glyceraldehyde 3 phosphate dehydrogenase gapdh Cell Signaling Technology Inc Search Results


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  • 99
    Cell Signaling Technology Inc anti bax
    Effect of the inhibition of p38 microtubule associated protein kinase/extracellular signal-regulated kinases (MAPK/ERK) signalling on the protective effects of dexmedetomidine (DEX) in an  in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) injury model. (a) Western blot analysis of p38 MAPK/ERK signalling proteins in OGD/R N2A cells, OGD/R N2A cells treated with DEX 500 ng/ml, and OGD/R N2A cells co-treated with DEX 500 ng/ml and the p38 MAPK/ERK signalling inhibitor CV-65 20 μM. (b) Quantitative analysis of protein levels shown in Figure 4a ( n  = 3). (c) Western blot analysis showing levels of Bax, Bcl-2 and cleaved caspase-3 in cell groups. (d) Quantitative analysis of protein levels shown in Figure 4c ( n  = 3). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the internal control. (e) Cell growth curves of cell groups measured using a Cell Counting Kit-8 assay ( n  = 3). (f) Early apoptotic cell counts of cell groups analysed with flow cytometry by measuring the percentage of Annexin V-stained cells ( n  = 3). Data are presented as mean ± SD. *** P
    Anti Bax, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 4268 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti bax
    Western blots for (A) HIF-1α, (B) BNIP3, (C) Beclin1, (D) LC3-II/LC3-I, (E) p62, (F) Bcl2, (G) <t>Bax</t> and <t>GAPDH.</t> The intensity of the protein blots was normalized with GAPDH. The data are expressed as the mean ± SEM. Optical density (%),
    Rabbit Anti Bax, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 995 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti bax antibody
    Analysis of <t>Beclin</t> 1 protein interactions and Bcl2 and <t>Bax</t> protein expression on 793 cells starved with and without anti-EGF antibody Western blot in ( A ), shows that: phospho-EGFR strongly binds Beclin 1 on 793 cells starved for 4 hrs; p-Bcl2 bind Beclin 1 up to 4 hrs of starvation and then detach; p-JNK and p-Bcl2 protein expressions increase at 4 and 8 hrs of starvation, respectively; Bcl2 and Bax were attached until 8 hrs of starvation and, afterwards, they were detached. ( B ) 793 cells starved and treated with anti-EGF antibody showed that: Beclin 1 and p-EGFR were detached; Beclin 1 and p-Bcl2 stayed attached during starvation; p-JNK and p-Bcl-2 expression levels were constantly down-regulated; p-Bcl2 and Bax were constantly attached. Time of exposure of p-Bcl2, p-EGFR, p-JNK, Beclin-1, Bax and tubulin were 7.5, 2, 15, 9.6, 5 and 4.3 s, respectively.
    Anti Bax Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 381 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc gapdh
    Deletion of <t>Ezh2</t> driven by Math5-Cre in mKO mice. (A) PCR genotyping of Ezh2 and Cre genes. (B) Representative result of Western blot of Ezh2 expression in RGCs purified from P0 WT and mKO mice. <t>GAPDH</t> was used as a loading control. A strongly reduced level of Ezh2 was found in mKO RGCs as compared to WT RGCs. ( C) Epifluorescence images of retinal sections taken from P0 WT and mKO mice that were immunolabeled for H3K27me3 (red) and nuclear marker 4’,6-Diamidino-2-Phenylindole (DAPI; blue). Note the higher level of H3K27me3 signals in the mKO retina compared to WT retina. Scale bar: 50 μm.
    Gapdh, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 30834 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti gapdh
    Proteasome inhibitors induce apoptosis in <t>HIV-infected</t> T cells, but not uninfected T cells, that depends upon procaspase 8, which is cleavable by HIV protease. (A) Chronically HIV-infected J-Lat 10.6 cells and uninfected parental Jurkat cells were treated with DMSO, bortezomib, or ixazomib and monitored for apoptosis by intracellular expression of active caspase 3. (B) Polled data depicting means (±SD) of the results of four independent experiments, as shown in panel A. (C) J-Lat 10.6 cells treated with DMSO, bortezomib, or ixazomib were analyzed by immunoblotting for HIV p24, cleaved caspase 3, cleaved PARP, and <t>GAPDH.</t> (D) Caspase 8-deficient JB-6 cells were transduced with VSV-G-pseudotyped HIV and then transfected with EGFP, EGFP-caspase 8, or EGFP-caspase8RN. Caspase 8 expression was confirmed by Western blotting (with a mutant of caspase 8 that is not cleaved by HIV protease and therefore does not generate Casp8p41). (E) HIV-infected JB-6 cells as in panel D were treated with ixazomib or control, and cell death was determined by active caspase 3 in caspase 8-expressing (GFP + ) and HIV-infected (p24 + ) cells. (F) Pooled data from three replicates of the experiment shown in panel E showing mean and SD. (G) Active caspase 3 expression was also compared in the subset of HIV p24-bright cells (boxed in panel E). *, P
    Anti Gapdh, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 9865 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cell Signaling Technology Inc rabbit polyclonal anti bax
    Western blot analysis of <t>BAX</t> in CC of COC cultured without the addition of drugs (control group, C), treated with GnRHa (A), phosphoramide mustard (P), and GnRHa+phosphoramide mustard (A+P). The rabbit <t>polyclonal</t> anti-BAX antibody was diluted 1:1000.
    Rabbit Polyclonal Anti Bax, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 149 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti glyceraldehyde 3 phosphate dehydrogenase gapdh
    Western blot analysis of <t>BAX</t> in CC of COC cultured without the addition of drugs (control group, C), treated with GnRHa (A), phosphoramide mustard (P), and GnRHa+phosphoramide mustard (A+P). The rabbit <t>polyclonal</t> anti-BAX antibody was diluted 1:1000.
    Rabbit Anti Glyceraldehyde 3 Phosphate Dehydrogenase Gapdh, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 189 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti glyceraldehyde 3 phosphate dehydrogenase
    Western blot analysis of <t>BAX</t> in CC of COC cultured without the addition of drugs (control group, C), treated with GnRHa (A), phosphoramide mustard (P), and GnRHa+phosphoramide mustard (A+P). The rabbit <t>polyclonal</t> anti-BAX antibody was diluted 1:1000.
    Rabbit Anti Glyceraldehyde 3 Phosphate Dehydrogenase, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti gapdh
    Cellular activity of <t>MLKL</t> bearing second brace helix mutations parallels behaviour of recombinant proteins. a Mlkl -/- MDFs reconstituted with wild-type MLKL were stimulated with TNF (T), Smac-mimetic (S) and Q-VD-OPh (Q) or emricasan/IDN-6556 (I), and cell death was quantified using propidium iodide (PI) staining and flow cytometry. b Western blot of Blue-Native PAGE of wild-type MDFs treated ± TSQ to measure localisation to cytoplasmic (C) or membrane (M) fractions and oligomerisation state. VDAC1 and <t>GAPDH</t> western blot reprobes were used to assess quality of fractionation. c Full-length MLKL constructs containing indicated alanine mutations were stably incorporated into MDFs via lentiviral infection, and protein expression induced by doxycycline (dox) treatment. Cells were either left untreated (UT), or treated with TSQ or TSI to induce necroptosis 3 h post dox induction. Cell death was measured using PI staining and flow cytometry 24 h after death stimulation. Responses to an apoptotic stimulus (TS), and death in the presence or absence of dox-induction, are shown in entirety in Supplementary Figure 2. d Western blot of Blue-Native PAGE on cell fractions was used to monitor the formation of MLKL higher-order species and determine cellular localisation in untreated or TSQ stimulated Mlkl -/- MDFs, expressing alanine mutant constructs. C; cytoplasmic fraction, M; membrane fraction. Western blots for VDAC1 (membrane associated) and GAPDH (cytoplasmic protein) were used to assess the quality of fractionation. BN-PAGE blots are representative of 2 independent experiments. Cell death data are the mean ± s.e.m. of three independent experiments with 2–3 biologically independent replicates per experiment ( n = 6–9). e ]. Mutation of residues in green (N163, K164 and K167, Q168) does not preclude oligomerisation, while those in red (E161, I162 and T165, L166) block oligomerisation. f Zoomed depiction of second brace helix structure in e . Hydrogen bonds between the second brace helix (E161 and T165) and C-lobe of the pseudokinase domain (T291 and S293) are indicated in black dashed lines with interatomic distances annotated. g Atoms from the first brace helix that form hydrophobic packing interactions with I162 and L166 from the second brace helix are coloured pink and shown in a surface representation
    Rabbit Anti Gapdh, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 3270 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc glyceraldehydes 3 phosphate dehydrogenase gapdh
    Cellular activity of <t>MLKL</t> bearing second brace helix mutations parallels behaviour of recombinant proteins. a Mlkl -/- MDFs reconstituted with wild-type MLKL were stimulated with TNF (T), Smac-mimetic (S) and Q-VD-OPh (Q) or emricasan/IDN-6556 (I), and cell death was quantified using propidium iodide (PI) staining and flow cytometry. b Western blot of Blue-Native PAGE of wild-type MDFs treated ± TSQ to measure localisation to cytoplasmic (C) or membrane (M) fractions and oligomerisation state. VDAC1 and <t>GAPDH</t> western blot reprobes were used to assess quality of fractionation. c Full-length MLKL constructs containing indicated alanine mutations were stably incorporated into MDFs via lentiviral infection, and protein expression induced by doxycycline (dox) treatment. Cells were either left untreated (UT), or treated with TSQ or TSI to induce necroptosis 3 h post dox induction. Cell death was measured using PI staining and flow cytometry 24 h after death stimulation. Responses to an apoptotic stimulus (TS), and death in the presence or absence of dox-induction, are shown in entirety in Supplementary Figure 2. d Western blot of Blue-Native PAGE on cell fractions was used to monitor the formation of MLKL higher-order species and determine cellular localisation in untreated or TSQ stimulated Mlkl -/- MDFs, expressing alanine mutant constructs. C; cytoplasmic fraction, M; membrane fraction. Western blots for VDAC1 (membrane associated) and GAPDH (cytoplasmic protein) were used to assess the quality of fractionation. BN-PAGE blots are representative of 2 independent experiments. Cell death data are the mean ± s.e.m. of three independent experiments with 2–3 biologically independent replicates per experiment ( n = 6–9). e ]. Mutation of residues in green (N163, K164 and K167, Q168) does not preclude oligomerisation, while those in red (E161, I162 and T165, L166) block oligomerisation. f Zoomed depiction of second brace helix structure in e . Hydrogen bonds between the second brace helix (E161 and T165) and C-lobe of the pseudokinase domain (T291 and S293) are indicated in black dashed lines with interatomic distances annotated. g Atoms from the first brace helix that form hydrophobic packing interactions with I162 and L166 from the second brace helix are coloured pink and shown in a surface representation
    Glyceraldehydes 3 Phosphate Dehydrogenase Gapdh, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effect of the inhibition of p38 microtubule associated protein kinase/extracellular signal-regulated kinases (MAPK/ERK) signalling on the protective effects of dexmedetomidine (DEX) in an  in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) injury model. (a) Western blot analysis of p38 MAPK/ERK signalling proteins in OGD/R N2A cells, OGD/R N2A cells treated with DEX 500 ng/ml, and OGD/R N2A cells co-treated with DEX 500 ng/ml and the p38 MAPK/ERK signalling inhibitor CV-65 20 μM. (b) Quantitative analysis of protein levels shown in Figure 4a ( n  = 3). (c) Western blot analysis showing levels of Bax, Bcl-2 and cleaved caspase-3 in cell groups. (d) Quantitative analysis of protein levels shown in Figure 4c ( n  = 3). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the internal control. (e) Cell growth curves of cell groups measured using a Cell Counting Kit-8 assay ( n  = 3). (f) Early apoptotic cell counts of cell groups analysed with flow cytometry by measuring the percentage of Annexin V-stained cells ( n  = 3). Data are presented as mean ± SD. *** P

