Determination of Caspase Activation by Western Blot

2020 ◽  
pp. 1-12
Author(s):  
Hussein Chehade ◽  
Alexandra Fox ◽  
Gil G. Mor ◽  
Ayesha B. Alvero
Keyword(s):  
Western Blot ◽  
Caspase Activation

scholarly journals Stimulation of Erythrocyte Cell Membrane Scrambling by C-Reactive Protein

2017 ◽  
Vol 41 (2) ◽  
pp. 806-818 ◽  
Author(s):  
Majed Abed ◽  
Christian Thiel ◽  
Syeda T. Towhid ◽  
Kousi Alzoubi ◽  
Sabina Honisch ◽  
...  
Keyword(s):  
Plasma Concentration ◽  
Healthy Volunteers ◽  
Annexin V ◽  
Caspase Activation ◽  
C Reactive Protein ◽  
Forward Scatter ◽  
Reactive Protein ◽  
Phosphatidylserine Translocation ◽  
Binding Cell

Background: Eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and phosphatidylserine-translocation, is triggered by fever and inflammation. Signaling includes increased cytosolic Ca2+-activity ([Ca2+]i), caspase activation, and ceramide. Inflammation is associated with increased plasma concentration of C-reactive protein (CRP). The present study explored whether CRP triggers eryptosis. Methods: Phosphatidylserine abundance at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ceramide abundance and caspase-3-activity utilizing FITC-conjugated antibodies. Moreover, blood was drawn from patients with acute appendicitis (9♀,11♂) and healthy volunteers (10♀,10♂) for determination of CRP, blood count and phosphatidylserine. Results: A 48h CRP treatment significantly increased the percentage of annexin-V-binding cells (≥5µg/ml), [Ca2+]i (≥5µg/ml), ceramide (20µg/ml) and caspase-activity (20µg/ml). Annexin-V-binding was significantly blunted by caspase inhibitor zVAD (10µM). The percentage of phosphatidylserine-exposing erythrocytes in freshly drawn blood was significantly higher in appendicitis patients (1.83±0.21%) than healthy volunteers (0.81±0.09%), and significantly higher following a 24h incubation of erythrocytes from healthy volunteers to patient plasma than to plasma from healthy volunteers. The percentage of phosphatidylserine-exposing erythrocytes correlated with CRP plasma concentration. Conclusion: C-reactive protein triggers eryptosis, an effect at least partially due to increase of [Ca2+]i, increase of ceramide abundance and caspase activation.

scholarly journals Prolactin Exerts a Prosurvival Effect on Human Spermatozoa via Mechanisms that Involve the Stimulation of Akt Phosphorylation and Suppression of Caspase Activation and Capacitation

Endocrinology ◽  
2009 ◽  
Vol 151 (3) ◽  
pp. 1269-1279 ◽  
Author(s):  
Dwi Ari Pujianto ◽  
Benjamin J. Curry ◽  
R. John Aitken
Keyword(s):  
Western Blot ◽  
Human Spermatozoa ◽  
Pcr Analysis ◽  
Dependent Manner ◽  
Sperm Capacitation ◽  
Caspase Activation ◽  
Apoptotic Pathway ◽  
Akt Phosphorylation ◽  
The Impact ◽  
Stimulation Of

The purpose of this study was to examine the impact of prolactin (PRL) on human sperm function, in light of a recent proteomic analysis indicating that these cells express the PRL receptor (PRLR). Immunocytochemical analyses confirmed the presence of PRLR in human spermatozoa and localized this receptor to the postacrosomal region of the sperm head as well as the neck, midpiece, and principal piece of the sperm tail. Nested PCR analysis indicated that these cells possess four splice variants of the PRLR: the long form and three short isoforms, one of which is reported for the first time. A combination of Western blot analyses and immunocytochemistry demonstrated that PRL inhibited sperm capacitation in a dose-dependent manner, suppressing SRC kinase activation and phosphotyrosine expression, two hallmarks of this process. The suppression of sperm capacitation was accompanied by a powerful prosurvival effect, supporting the prolonged motility of these cells and preventing the formation of spontaneous DNA strand breaks via mechanisms that involved the concomitant suppression of caspase activation. Western blot analyses indicated that the prosurvival effect of PRL on human spermatozoa involved the stimulation of Akt phosphorylation, whereas inhibitors of phosphatidylinositol-3-OH kinase and Akt negated this effect, as did the direct induction of sperm capacitation with cAMP analogues. We conclude that PRL is a prosurvival factor for human spermatozoa that prevents these cells from defaulting to an intrinsic apoptotic pathway associated with cell senescence. These findings have implications for preservation of sperm integrity in vivo and in vitro.

The Combination of a Histone Deacetylase (HDAC) Inhibitor with the BCL-2 Inhibitor GX15-070 Has Synergistic Antileukemia Effect by Inducing Both Apoptotic and Autophagic Pathways.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1633-1633
Author(s):  
Yue Wei ◽  
Tapan Kadia ◽  
Weigang Tong ◽  
Susan O’Brien ◽  
Jean Viallet ◽  
...  
Keyword(s):  
Synergistic Effect ◽  
Western Blot ◽  
Blot Analysis ◽  
Hdac Inhibitor ◽  
Caspase 3 ◽  
Single Agent ◽  
Apoptotic Cells ◽  
Caspase Activation ◽  
Apoptotic Protein ◽  
Induction Of Apoptosis

