Journal: The Journal of Experimental Medicine

Article Title: Mitochondrial Release of Caspase-2 and -9 during the Apoptotic Process

doi:

Figure Lengend Snippet: Identification of the Z-VAD– binding activity as caspase-2 and -9. (A) Proteins reacting with biotinylated VAD.fmk in the intermembrane space of liver mitochondria. The supernatant of control mitochondria (lane 1) or of Atr-treated mitochondria (lanes 2 and 3), the flow-through of the MiniS column (see Fig. and Fig. A; lanes 4 and 5), purified AIF (lane 6), or the Z-VAD.afc–cleaving activity eluting at 280 mM from the MiniQ column (see Fig. and Fig. A; lanes 7 and 8) were allowed to react with biotinylated VAD.fmk, either without pretreatment (lanes 1, 2, 4, 6, and 7) or after preincubation with Z-VAD.fmk (lanes 3, 5, and 8). Note that Z-VAD.fmk has been added to mitochondria before Atr (line 3). In addition, the proteins reacting with biotinylated VAD.fmk retained on an avidin column were purified (lane 9). These proteins, which contained approximately similar levels of Z-VAD.afc–cleaving activity (10 U) or ∼100 ng purified protein (lane 6) were separated by SDS-PAGE, blotted onto nitrocellulose, and subjected to the detection of biotinylated VAD.fmk using an avidin-based detection system. (B and C) The same blot as in A was subjected to immunodetection with antibodies specific for caspase-2 (B) or -9 (C). (D and E) Mitochondria from different organs were purified and cultured for 30 min in the presence or absence of 5 mM Atr, followed by immunoblot detection of caspase-2 (D) or -9 (E). Results are representative of two to four independent experiments. (F and G) Specificity control of caspase-2– and caspase-9–specific antisera. Recombinant caspase-2 (lane 1), -3 (lane 2), or -9 (lane 3) was immunoblotted (100 ng/lane), followed by immunodetection with the caspase-2 (F) or caspase-9 (G)–specific antibody. Similarly, caspase-2– and caspase-9–specific antibodies fail to recognize caspase-6 and -7 (not shown).

Article Snippet: Immunoelectron microscopy was performed using the rabbit anti– caspase-9 antibody and an Immunogold (5 nm) anti–rabbit Ig conjugate ( Amersham Pharmacia Biotech ).

Techniques: Binding Assay, Activity Assay, Purification, Avidin-Biotin Assay, SDS Page, Immunodetection, Cell Culture, Western Blot, Recombinant

Journal: The Journal of Experimental Medicine

Article Title: Mitochondrial Release of Caspase-2 and -9 during the Apoptotic Process

doi:

Figure Lengend Snippet: Submitochondrial localization of caspase-2 and -9. Purified mitochondria were either lysed by osmotic shock (total preparation, lane 1) or were subjected to fractionation into matrix (lanes 2 and 6), inner membrane (lanes 3 and 7), intermembrane (lanes 4 and 8), or outer membrane (lanes 5 and 9) proteins, in the absence (lanes 2–5) or presence (lanes 6–9) of 100 μM Z-VAD.fmk throughout each single step of the fractionation procedure. Thereafter, equivalent amounts of protein (15 μg/lane) were subjected to immunoblot detection of caspase-2 (A) and -9 (B). A representative immunoelectron micrograph of mitochondria labeled with a specific caspase-9 antibody is also shown (C).

Article Snippet: Immunoelectron microscopy was performed using the rabbit anti– caspase-9 antibody and an Immunogold (5 nm) anti–rabbit Ig conjugate ( Amersham Pharmacia Biotech ).