    Journal: The Journal of International Medical Research

    Article Title: Dexmedetomidine protects against oxygen-glucose deprivation/reoxygenation injury-induced apoptosis via the p38 MAPK/ERK signalling pathway

    doi: 10.1177/0300060517734460

    Figure Lengend Snippet: Effect of the inhibition of p38 microtubule associated protein kinase/extracellular signal-regulated kinases (MAPK/ERK) signalling on the protective effects of dexmedetomidine (DEX) in an in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) injury model. (a) Western blot analysis of p38 MAPK/ERK signalling proteins in OGD/R N2A cells, OGD/R N2A cells treated with DEX 500 ng/ml, and OGD/R N2A cells co-treated with DEX 500 ng/ml and the p38 MAPK/ERK signalling inhibitor CV-65 20 μM. (b) Quantitative analysis of protein levels shown in Figure 4a ( n  = 3). (c) Western blot analysis showing levels of Bax, Bcl-2 and cleaved caspase-3 in cell groups. (d) Quantitative analysis of protein levels shown in Figure 4c ( n  = 3). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the internal control. (e) Cell growth curves of cell groups measured using a Cell Counting Kit-8 assay ( n  = 3). (f) Early apoptotic cell counts of cell groups analysed with flow cytometry by measuring the percentage of Annexin V-stained cells ( n  = 3). Data are presented as mean ± SD. *** P

    Article Snippet: After blocking with 5% fat-free milk for 2 h at room temperature, the membranes were incubated with primary antibodies against p-p38 MAPK (1:500 dilution), p38 MAPK (1:500 dilution), p-ERK1/2 (1:500 dilution), ERK1/2 (1:500 dilution), Bax (1:500 dilution), Bcl-2 (1:500 dilution), caspase-3 (1:500 dilution) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:2000 dilution) (Cell Signaling Technology®, Danvers, MA, USA) at 4 °C overnight.

    Techniques: Inhibition, In Vitro, Western Blot, Cell Counting, Flow Cytometry, Cytometry, Staining

    Effect of dexmedetomidine (DEX) on p38 microtubule associated protein kinase/extracellular signal-regulated kinases (MAPK/ERK) signalling and apoptosis-related molecules in an  in vitro  oxygen-glucose deprivation/reoxygenation (OGD/R) injury model. (a) Western blot analysis of p38 MAPK/ERK signalling proteins in cell groups. (b) Quantitative analysis of protein levels shown in Figure 3a ( n  = 3). (c) Western blot analysis showing levels of Bax, Bcl-2 and cleaved caspase-3 in cell groups. (d) Quantitative analysis of protein levels shown in Figure 3c ( n  = 3). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the internal control. Data are presented as mean ± SD. *** P

    Journal: The Journal of International Medical Research

    Article Title: Dexmedetomidine protects against oxygen-glucose deprivation/reoxygenation injury-induced apoptosis via the p38 MAPK/ERK signalling pathway

    doi: 10.1177/0300060517734460

    Figure Lengend Snippet: Effect of dexmedetomidine (DEX) on p38 microtubule associated protein kinase/extracellular signal-regulated kinases (MAPK/ERK) signalling and apoptosis-related molecules in an in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) injury model. (a) Western blot analysis of p38 MAPK/ERK signalling proteins in cell groups. (b) Quantitative analysis of protein levels shown in Figure 3a ( n  = 3). (c) Western blot analysis showing levels of Bax, Bcl-2 and cleaved caspase-3 in cell groups. (d) Quantitative analysis of protein levels shown in Figure 3c ( n  = 3). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the internal control. Data are presented as mean ± SD. *** P

    Article Snippet: After blocking with 5% fat-free milk for 2 h at room temperature, the membranes were incubated with primary antibodies against p-p38 MAPK (1:500 dilution), p38 MAPK (1:500 dilution), p-ERK1/2 (1:500 dilution), ERK1/2 (1:500 dilution), Bax (1:500 dilution), Bcl-2 (1:500 dilution), caspase-3 (1:500 dilution) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:2000 dilution) (Cell Signaling Technology®, Danvers, MA, USA) at 4 °C overnight.

    Techniques: In Vitro, Western Blot

    Enrichment of HA-KCNQ3/KCNQ2 channels and CD4-Q2C at the axonal surface. Schematic drawings (not to scale) of a human KCNQ2 subunit (accession #Y15065), including the subunit interaction domain (Sid, amino acids 580–623) [52] , [53] , and CaM-binding domain (amino acids 323–579) [22] , [23] . (B) Immunoblot analysis of CaM in cultured rat hippocampal neurons at 5–9 days in vitro (DIV). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a loading control. (C) Schematic drawings (not to scale) of a human CD4 protein, and CD4 fused to KCNQ2 C-terminal tail (CD4-Q2C, [18] ). (D) Surface immunostaining of hippocampal neurons (DIV 7) transfected with KCNQ2 and KCNQ3 containing an extracellular hemagglutinin (HA) epitope (HA-KCNQ3). Neuronal soma and dendrites were visualized by immunostaining for MAP2. Surface HA-KCNQ3/KCNQ2 channels are enriched on a MAP2-negative neurite that originates directly from the soma. (E) Pseudo-color image of the inset in Fig. 1D displays differences in the surface HA intensity. Surface HA-KCNQ3/KCNQ2 channels are enriched at the initial segment of an axon. (F) Surface immunostaining of hippocampal neurons (DIV 8) transfected with CD4, or CD4-Q2C. (G) Pseudo-color images of the insets in Fig. 1F display differences in the surface CD4 intensity. Fusion of KCNQ2 C-terminal tail enriches CD4 at the axonal surface. Camera lucida drawings of the neuronal images (D, F) show the soma and dendrites (gray) and an axon (black). Arrows mark the main axon. Scale bars are 20 µm.

    Journal: PLoS ONE

    Article Title: Polarized Axonal Surface Expression of Neuronal KCNQ Potassium Channels Is Regulated by Calmodulin Interaction with KCNQ2 Subunit

    doi: 10.1371/journal.pone.0103655

    Figure Lengend Snippet: Enrichment of HA-KCNQ3/KCNQ2 channels and CD4-Q2C at the axonal surface. Schematic drawings (not to scale) of a human KCNQ2 subunit (accession #Y15065), including the subunit interaction domain (Sid, amino acids 580–623) [52] , [53] , and CaM-binding domain (amino acids 323–579) [22] , [23] . (B) Immunoblot analysis of CaM in cultured rat hippocampal neurons at 5–9 days in vitro (DIV). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a loading control. (C) Schematic drawings (not to scale) of a human CD4 protein, and CD4 fused to KCNQ2 C-terminal tail (CD4-Q2C, [18] ). (D) Surface immunostaining of hippocampal neurons (DIV 7) transfected with KCNQ2 and KCNQ3 containing an extracellular hemagglutinin (HA) epitope (HA-KCNQ3). Neuronal soma and dendrites were visualized by immunostaining for MAP2. Surface HA-KCNQ3/KCNQ2 channels are enriched on a MAP2-negative neurite that originates directly from the soma. (E) Pseudo-color image of the inset in Fig. 1D displays differences in the surface HA intensity. Surface HA-KCNQ3/KCNQ2 channels are enriched at the initial segment of an axon. (F) Surface immunostaining of hippocampal neurons (DIV 8) transfected with CD4, or CD4-Q2C. (G) Pseudo-color images of the insets in Fig. 1F display differences in the surface CD4 intensity. Fusion of KCNQ2 C-terminal tail enriches CD4 at the axonal surface. Camera lucida drawings of the neuronal images (D, F) show the soma and dendrites (gray) and an axon (black). Arrows mark the main axon. Scale bars are 20 µm.

    Article Snippet: Reagents used in immunoprecipitation and western blotting include human embryonic kidney (HEK) 293T cell lines (ATCC, CRL-11268) and antibodies including anti-CD4 (Santa Cruz Biotech, sc-13573), anti-HA (Covance, MMS-101P, Cell Signaling, 3724), anti-KCNQ2 (Alamone, APC-050; Neuromab, N26A/23), anti-CaM (Novus Biologicals, NB110-55649; Santa Cruz, SC137079; Cell Signaling, 4830), anti-HA, anti-β-actin, and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (Cell Signaling, 3724, 4967, and 2118).

