Journal: Molecular and Cellular Biology

Article Title: Oligomerization of the Mitochondrial Protein Voltage-Dependent Anion Channel Is Coupled to the Induction of Apoptosis

doi: 10.1128/mcb.00165-10

Figure Lengend Snippet: FIG. 3. STS induces VDAC oligomerization in all cell types used. (A) T-REx-293, HeLa, and T47D cells (2.5 mg/ml) were incubated in the absence or presence of STS (1.25 M; 5 h) and were subjected to cross-linking with EGS (250 M) and to immunoblotting using anti-VDAC antibodies. The positions of molecular size protein standards are provided. (B) Quantitative analysis of apoptosis (assayed via AcrOr-EthBro staining) (n 3). (C) T-REx-293, HeLa, and human peripheral blood mononuclear cells (PBMCs; isolated using Ficoll-Paque density gradient centrifugation) (30 and 60 g) were subjected to immunoblotting using anti-Bax, anti-Bak, anti-Bid, and anti-VDAC antibodies. As a loading control, actin levels in the samples were determined using antiactin antibodies. Note that the increase in the amount of protein loaded was seen for all immunoblotted proteins, except for VDAC, due to the high affinity of the antibodies used, which yielded a saturated signal over 30 g.

Article Snippet: Monoclonal anti-Bax antibodies were obtained from Trevigen (Gaithersburg, Maryland).

Techniques: Incubation, Western Blot, Staining, Isolation, Gradient Centrifugation, Control

Journal: International Journal of Molecular Sciences

Article Title: GSK3β Inhibition Is the Molecular Pivot That Underlies the Mir-210-Induced Attenuation of Intrinsic Apoptosis Cascade during Hypoxia

doi: 10.3390/ijms23169375

Figure Lengend Snippet: miR-210 attenuates the hypoxia-driven intrinsic apoptosis pathway through the inhibition of GSK3β kinase activity. ( A , B ) Quantitative ELISA immunoassays determining the Cytochrome C abundance in the mitochondrial fractions (A) and cytosolic fractions ( B ), expressed as experimental blank-corrected absorbances (O.D) measured at λ 450 (450 nm). ( C – F ) Quantitative ELISA immunoassays determining the abundance of BAX ( C , D ) and BAK ( E , F ) in the mitochondrial fractions ( C , E ) and cytosolic fractions ( D , F ), expressed as experimental blank-corrected absorbances (O.D) measured at λ 450 (450 nm) normalized to fold-change values. Quantitative ELISA immunoassays determining the abundance of BAX and BAK in whole-cell fractions are reported in . The validity of the integrity of the respective subcellular compartments is reported in . miR-210 expression levels in the respective cell lysates were determined by the miR-210 hybridization immunoassay (as described in ) and are reported in . The validation of the ectopic expression of the HA-tagged ca -GSK3β-S9A mutant in the pertinent experimental groups was performed by ELISA immunoassay and is reported in . GSK3β kinase activity (as described in ) was measured in all experimental groups to corroborate and validate the translative effects of the ectopic expression of the ca -GSK3β-S9A mutant (reported in ). All data are expressed as mean ± S.D from three technical replicates for each of the four biological replicates belonging to each experimental group ( n = 4). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns: not significant ( p > 0.05). OE: miR-210 overexpression; O.D: optical density; S.D: standard deviation.

Article Snippet: BAX , ELISA detection , 20 ng/well , Rabbit , Novus Biologicals , NBP1-88682 , AB_11014342.

Techniques: Inhibition, Activity Assay, Enzyme-linked Immunosorbent Assay, Expressing, Hybridization, Biomarker Discovery, Mutagenesis, Over Expression, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: GSK3β Inhibition Is the Molecular Pivot That Underlies the Mir-210-Induced Attenuation of Intrinsic Apoptosis Cascade during Hypoxia