Abstract HDAC inhibitors have limited single agent clinical activity in leukemia, a probable effect of dysregulated proapoptotic pathways. We hypothesized that the combination of a HDAC inhibitor with a Bcl-2 inhibitor may result in synergistic antileukemia activity. To test this concept, we modeled in vitro the combination of MGCD0103, a class 1 specific HDAC inhibitor, with GX15-070 (Obatoclax), a novel Bcl-2 homology domain-3 (BH3) mimetic The cytotoxic potential of the combination was first assessed measuring cell viability using trypan blue extraction assays in leukemia cell lines HL60, U937, THP1, and Molt4. Mathematical modeling analysis indicated a strong synergistic effect of the combination of MGCD0103 and GX15-070 in terms of growth inhibition. Inhibitory effect of the combination at concentrations of MGCD0103 600 nM and GX15-070 200 nM was 64 fold the effect of single agent MGCD0103 and 16 fold that of GX15-070. Analysis of sequence of treatment indicated that the optimal sequence was addition of GX15-070 first followed by MGCD0103. We then analyzed the effect of the combination on the induction of apoptosis using Annexin V flow assays. The combination also showed a marked synergistic effect in terms of apoptosis induction (the combination was 217% more potent than single agent MGCD0103 and 79% more than GX15-070). Western blot analysis indicated that the use of the combination substantially enhanced the cleavage/activation of caspase-3 and the subsequent cleavage of caspase-3 substrate PARP. As demonstrated in AML and other cell types previously, we showed that GX15-070 dissociated the pro-apoptotic protein BAK from MCL-1. The release of BAK has been demonstrated to play an important role in caspase activation. Western blot analysis also demonstrated that while anti-apoptotic protein XIAP, the major inhibitor of caspase 3, was reduced in MGCD0103 treated cells, the reduction was enhanced with the combination. These results suggest that GX15-070 may sensitize XIAP to the down-regulation effect of MGCD0103, which in turn further activates caspase 3. The combinatory effect of BAK release by GX-15-070, with the suppression of XIAP by MGCD0103, that is potentiated by GX15-070, may play a critical role in the synergistic effect of these two agents on caspase activation, which then results in increased induction of apoptosis. Because in initial experiments we evidenced a discrepancy between cell viability and apoptosis, we also studied the effect of the combination on the induction of autophagy using electron microscopy: non-apoptotic cells after MGCD0103 treatment did not show increase autophagic characteristics compared to control cells, while more non-apoptotic cells under GX15-070 evidenced swollen endoreticulum, a feature of early autophagy. Most non-apoptotic cells treated with MGCD0103 and GX15-070 combination showed accumulation of macro-autophagosomes, a marker of late autophagy, indicating that more severe autophagy was induced by the combination. Furthermore, induction of LC3-II, a marker of autophagy, was observed after combination. We confirmed these results (both in terms of apoptosis and autophagy) ex vivo in primary cells (n=8) obtained from patients with relapsed/refractory AML. In conclusion, the combination of an HDAC inhibitor with GX15-070 has synergistic antileukemia activity and should be studied in human clinical trials.

Comparison of Four Enzyme Immunoassays With a Western Blot Assay for the Determination of Type-Specific Antibodies to Herpes Simplex Virus

2001 ◽  
Vol 115 (2) ◽  
pp. 272-277 ◽  
Author(s):  
Thomas B. Martins ◽  
Ryan D. Woolstenhulme ◽  
Troy D. Jaskowski ◽  
Harry R. Hill ◽  
Christine M. Litwin
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Western Blot ◽  
Western Blot Assay ◽  
Specific Antibodies ◽  
Enzyme Immunoassays ◽  
Simplex Virus

scholarly journals Gastrodia elata powder capsule enhances anti-epileptic effect of carbamazepine by decreasing P-gp expression

2021 ◽  
Vol 18 (9) ◽  
pp. 1859-1865
Author(s):  
Xiangji Dang ◽  
Pei Zhao ◽  
Yan Liu ◽  
Long Qin ◽  
Haisheng Jiao
Keyword(s):  
Drug Resistance ◽  
Western Blot ◽  
Reverse Transcription ◽  
Normal Saline ◽  
Control Group ◽  
Behavioral Characteristics ◽  
Gastrodia Elata ◽  
Reverse Transcription Pcr ◽  
Protein Expressions

Purpose: To investigate the influence of Gastrodia elata powder capsule (GC) or gastrodin (GTD) on the anti-epileptic effect of carbamazepine (CBZ) on penicillin (PG)-induced epilepsy in rats. Methods: A total 116 rats were used in this study. Rats in the control group (n = 8) were injected with normal saline (NS) in place PG. Epilepsy was induced in the remaining 108 rats on the first day via PG injection. The rats were then divided randomly into six groups (18 rats per group): PG group, CBZ group, CBZ + GC group, CBZ + GTD group, GC group, and GTD group, which were given (p.o.) NS, CBZ (100 mg/kg), CBZ (100 mg/kg.) + GC (350 mg/kg), CBZ (100 mg/kg) + GTD (100 mg/kg), GC (350 mg/kg), and GTD (100 mg/kg), respectively, once a day for 15 days. The behavioral characteristics of the rats were observed and used to assess the anti-epileptic effect of the test drugs. Real-time quantitative reverse transcription-PCR and Western blot assays were employed for the determination of the effect of CBZ, GC and GTD on the expression levels of P-gp. Results: CBZ significantly reduced the symptoms of epilepsy, while GC and GTD enhanced the antiepileptic effect of CBZ, and reversed the CBZ-induced increases in the protein expressions of mrd1a and P-gp (p < 0.05). Conclusion: GC reverses CBZ drug resistance, probably through downregulation of P-gp expression. This finding indicates that GC is a potential anti-epilepsy drug, but it merits further studies.

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