    Techniques: Chick Chorioallantoic Membrane Assay, Binding Assay, Cell Culture, In Vitro, Immunostaining, Transfection

    Anesthesia with 2.5% sevoflurane for 2 hours in pregnant mice on the 15th day of gestation induced caspase-3 activation in the cochlear tissues of fetal mice. Notes: Representative Western blot images of caspase-3 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in fetal tissue samples from the sevoflurane and control groups ( A ). Semiquantification of the Western blot showed that compared with the control conditions, sevoflurane anesthesia was associated with greater quantities of caspase-3 in fetal mice cochlear tissues ( B ). Expression of Bcl-2 in fetal cochlear tissues was higher in the sevoflurane group ( C and D ). *** P

    Journal: Drug Design, Development and Therapy

    Article Title: Sevoflurane anesthesia during pregnancy in mice induces hearing impairment in the offspring

    doi: 10.2147/DDDT.S156040

    Figure Lengend Snippet: Anesthesia with 2.5% sevoflurane for 2 hours in pregnant mice on the 15th day of gestation induced caspase-3 activation in the cochlear tissues of fetal mice. Notes: Representative Western blot images of caspase-3 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in fetal tissue samples from the sevoflurane and control groups ( A ). Semiquantification of the Western blot showed that compared with the control conditions, sevoflurane anesthesia was associated with greater quantities of caspase-3 in fetal mice cochlear tissues ( B ). Expression of Bcl-2 in fetal cochlear tissues was higher in the sevoflurane group ( C and D ). *** P

    Article Snippet: Caspase-3 antibody (1:1,000, CY3457; Abways Technology, Beijing, People’s Republic of China), Bcl-2 (1:1,000, ab32124; Abcam, Cambridge, MA, USA), COX-2 (1:1,000, ab15191; Abcam), iNOS (1:1,000, ab15323; Abcam), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (1:1,000, rabbit polyclonal; Cell Signaling Technology, Beverly, MA, USA), and β-actin (1:1,000, ab8226; Abcam) were the primary antibodies.

    Techniques: Mouse Assay, Activation Assay, Western Blot, Expressing

    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

    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

    doi: 10.1155/2017/8325754

    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: ERK1/2 and SAPK/JNK antibodies were analyzed by blotting their respective phosphorylated and total fractions; all Western blots were blotted a second time using a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (rabbit, 37 kDa, Cell Signaling Technology, USA) in order to achieve a reliable loading control.

    Techniques: Western Blot, Activity Assay, Activation Assay

    RASSF9 siRNAs promote the proliferation of human GC cells. a The results showed the knockdown efficiency of RASSF9 siRNA-1/2 in AGS/MKN-45 cells at 24 h after transfection. b MTT assay showed that RASSF9 siRNA-1/2 increased the activity of AGS/MKN-45 cells at 48 and 72 h. c Cell counting assay revealed that RASSF9 siRNA-1/2 promoted AGS/MKN-45 cell proliferation at 48 and 72 h. d Flow cytometric analysis showed the percentage of AGS/MKN-45 cells in the G0/G1, S and G2/M phases. The proportion of G0/G1 phase cells decreased after RASSF9 siRNA-1/2 transfection, and the proportion of S and G2/M phase cells increased. e The data showed the percentage of early and late apoptosis after RASSF9 siRNA-1/2 transfection. f RASSF9, p-AKT, Cyclin D1, CDK2, Bcl-2 and Bax protein expression levels were measured 48 h after RASSF9 siRNA-1/2 transfection. Wilcoxon test, *p

    Journal: Cancer Cell International

    Article Title: MicroRNA-1269 promotes cell proliferation via the AKT signaling pathway by targeting RASSF9 in human gastric cancer

    doi: 10.1186/s12935-019-1026-4

    Figure Lengend Snippet: RASSF9 siRNAs promote the proliferation of human GC cells. a The results showed the knockdown efficiency of RASSF9 siRNA-1/2 in AGS/MKN-45 cells at 24 h after transfection. b MTT assay showed that RASSF9 siRNA-1/2 increased the activity of AGS/MKN-45 cells at 48 and 72 h. c Cell counting assay revealed that RASSF9 siRNA-1/2 promoted AGS/MKN-45 cell proliferation at 48 and 72 h. d Flow cytometric analysis showed the percentage of AGS/MKN-45 cells in the G0/G1, S and G2/M phases. The proportion of G0/G1 phase cells decreased after RASSF9 siRNA-1/2 transfection, and the proportion of S and G2/M phase cells increased. e The data showed the percentage of early and late apoptosis after RASSF9 siRNA-1/2 transfection. f RASSF9, p-AKT, Cyclin D1, CDK2, Bcl-2 and Bax protein expression levels were measured 48 h after RASSF9 siRNA-1/2 transfection. Wilcoxon test, *p

    Article Snippet: The membranes were incubated at 4 °C with primary antibodies that included mouse monoclonal anti-RASSF9 (1:1000; Cell Signaling Technology, USA), mouse monoclonal anti-p-AKT (1:1000; Cell Signaling Technology, USA), rabbit monoclonal anti-AKT (1:1000; Cell Signaling Technology, USA), rabbit monoclonal anti-Cyclin D1 (1:1000; Cell Signaling Technology, USA), rabbit monoclonal anti-CDK2 (1:1000; Cell Signaling Technology, USA), rabbit monoclonal anti-Bcl-2 (1:2000; Cell Signaling Technology, USA), rabbit monoclonal anti-Bax (1:2000; Cell Signaling Technology, USA), and mouse monoclonal anti-GAPDH (1:5000; Santa Cruz Biotechnology, CA, USA).

    Techniques: Transfection, MTT Assay, Activity Assay, Cell Counting, Flow Cytometry, Expressing

    RASSF9 overexpression inhibits the proliferation of human GC cells. a RASSF9 overexpression promoted RASSF9 mRNA levels in AGS/MKN-45 cells. b MTT assay showed that RASSF9 overexpression decreased AGS/MKN-45 cell activity at 48 and 72 h. c Cell counting assay showed that RASSF9 overexpression inhibited AGS/MKN-45 cell growth. d The cell cycle was measured in AGS/MKN-45 cells at 48 h. e Apoptosis was examined in AGS/MKN-451 cells at 48 h. f RASSF9, p-AKT, AKT, Cyclin D1, CDK2, Bcl-2 and Bax expression levels were detected after transfection with the RASSF9 overexpression vector. Wilcoxon test, *p

    Journal: Cancer Cell International

    Article Title: MicroRNA-1269 promotes cell proliferation via the AKT signaling pathway by targeting RASSF9 in human gastric cancer

    doi: 10.1186/s12935-019-1026-4

    Figure Lengend Snippet: RASSF9 overexpression inhibits the proliferation of human GC cells. a RASSF9 overexpression promoted RASSF9 mRNA levels in AGS/MKN-45 cells. b MTT assay showed that RASSF9 overexpression decreased AGS/MKN-45 cell activity at 48 and 72 h. c Cell counting assay showed that RASSF9 overexpression inhibited AGS/MKN-45 cell growth. d The cell cycle was measured in AGS/MKN-45 cells at 48 h. e Apoptosis was examined in AGS/MKN-451 cells at 48 h. f RASSF9, p-AKT, AKT, Cyclin D1, CDK2, Bcl-2 and Bax expression levels were detected after transfection with the RASSF9 overexpression vector. Wilcoxon test, *p

    Article Snippet: The membranes were incubated at 4 °C with primary antibodies that included mouse monoclonal anti-RASSF9 (1:1000; Cell Signaling Technology, USA), mouse monoclonal anti-p-AKT (1:1000; Cell Signaling Technology, USA), rabbit monoclonal anti-AKT (1:1000; Cell Signaling Technology, USA), rabbit monoclonal anti-Cyclin D1 (1:1000; Cell Signaling Technology, USA), rabbit monoclonal anti-CDK2 (1:1000; Cell Signaling Technology, USA), rabbit monoclonal anti-Bcl-2 (1:2000; Cell Signaling Technology, USA), rabbit monoclonal anti-Bax (1:2000; Cell Signaling Technology, USA), and mouse monoclonal anti-GAPDH (1:5000; Santa Cruz Biotechnology, CA, USA).