Techniques: Purification, Fractionation, Western Blot, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Mitochondrial Release of Caspase-2 and -9 during the Apoptotic Process

doi:

Figure Lengend Snippet: Apoptosis induction by microinjection of recombinant caspases. Rat-1 fibroblasts were left untreated or cultured with etoposide (1 μM, 6 h), in the absence or presence of Z-VAD.fmk (100 μM), to obtain a positive or negative control of apoptotic morphology, respectively. Alternatively, cells pretreated with Z-VAD.fmk (100 μM, 60 min) or left untreated were microinjected with buffer only or with recombinant caspase-2 or -9 (3 U/μl). Microinjected viable cells (100–200 per session, two to five independent sessions of injection) could be identified because the injectate contained FITC-dextran (0.25% [wt/vol], green fluorescence; not shown). Microphotographs representing the dominant (>70%) phenotype of microinjected cells stained either with Hoechst 33342 (blue fluorescence, A) or with Annexin V (red fluorescence, B) are shown after 90 min of culture. Z-VAD. fmk pretreatment prevented phosphatidylserine exposure induced by etoposide, caspase-2, or caspase-9, as measured with Annexin V (not shown).

Article Snippet: Immunoelectron microscopy was performed using the rabbit anti– caspase-9 antibody and an Immunogold (5 nm) anti–rabbit Ig conjugate ( Amersham Pharmacia Biotech ).

Techniques: Recombinant, Cell Culture, Negative Control, Injection, Fluorescence, Staining

Journal: The Journal of Experimental Medicine

Article Title: Mitochondrial Release of Caspase-2 and -9 during the Apoptotic Process

doi:

Figure Lengend Snippet: Loss of subcellular compartmentalization of cytochrome c , caspase-2, and caspase-9. Rat-1 cells were left untreated or were cultured in the presence of staurosporine (4 h, 1 μM), fixed, and stained to determine the subcellular distribution of the indicated molecules by indirect immunofluorescence analysis.

Article Snippet: Immunoelectron microscopy was performed using the rabbit anti– caspase-9 antibody and an Immunogold (5 nm) anti–rabbit Ig conjugate ( Amersham Pharmacia Biotech ).

Techniques: Cell Culture, Staining, Immunofluorescence

Journal: The Journal of Experimental Medicine

Article Title: Mitochondrial Release of Caspase-2 and -9 during the Apoptotic Process

doi:

Figure Lengend Snippet: Redistribution of caspase-2 and -9 from the mitochondrion. T cell hybridoma cells transfected with a Neo control vector or with Bcl-2 were cultured in the absence or presence of 1 μM DEX, followed by subcellular fractionation, as described in Materials and Methods. Equivalent amounts of proteins were subjected to immunoblot analysis in order to determine the subcellular localization and activation of cytochrome c (A), caspase-2 (B), or caspase-9 (C). Results are representative of three independent determinations.

Article Snippet: Immunoelectron microscopy was performed using the rabbit anti– caspase-9 antibody and an Immunogold (5 nm) anti–rabbit Ig conjugate ( Amersham Pharmacia Biotech ).

Techniques: Transfection, Plasmid Preparation, Cell Culture, Fractionation, Western Blot, Activation Assay

Journal: PLoS ONE

Article Title: γ-Synuclein Antibodies Have Neuroprotective Potential on Neuroretinal Cells via Proteins of the Mitochondrial Apoptosis Pathway

doi: 10.1371/journal.pone.0090737

Figure Lengend Snippet: List of used abs.

Article Snippet: polyclonal anti Caspase 9 , Rabbit , Not available , Bioworld Technology.

Techniques: Recombinant

Journal: Oncology Letters

Article Title: A LY-15, a novel cyclic pentapeptide that inhibits B16 cell proliferation and migration and induces cell apoptosis

doi: 10.3892/ol.2018.8023

Figure Lengend Snippet: No significant difference was identified in the protein grayscale value of the 5 µM group compared with that of the control group (P>0.05). Besides, there was no significant difference between 15 and 25 µM (P>0.05). Compared with the control group, 15 and 25 µM groups present significant differences. For Bax and Bcl-2, P<0.001. For caspase-3 and caspase-9, P<0.01. The overall average is not exactly equal (P<0.001). **P<0.01; ***P<0.001.

Article Snippet: The antibodies against B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), caspase-3 and caspase-9 (all polyclonal rabbit anti-mouse) were purchased from Arigo Bio (Taiwan, Xinzhu).

Techniques: