionally treated with the proteasome inhibitor MG-132 (52.6 μM) as indicated. PARP-1 was detected with antibody 556362. d The expression levels of PARP-1 mRNA were determined by qRT-PCR in parallel to protein levels (shown below, PARP-1 was detected with antibody 9542). The expression of PARP-1 is shown relative to untreated cells. Comparable results were obtained with a different primer pair. e Following detection of PARP-1 (antibody 9542), the membrane was reprobed with an antibody for PAR to reveal the presence of poly(ADP-ribosyl)ated proteins. f Lysates from cells treated with TNF for the indicated times were additionally incubated for the indicated times in the presence of 10 mM β-mercaptoethanol and 0.05 U PARG before PARP-1 was detected with antibody 9542 and reprobing of the membrane with an antibody for PAR. g L929Ts, L929ATCC, and L929sA cells were left untreated, treated with TNF as above, or treated with 0.5 mM MNNG for 15 min and further incubated with fresh medium without MNNG for the indicated times before PARP-1 was detected with antibody 9542. h Lysates from L929Ts cells treated with MNNG as in ( g ) for the indicated times were incubated with PARG and analyzed as in ( f ). For all Western blot panels, detection of actin served as a loading control. For all figures, representative data from one out of at least two or more experiments with similar results are shown ( n ≥ 2) and error bars indicate the standard deviations (SD) from at least triplicate determinations ( n ≥ 3)

Article Snippet: After electrophoretic transfer to nitrocellulose, reactive proteins were detected using antisera specific for actin (sc-1615, Santa Cruz, Heidelberg, Germany; A1978, Sigma), PARP-1 (556362, Becton–Dickinson, Heidelberg, Germany; 9542, Cell Signaling, Danvers, MA, USA), TRADD (3684, Cell Signaling), FADD (7B5, Enzo), Caspase-8 (4927, Cell Signaling), RIP1 (610458, Becton–Dickinson), PAR (551813, Becton–Dickinson), AIF (sc-13116, Santa Cruz), COXIV (sc-58348, Santa Cruz), histone (ab1791, Abcam, Cambridge, UK), JNK1 (554286, Becton–Dickinson), JNK2 (sc-271133, Santa Cruz), phospho-JNK (AF1205, R&D Systems, Wiesbaden, Germany), TNF-R1 (sc-8436, Santa Cruz), human (PAB0287, Abnova, Heidelberg, Germany) or murine RIP3 (ADI-905-242, Enzo), GFP (8369-1, Takara), and the ECL detection kit (GE Healthcare, Munich, Germany).

Techniques: Western Blot, Control, Expressing, Quantitative RT-PCR, Membrane, Incubation