    Techniques: Over Expression, MTT Assay, Activity Assay, Cell Counting, Expressing, Transfection, Plasmid Preparation

    miR-1269 regulates the AKT and Bcl-2/Bax signaling pathways in human GC cells by targeting RASSF9. a RASSF9 mRNA and protein expression levels were significantly decreased in GC cell lines (BGC-823, SGC-7901, MKN-45 and AGS) compared with a normal gastric epithelial cell line (GES-1). b RASSF9 mRNA was examined in AGS/MKN-45 cells after miR-1269 overexpression. c RASSF9 mRNA was determined in AGS/MKN-45 cells after anti-miR-1269 treatment. d miR-1269 overexpression promoted the expression of the p-AKT, Cyclin D1, CDK2 and Bcl-2 proteins and inhibited RASSF9 and Bax expression in AGS/MKN-45 cells. e Anti-miR-1269 inhibited p-AKT, Cyclin D1, CDK2 and Bcl-2 protein expression and increased RASSF9 and Bax expression. Wilcoxon test, *p

    Journal: Cancer Cell International

    Article Title: MicroRNA-1269 promotes cell proliferation via the AKT signaling pathway by targeting RASSF9 in human gastric cancer

    doi: 10.1186/s12935-019-1026-4

    Figure Lengend Snippet: miR-1269 regulates the AKT and Bcl-2/Bax signaling pathways in human GC cells by targeting RASSF9. a RASSF9 mRNA and protein expression levels were significantly decreased in GC cell lines (BGC-823, SGC-7901, MKN-45 and AGS) compared with a normal gastric epithelial cell line (GES-1). b RASSF9 mRNA was examined in AGS/MKN-45 cells after miR-1269 overexpression. c RASSF9 mRNA was determined in AGS/MKN-45 cells after anti-miR-1269 treatment. d miR-1269 overexpression promoted the expression of the p-AKT, Cyclin D1, CDK2 and Bcl-2 proteins and inhibited RASSF9 and Bax expression in AGS/MKN-45 cells. e Anti-miR-1269 inhibited p-AKT, Cyclin D1, CDK2 and Bcl-2 protein expression and increased RASSF9 and Bax expression. Wilcoxon test, *p

    Article Snippet: The membranes were incubated at 4 °C with primary antibodies that included mouse monoclonal anti-RASSF9 (1:1000; Cell Signaling Technology, USA), mouse monoclonal anti-p-AKT (1:1000; Cell Signaling Technology, USA), rabbit monoclonal anti-AKT (1:1000; Cell Signaling Technology, USA), rabbit monoclonal anti-Cyclin D1 (1:1000; Cell Signaling Technology, USA), rabbit monoclonal anti-CDK2 (1:1000; Cell Signaling Technology, USA), rabbit monoclonal anti-Bcl-2 (1:2000; Cell Signaling Technology, USA), rabbit monoclonal anti-Bax (1:2000; Cell Signaling Technology, USA), and mouse monoclonal anti-GAPDH (1:5000; Santa Cruz Biotechnology, CA, USA).

    Techniques: Expressing, Over Expression

    Blue honeysuckle extract (BHE) improved hepatic antioxidant capacity. (A) The level of thiobarbituric acid reactive substances (TBARS) in liver. (B) The representative blots of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), heme oxygenase-1 (HO-1), and manganese-dependent superoxide dismutase (MnSOD) protein in liver by Western blotting. The induction folds of the proteins were calculated as the intensity of the treatment relative to that of control normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by densitometry. Data represent means ± SD of 4 mice, and the blots are representatives of 4 samples. Bars with different letters differ significantly ( P

    Journal: Animal Nutrition

    Article Title: Inhibitory effect of blue honeysuckle extract on high-fat-diet-induced fatty liver in mice

    doi: 10.1016/j.aninu.2018.06.001

    Figure Lengend Snippet: Blue honeysuckle extract (BHE) improved hepatic antioxidant capacity. (A) The level of thiobarbituric acid reactive substances (TBARS) in liver. (B) The representative blots of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), heme oxygenase-1 (HO-1), and manganese-dependent superoxide dismutase (MnSOD) protein in liver by Western blotting. The induction folds of the proteins were calculated as the intensity of the treatment relative to that of control normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by densitometry. Data represent means ± SD of 4 mice, and the blots are representatives of 4 samples. Bars with different letters differ significantly ( P

    Article Snippet: Antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and corresponding secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Derivative Assay, Western Blot, Mouse Assay

    Differences in the expression levels of vinculin and integrin-linked kinase-1 (ILK-1) between the MCF-7 and MCR-7/ADR cells. (A) Representative western blotting images of vinculin and ILK-1 proteins. (B) Quantitative evaluation of vinculin and ILK-1 expression levels in the MCF-7/ADR cells normalized to those in the MCF-7 cells. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. * P

    Journal: Journal of Cancer Prevention

    Article Title: Mechanical Alteration Associated With Chemotherapeutic Resistance of Breast Cancer Cells

    doi: 10.15430/JCP.2018.23.2.87

    Figure Lengend Snippet: Differences in the expression levels of vinculin and integrin-linked kinase-1 (ILK-1) between the MCF-7 and MCR-7/ADR cells. (A) Representative western blotting images of vinculin and ILK-1 proteins. (B) Quantitative evaluation of vinculin and ILK-1 expression levels in the MCF-7/ADR cells normalized to those in the MCF-7 cells. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. * P

    Article Snippet: The blots were detected with anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH), anti-vinculin, and anti-ILK-1 primary antibodies bought from Cell Signaling Technology (Danvers, MA, USA) or Abcam (Cambridge, UK).

    Techniques: Expressing, Western Blot

    Western blots for (A) HIF-1α, (B) BNIP3, (C) Beclin1, (D) LC3-II/LC3-I, (E) p62, (F) Bcl2, (G) Bax and GAPDH. The intensity of the protein blots was normalized with GAPDH. The data are expressed as the mean ± SEM. Optical density (%),

    Journal: Molecular Medicine Reports

    Article Title: Autophagy may play an important role in varicocele

    doi: 10.3892/mmr.2017.7253

    Figure Lengend Snippet: Western blots for (A) HIF-1α, (B) BNIP3, (C) Beclin1, (D) LC3-II/LC3-I, (E) p62, (F) Bcl2, (G) Bax and GAPDH. The intensity of the protein blots was normalized with GAPDH. The data are expressed as the mean ± SEM. Optical density (%),

    Article Snippet: Rabbit anti Bax (CST 14796) and rabbit anti GAPDH, AB-P-R 001, were provided by Hangzhou Goodhere Biotechnology Co. (LTD, Hangzhou, China).

    Techniques: Western Blot

    Western blots for HIF-1α, Beclin1, p62, Bcl2, Bax, BNIP3, LC3-II, LC3-I, and GAPDH. HIF-1α, hypoxia-inducible factor 1α; Beclin1, BCL2 interacting protein.