doi: 10.3390/ijms23169375

Figure Lengend Snippet: miR-210 mitigates the hypoxia-induced oligomerization and insertion of BAX and BAK into the outer mitochondrial membrane ( OMM ). Isolated mitochondria were subjected to a 0.1 M sodium carbonate (Na 2 CO 3 ) treatment to produce the alkali-resistant OMM-inserted ( OMM -embedded) protein fraction and the alkali-soluble OMM-tethered ( OMM -anchored) protein fraction. ( A – D ) ELISA immunoassays determining the abundance of BAX ( A , C ) and BAK ( B , D ) performed on the denatured lysates from the respective OMM-inserted protein fraction ( A , B ) and OMM-tethered protein fraction ( C , D ). Experimental blank-corrected absorbances (O.D) measured at λ 450 (450 nm) were first expressed as fold-change and subsequently normalized to either TOM40 expression levels (for the OMM-inserted protein fraction) or HK2 expression levels ( OMM-tethered protein fraction). The expression levels of TOM40 and HK2 in the respective fractions are reported in . miR-210 expression levels in the respective whole-cell lysates were determined by the miR-210 hybridization immunoassay (as described in ) and are reported in . The validation of the ectopic expression of the HA-tagged ca -GSK3β-S9A mutant in the pertinent experimental groups was performed by ELISA immunoassay and is reported in . GSK3β kinase activity (as described in ) was measured in all experimental groups to corroborate and validate the translative effects of the ectopic expression of the ca -GSK3β-S9A mutant (reported in ). Data are represented as double-normalized ratiometric values (BAX/TOM40 and BAX/HK2 as well as BAK/TOM40 and BAK/HK2), expressed as mean ± S.D fold-change , from three technical replicates for each of the four biological replicates belonging to each experimental group ( n = 4). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns: not significant ( p > 0.05). OE: miR-210 overexpression; O.D: optical density; S.D: standard deviation; OMM : outer mitochondrial membrane.

Article Snippet: BAX , ELISA detection , 20 ng/well , Rabbit , Novus Biologicals , NBP1-88682 , AB_11014342.

Techniques: Membrane, Isolation, Enzyme-linked Immunosorbent Assay, Expressing, Hybridization, Biomarker Discovery, Mutagenesis, Activity Assay, Over Expression, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: GSK3β Inhibition Is the Molecular Pivot That Underlies the Mir-210-Induced Attenuation of Intrinsic Apoptosis Cascade during Hypoxia

doi: 10.3390/ijms23169375

Figure Lengend Snippet: List of antibodies and antibody-blocking peptides used in the study.

Article Snippet: BAX , ELISA detection , 20 ng/well , Rabbit , Novus Biologicals , NBP1-88682 , AB_11014342.

Techniques: Enzyme-linked Immunosorbent Assay, Blocking Assay, Peptide ELISA, Activity Assay

Journal: Scientific Reports

Article Title: Surface modification of pig endothelial cells with a branched heparin conjugate improves their compatibility with human blood

doi: 10.1038/s41598-017-04898-w

Figure Lengend Snippet: CHC coating of microbead-borne PAEC reduces clotting time of whole human blood. ( A ) Binding of FITC-labeled CHC (100 µg/ml, 15 min) to PAEC grown on microbeads (top panel), analyzed by confocal microscopy. Minimal binding of FITC-CHC to control beads without PAEC was observed (bottom panel). Nuclei were stained with DAPI. Scale bar: 100 µm. ( B ) Clotting time of non-anticoagulated whole human blood incubated with and without microbeads. Microbeads carrying GTKO.hCD46.hTBM PAEC significantly prolonged clotting time compared to microbeads carrying WT PAEC. Coating of WT or GTKO.hCD46.hTBM PAEC on microbeads with CHC significantly prolonged clotting time compared to uncoated PAEC. Statistical analysis was carried out by one-way ANOVA with Bonferroni correction (*p < 0.05, **p < 0.01, ***p < 0.001). Data are mean ± SD of four independent experiments per group. ( C,D ) CHC coating of microbead-borne PAEC reduces deposition of human platelets during incubation with human blood. ( C ) PAEC-bead samples were collected from the whole blood coagulation assays at 20 min and stained for expression of CD31 (red) and deposition of CD41-positive human platelets (green). Nuclei stained with DAPI (blue). Scale bar: 100 µm. ( D ) Platelet deposition was determined by counting the number of platelets on 20 randomly selected beads, and is presented as % platelet deposition relative to that observed with uncoated WT PAEC. Coating of WT and GTKO.hCD46.hTBM PAEC with CHC significantly reduced platelet deposition at 20 min and 60 min, respectively. GTKO.hCD46.hTBM PAEC showed significantly reduced platelet deposition compared to WT PAEC irrespective of CHC coating. Data are mean ± SD of at least three independent experiments. Significance was tested using one-way ANOVA with Bonferroni correction (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Following washing, the beads were stained with rat anti-porcine CD31 (MAB88371, R&D Systems) plus goat anti-rat IgG Alexa Fluor 546 (Thermo Fisher Scientific) and mouse anti-human CD41 FITC antibody.

Techniques: Coagulation, Binding Assay, Labeling, Confocal Microscopy, Control, Staining, Incubation, Expressing