    Journal: Molecular Medicine Reports

    Article Title: Autophagy may play an important role in varicocele

    doi: 10.3892/mmr.2017.7253

    Figure Lengend Snippet: Western blots for HIF-1α, Beclin1, p62, Bcl2, Bax, BNIP3, LC3-II, LC3-I, and GAPDH. HIF-1α, hypoxia-inducible factor 1α; Beclin1, BCL2 interacting protein.

    Article Snippet: Rabbit anti Bax (CST 14796) and rabbit anti GAPDH, AB-P-R 001, were provided by Hangzhou Goodhere Biotechnology Co. (LTD, Hangzhou, China).

    Techniques: Western Blot

    Analysis of Beclin 1 protein interactions and Bcl2 and Bax protein expression on 793 cells starved with and without anti-EGF antibody Western blot in ( A ), shows that: phospho-EGFR strongly binds Beclin 1 on 793 cells starved for 4 hrs; p-Bcl2 bind Beclin 1 up to 4 hrs of starvation and then detach; p-JNK and p-Bcl2 protein expressions increase at 4 and 8 hrs of starvation, respectively; Bcl2 and Bax were attached until 8 hrs of starvation and, afterwards, they were detached. ( B ) 793 cells starved and treated with anti-EGF antibody showed that: Beclin 1 and p-EGFR were detached; Beclin 1 and p-Bcl2 stayed attached during starvation; p-JNK and p-Bcl-2 expression levels were constantly down-regulated; p-Bcl2 and Bax were constantly attached. Time of exposure of p-Bcl2, p-EGFR, p-JNK, Beclin-1, Bax and tubulin were 7.5, 2, 15, 9.6, 5 and 4.3 s, respectively.

    Journal: Oncotarget

    Article Title: Autophagy processes are dependent on EGF receptor signaling

    doi: 10.18632/oncotarget.25708

    Figure Lengend Snippet: Analysis of Beclin 1 protein interactions and Bcl2 and Bax protein expression on 793 cells starved with and without anti-EGF antibody Western blot in ( A ), shows that: phospho-EGFR strongly binds Beclin 1 on 793 cells starved for 4 hrs; p-Bcl2 bind Beclin 1 up to 4 hrs of starvation and then detach; p-JNK and p-Bcl2 protein expressions increase at 4 and 8 hrs of starvation, respectively; Bcl2 and Bax were attached until 8 hrs of starvation and, afterwards, they were detached. ( B ) 793 cells starved and treated with anti-EGF antibody showed that: Beclin 1 and p-EGFR were detached; Beclin 1 and p-Bcl2 stayed attached during starvation; p-JNK and p-Bcl-2 expression levels were constantly down-regulated; p-Bcl2 and Bax were constantly attached. Time of exposure of p-Bcl2, p-EGFR, p-JNK, Beclin-1, Bax and tubulin were 7.5, 2, 15, 9.6, 5 and 4.3 s, respectively.

    Article Snippet: For each sample, total cell lysate containing 120 μg protein was incubated with 5 μl of anti-Beclin or anti-Bax antibody (Cell Signaling Technologies, Danvers, MA, USA) in rotation overnight at 4° C. Then 30 μl of Protein G Agarose beads were added to the mix of protein lysate and anti-Beclin or anti-Bax antibody, and incubation was performed overnight at 4° C. The IP complex was washed 6 times with NP40 lysis buffer.

    Techniques: Expressing, Western Blot

    Deletion of Ezh2 driven by Math5-Cre in mKO mice. (A) PCR genotyping of Ezh2 and Cre genes. (B) Representative result of Western blot of Ezh2 expression in RGCs purified from P0 WT and mKO mice. GAPDH was used as a loading control. A strongly reduced level of Ezh2 was found in mKO RGCs as compared to WT RGCs. ( C) Epifluorescence images of retinal sections taken from P0 WT and mKO mice that were immunolabeled for H3K27me3 (red) and nuclear marker 4’,6-Diamidino-2-Phenylindole (DAPI; blue). Note the higher level of H3K27me3 signals in the mKO retina compared to WT retina. Scale bar: 50 μm.

    Journal: PLoS ONE

    Article Title: Ezh2 does not mediate retinal ganglion cell homeostasis or their susceptibility to injury

    doi: 10.1371/journal.pone.0191853

    Figure Lengend Snippet: Deletion of Ezh2 driven by Math5-Cre in mKO mice. (A) PCR genotyping of Ezh2 and Cre genes. (B) Representative result of Western blot of Ezh2 expression in RGCs purified from P0 WT and mKO mice. GAPDH was used as a loading control. A strongly reduced level of Ezh2 was found in mKO RGCs as compared to WT RGCs. ( C) Epifluorescence images of retinal sections taken from P0 WT and mKO mice that were immunolabeled for H3K27me3 (red) and nuclear marker 4’,6-Diamidino-2-Phenylindole (DAPI; blue). Note the higher level of H3K27me3 signals in the mKO retina compared to WT retina. Scale bar: 50 μm.

    Article Snippet: The blots were incubated with primary antibodies against Ezh2 (1:200; Cat. No. 5246S; Cell Signaling Technology, Danvers, MA) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase (1:1000; Cat. No. 3683S; Cell Signaling Technology) as a loading control in a solution containing 5% non-fat dry milk and 0.05% Tween-20 overnight.

    Techniques: Mouse Assay, Polymerase Chain Reaction, Western Blot, Expressing, Purification, Immunolabeling, Marker

    Proteasome inhibitors induce apoptosis in HIV-infected T cells, but not uninfected T cells, that depends upon procaspase 8, which is cleavable by HIV protease. (A) Chronically HIV-infected J-Lat 10.6 cells and uninfected parental Jurkat cells were treated with DMSO, bortezomib, or ixazomib and monitored for apoptosis by intracellular expression of active caspase 3. (B) Polled data depicting means (±SD) of the results of four independent experiments, as shown in panel A. (C) J-Lat 10.6 cells treated with DMSO, bortezomib, or ixazomib were analyzed by immunoblotting for HIV p24, cleaved caspase 3, cleaved PARP, and GAPDH. (D) Caspase 8-deficient JB-6 cells were transduced with VSV-G-pseudotyped HIV and then transfected with EGFP, EGFP-caspase 8, or EGFP-caspase8RN. Caspase 8 expression was confirmed by Western blotting (with a mutant of caspase 8 that is not cleaved by HIV protease and therefore does not generate Casp8p41). (E) HIV-infected JB-6 cells as in panel D were treated with ixazomib or control, and cell death was determined by active caspase 3 in caspase 8-expressing (GFP + ) and HIV-infected (p24 + ) cells. (F) Pooled data from three replicates of the experiment shown in panel E showing mean and SD. (G) Active caspase 3 expression was also compared in the subset of HIV p24-bright cells (boxed in panel E). *, P

    Journal: Journal of Virology

    Article Title: HIV Protease-Generated Casp8p41, When Bound and Inactivated by Bcl2, Is Degraded by the Proteasome

    doi: 10.1128/JVI.00037-18

    Figure Lengend Snippet: Proteasome inhibitors induce apoptosis in HIV-infected T cells, but not uninfected T cells, that depends upon procaspase 8, which is cleavable by HIV protease. (A) Chronically HIV-infected J-Lat 10.6 cells and uninfected parental Jurkat cells were treated with DMSO, bortezomib, or ixazomib and monitored for apoptosis by intracellular expression of active caspase 3. (B) Polled data depicting means (±SD) of the results of four independent experiments, as shown in panel A. (C) J-Lat 10.6 cells treated with DMSO, bortezomib, or ixazomib were analyzed by immunoblotting for HIV p24, cleaved caspase 3, cleaved PARP, and GAPDH. (D) Caspase 8-deficient JB-6 cells were transduced with VSV-G-pseudotyped HIV and then transfected with EGFP, EGFP-caspase 8, or EGFP-caspase8RN. Caspase 8 expression was confirmed by Western blotting (with a mutant of caspase 8 that is not cleaved by HIV protease and therefore does not generate Casp8p41). (E) HIV-infected JB-6 cells as in panel D were treated with ixazomib or control, and cell death was determined by active caspase 3 in caspase 8-expressing (GFP + ) and HIV-infected (p24 + ) cells. (F) Pooled data from three replicates of the experiment shown in panel E showing mean and SD. (G) Active caspase 3 expression was also compared in the subset of HIV p24-bright cells (boxed in panel E). *, P

    Article Snippet: The following antibodies were used for immunoblotting or immunoprecipitation: anti-HA peroxidase (3F10) rat monoclonal antibody (catalog number 12013819001; Roche, Indianapolis, IN), mouse anti-caspase 8 antibody (received from Marcus E. Peter, Northwestern University, Chicago, IL), mouse monoclonal anti-ubiquitin (P4D1) antibody (sc-8017; Santa Cruz), anti-Bcl-2 (C21) rabbit polyclonal IgG (catalog number sc-783; Santa Cruz), monoclonal anti-HIV p24 (catalog number 530; NIH AIDS Reagent Program), anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (14C10) rabbit MAb (catalog number 2118; Cell Signaling), histone deacetylase (HDAC) antibody sampler kit (catalog number 9928; Cell Signaling), anti-phospho-NF-κB-p65 (Ser 536) rabbit MAb (catalog number 3033; Cell Signaling), anti-NF-κBp65 (D14E12) rabbit MAb (catalog number 8242; Cell Signaling), anti-phospho-IκBα (Ser32) (14D4) rabbit MAb (catalog number 2859; Cell Signaling), anti-IκBα (L35A5) mouse MAb (amino-terminal antigen; catalog number 4814; Cell Signaling), anti-cleaved caspase 3 (Asp175) antibody (catalog number 9661; Cell Signaling), and mouse anti-human PARP (catalog number 556494; BD Pharmingen).

    Techniques: Infection, Expressing, Transduction, Transfection, Western Blot, Mutagenesis

    Western blot analysis of BAX in CC of COC cultured without the addition of drugs (control group, C), treated with GnRHa (A), phosphoramide mustard (P), and GnRHa+phosphoramide mustard (A+P). The rabbit polyclonal anti-BAX antibody was diluted 1:1000.

    Journal: International Journal of Molecular Sciences

    Article Title: Gonadotropin Releasing Hormone Agonists Have an Anti-apoptotic Effect on Cumulus Cells

    doi: 10.3390/ijms20236045

    Figure Lengend Snippet: Western blot analysis of BAX in CC of COC cultured without the addition of drugs (control group, C), treated with GnRHa (A), phosphoramide mustard (P), and GnRHa+phosphoramide mustard (A+P). The rabbit polyclonal anti-BAX antibody was diluted 1:1000.

    Article Snippet: Membranes were blocked with 1% no fat milk in PBS containing 0.05% Tween 20 for 1 h and probed with rabbit polyclonal anti-BAX (Cell Signaling Technology) antibody overnight at +4 °C.

    Techniques: Western Blot, Cell Culture

    Cellular activity of MLKL bearing second brace helix mutations parallels behaviour of recombinant proteins. a Mlkl -/- MDFs reconstituted with wild-type MLKL were stimulated with TNF (T), Smac-mimetic (S) and Q-VD-OPh (Q) or emricasan/IDN-6556 (I), and cell death was quantified using propidium iodide (PI) staining and flow cytometry. b Western blot of Blue-Native PAGE of wild-type MDFs treated ± TSQ to measure localisation to cytoplasmic (C) or membrane (M) fractions and oligomerisation state. VDAC1 and GAPDH western blot reprobes were used to assess quality of fractionation. c Full-length MLKL constructs containing indicated alanine mutations were stably incorporated into MDFs via lentiviral infection, and protein expression induced by doxycycline (dox) treatment. Cells were either left untreated (UT), or treated with TSQ or TSI to induce necroptosis 3 h post dox induction. Cell death was measured using PI staining and flow cytometry 24 h after death stimulation. Responses to an apoptotic stimulus (TS), and death in the presence or absence of dox-induction, are shown in entirety in Supplementary Figure 2. d Western blot of Blue-Native PAGE on cell fractions was used to monitor the formation of MLKL higher-order species and determine cellular localisation in untreated or TSQ stimulated Mlkl -/- MDFs, expressing alanine mutant constructs. C; cytoplasmic fraction, M; membrane fraction. Western blots for VDAC1 (membrane associated) and GAPDH (cytoplasmic protein) were used to assess the quality of fractionation. BN-PAGE blots are representative of 2 independent experiments. Cell death data are the mean ± s.e.m. of three independent experiments with 2–3 biologically independent replicates per experiment ( n = 6–9). e ]. Mutation of residues in green (N163, K164 and K167, Q168) does not preclude oligomerisation, while those in red (E161, I162 and T165, L166) block oligomerisation. f Zoomed depiction of second brace helix structure in e . Hydrogen bonds between the second brace helix (E161 and T165) and C-lobe of the pseudokinase domain (T291 and S293) are indicated in black dashed lines with interatomic distances annotated. g Atoms from the first brace helix that form hydrophobic packing interactions with I162 and L166 from the second brace helix are coloured pink and shown in a surface representation

    Journal: Cell Death and Differentiation

    Article Title: The brace helices of MLKL mediate interdomain communication and oligomerisation to regulate cell death by necroptosis

    doi: 10.1038/s41418-018-0061-3

    Figure Lengend Snippet: Cellular activity of MLKL bearing second brace helix mutations parallels behaviour of recombinant proteins. a Mlkl -/- MDFs reconstituted with wild-type MLKL were stimulated with TNF (T), Smac-mimetic (S) and Q-VD-OPh (Q) or emricasan/IDN-6556 (I), and cell death was quantified using propidium iodide (PI) staining and flow cytometry. b Western blot of Blue-Native PAGE of wild-type MDFs treated ± TSQ to measure localisation to cytoplasmic (C) or membrane (M) fractions and oligomerisation state. VDAC1 and GAPDH western blot reprobes were used to assess quality of fractionation. c Full-length MLKL constructs containing indicated alanine mutations were stably incorporated into MDFs via lentiviral infection, and protein expression induced by doxycycline (dox) treatment. Cells were either left untreated (UT), or treated with TSQ or TSI to induce necroptosis 3 h post dox induction. Cell death was measured using PI staining and flow cytometry 24 h after death stimulation. Responses to an apoptotic stimulus (TS), and death in the presence or absence of dox-induction, are shown in entirety in Supplementary Figure 2. d Western blot of Blue-Native PAGE on cell fractions was used to monitor the formation of MLKL higher-order species and determine cellular localisation in untreated or TSQ stimulated Mlkl -/- MDFs, expressing alanine mutant constructs. C; cytoplasmic fraction, M; membrane fraction. Western blots for VDAC1 (membrane associated) and GAPDH (cytoplasmic protein) were used to assess the quality of fractionation. BN-PAGE blots are representative of 2 independent experiments. Cell death data are the mean ± s.e.m. of three independent experiments with 2–3 biologically independent replicates per experiment ( n = 6–9). e ]. Mutation of residues in green (N163, K164 and K167, Q168) does not preclude oligomerisation, while those in red (E161, I162 and T165, L166) block oligomerisation. f Zoomed depiction of second brace helix structure in e . Hydrogen bonds between the second brace helix (E161 and T165) and C-lobe of the pseudokinase domain (T291 and S293) are indicated in black dashed lines with interatomic distances annotated. g Atoms from the first brace helix that form hydrophobic packing interactions with I162 and L166 from the second brace helix are coloured pink and shown in a surface representation

    Article Snippet: Antibodies used were rat anti-MLKL [ ] (1/1000; clone 3H1, available as cat. MABC604 from Millipore), rabbit anti-GAPDH (1/1000; glyceraldehyde-3-phosphate dehydrogenase; Cell Signalling Technology), mouse anti-VDAC1 (1/500; Voltage Dependent Anion Channel 1; Merck, cat. MABN504) and mouse anti-actin (1/3000; Sigma).

    Techniques: Activity Assay, Recombinant, Staining, Flow Cytometry, Cytometry, Western Blot, Blue Native PAGE, Fractionation, Construct, Stable Transfection, Infection, Expressing, Mutagenesis, Polyacrylamide Gel Electrophoresis, Blocking Assay

    Chimeric mouse:human MLKL constructs reveal key determinants for constitutive killing of human and mouse cell lines. a Sequence alignment of MLKL brace regions from multiple species. Residue number for mouse and a schematic of the position of brace helices in the mouse structure are presented above the alignment. b Schematics of mouse (orange) and human (blue) MLKL chimeras. c Mouse m4HB + Brace (residues 1–180), mouse 4HB (1–125), and (undimerised) gyrase fusions of full-length hMLKL (1–471), h4HB + Brace (1–180) and h4HB (1–125) were inducibly expressed in Mlk / -/- ], and are included for comparison with the chimera and mutant data reported herein. d – h Wild-type, Mlkl -/- and Mlkl -/- Ripk3 -/- MDFs, HT29, and U937 cells were stably transfected with doxycycline inducible chimeric constructs. Following induction of protein expression with dox, cells were either left untreated (UT) or stimulated with TSQ/TSI. MDF cell death was quantified by PI uptake after 24 h; data are the mean ± s.e.m. of three independent experiments with 2–3 biologically independent replicates per experiment ( n = 6–9). Death assays on the human cell lines, HT29 and U937, were performed similarly to MDFs, except that cell death was measured at 48 h post-stimulation. Human cell line data are the mean ± s.e.m. of three independent experiments ( n = 3), except for those in g , where n = 2. Responses to an apoptotic stimulus (TS), and death in the presence or absence of dox-induction, are shown in entirety in Supplementary Figure 4. ( i ) Western blot of Blue-Native PAGE on fractionated Mlkl -/- MDFs expressing inactive and constitutively-activated chimeras treated ± TSQ. h4HB-hBrace-mPsK cells were harvested 18 h after dox treatment; h4HB-mBrace-mPsK were harvested at 6 h. C cytoplasmic fraction, M membrane fraction. VDAC1 (membrane associated) and GAPDH (cytoplasmic protein) blots were used as fractionation controls. All BN-PAGE blots are representative of two independent experiments

    Journal: Cell Death and Differentiation

    Article Title: The brace helices of MLKL mediate interdomain communication and oligomerisation to regulate cell death by necroptosis

    doi: 10.1038/s41418-018-0061-3

    Figure Lengend Snippet: Chimeric mouse:human MLKL constructs reveal key determinants for constitutive killing of human and mouse cell lines. a Sequence alignment of MLKL brace regions from multiple species. Residue number for mouse and a schematic of the position of brace helices in the mouse structure are presented above the alignment. b Schematics of mouse (orange) and human (blue) MLKL chimeras. c Mouse m4HB + Brace (residues 1–180), mouse 4HB (1–125), and (undimerised) gyrase fusions of full-length hMLKL (1–471), h4HB + Brace (1–180) and h4HB (1–125) were inducibly expressed in Mlk / -/- ], and are included for comparison with the chimera and mutant data reported herein. d – h Wild-type, Mlkl -/- and Mlkl -/- Ripk3 -/- MDFs, HT29, and U937 cells were stably transfected with doxycycline inducible chimeric constructs. Following induction of protein expression with dox, cells were either left untreated (UT) or stimulated with TSQ/TSI. MDF cell death was quantified by PI uptake after 24 h; data are the mean ± s.e.m. of three independent experiments with 2–3 biologically independent replicates per experiment ( n = 6–9). Death assays on the human cell lines, HT29 and U937, were performed similarly to MDFs, except that cell death was measured at 48 h post-stimulation. Human cell line data are the mean ± s.e.m. of three independent experiments ( n = 3), except for those in g , where n = 2. Responses to an apoptotic stimulus (TS), and death in the presence or absence of dox-induction, are shown in entirety in Supplementary Figure 4. ( i ) Western blot of Blue-Native PAGE on fractionated Mlkl -/- MDFs expressing inactive and constitutively-activated chimeras treated ± TSQ. h4HB-hBrace-mPsK cells were harvested 18 h after dox treatment; h4HB-mBrace-mPsK were harvested at 6 h. C cytoplasmic fraction, M membrane fraction. VDAC1 (membrane associated) and GAPDH (cytoplasmic protein) blots were used as fractionation controls. All BN-PAGE blots are representative of two independent experiments

    Article Snippet: Antibodies used were rat anti-MLKL [ ] (1/1000; clone 3H1, available as cat. MABC604 from Millipore), rabbit anti-GAPDH (1/1000; glyceraldehyde-3-phosphate dehydrogenase; Cell Signalling Technology), mouse anti-VDAC1 (1/500; Voltage Dependent Anion Channel 1; Merck, cat. MABN504) and mouse anti-actin (1/3000; Sigma).

    Techniques: Construct, Sequencing, Mutagenesis, Stable Transfection, Transfection, Expressing, Western Blot, Blue Native PAGE, Fractionation, Polyacrylamide Gel Electrophoresis