Journal: Scientific Reports

Article Title: In vitro rejuvenation of brain mitochondria by the inhibition of actin polymerization

doi: 10.1038/s41598-018-34006-5

Figure Lengend Snippet: Inhibiting actin polymerization reduces the amount of CIV-bound cyt c. ( A ) Immunoblot analysis of the immunoprecipitates with anti-MTCO1 antibody from DMSO- and CB-treated mitochondria, followed by immunoblotting with antibodies against the indicated proteins. The relative amounts of the MTCO1-associated proteins to MTCO1 were determined (n = 6, mean ± S.D.). p

Article Snippet: The in-gel CcO activity of CIV was visualized by the precipitation of 3, 3′-diaminobenzidine oxides and indamine polymers in test buffer containing 50 mM sodium phosphate, 0.5 mg/ml 3, 3′-diaminobenzidine-tetrahydrochloride (D5905; Sigma), 0.25 mg/l cyt c (C2037; Sigma), and 75 mg/ml sucrose, pH 7.4 .

Techniques:

Journal: Scientific Reports

Article Title: In vitro rejuvenation of brain mitochondria by the inhibition of actin polymerization

doi: 10.1038/s41598-018-34006-5

Figure Lengend Snippet: Inhibiting actin polymerization increases the amount of CIII-bound cyt c. ( A ) Isolated brain mitochondria treated with DMSO or CB were lysed and immunoprecipitated with anti-UQCRC2 antibody, followed by immunoblotting for the indicated proteins. The relative amounts of coprecipitated proteins to UQCRC2 were determined (n = 8, mean ± S.D.). p

Article Snippet: The in-gel CcO activity of CIV was visualized by the precipitation of 3, 3′-diaminobenzidine oxides and indamine polymers in test buffer containing 50 mM sodium phosphate, 0.5 mg/ml 3, 3′-diaminobenzidine-tetrahydrochloride (D5905; Sigma), 0.25 mg/l cyt c (C2037; Sigma), and 75 mg/ml sucrose, pH 7.4 .

Techniques: Isolation, Immunoprecipitation

Journal: International Journal of Oncology

Article Title: miR-125a induces apoptosis, metabolism disorder and migration impairment in pancreatic cancer cells by targeting Mfn2-related mitochondrial fission

doi: 10.3892/ijo.2018.4380

Figure Lengend Snippet: Overexpression of miR-125a promotes cell death by inducing mitochondrial fission-related mitochondrial apoptosis pathways. (A) Change of the mitochondrial morphology via Tomm20 staining. (B) Western blot analysis was performed and densitometry performed for expression of (C) caspase-9, (D) Bax, (E) Bad, (F) Bcl-2 and (G) x-IAP, and (H) Bax/Bcl-2 ratio was calculated. (I) Mitochondrial membrane potential was measured by JC-1. (J) mPTP opening rate increased upon application of agmir-125a, but decreased when treated with fission inhibitors. (K) cyt-c immunofluorescence showed cyt-c leakage from mitochondria into the cytoplasm induced by agmir-125a, and some cyt-c even migrated into the nucleus. * P

Article Snippet: The primary antibodies used for cell immunofluorescence were cyt-c (1:500; cat. no. ab90529), translocase of outer mitochondrial membrane 20 (1:500; cat. no. ab56783) and F-actin (1:500; cat. no. ab205) from Abcam.

Techniques: Over Expression, Staining, Western Blot, Expressing, Immunofluorescence

Journal: International Journal of Molecular Medicine

Article Title: Mitochondria-targeted antioxidant therapy for an animal model of PCOS-IR

doi: 10.3892/ijmm.2018.3977

Figure Lengend Snippet: Relative expression of Cyt C, Bcl-xL, Bcl-2 and Bax in the ovaries of the Control, PCOS-IR and MitoQ 10 treatment groups. PCOS-IR, polycystic ovary syndrome and insulin resistance; Cyt C, cytochrome c ; Bcl-2, B-cell lymphoma 2; Bcl-xL, Bcl-extra large; Bax, Bcl-2-associated X protein.

Article Snippet: Following incubation of the PVDF membrane with blocking solution, the protein bands were mixed with special anti-Bax (cat. no. 34260; 1:500; Signalway Antibody LLC, College Park, MD, USA), anti-Bcl-xL (cat. no. 21061; 1:500; Signalway Antibody LLC), anti-Cyto C (cat. no. ab33484; 1:500; Abcam) and anti-Bcl-2 (cat. no. ab59348; 1:500; Abcam) antibodies overnight at 4°C.

Techniques: Expressing

Journal: International Journal of Molecular Medicine

Article Title: Mitochondria-targeted antioxidant therapy for an animal model of PCOS-IR

doi: 10.3892/ijmm.2018.3977

Figure Lengend Snippet: Qualification of the expression levels of Cyt C, Bcl-xL, Bcl-2 and Bax in the ovaries of the Control, PCOS-IR and MitoQ 10 treatment groups. ## P

Article Snippet: Following incubation of the PVDF membrane with blocking solution, the protein bands were mixed with special anti-Bax (cat. no. 34260; 1:500; Signalway Antibody LLC, College Park, MD, USA), anti-Bcl-xL (cat. no. 21061; 1:500; Signalway Antibody LLC), anti-Cyto C (cat. no. ab33484; 1:500; Abcam) and anti-Bcl-2 (cat. no. ab59348; 1:500; Abcam) antibodies overnight at 4°C.

Techniques: Expressing

Journal: Scientific Reports

Article Title: A systems pharmacology-oriented discovery of a new therapeutic use of the TCM formula Liuweiwuling for liver failure

doi: 10.1038/s41598-018-21515-6

Figure Lengend Snippet: Liuweiwuling administration suppresses TNF-α, FADD and Cyt C protein expression in the hepatocytes of GalN/LPS-treated mice by immunohistochemistry. For the abbreviation of groups in experimental design, control group (Con), model group (Mod), positive drug group (Bic), 0.5 mg/g Liuweiwuling group (LW1), 2 mg/g Liuweiwuling group (LW2), 8 mg/g Liuweiwuling group (LW3), and 16 mg/g Liuweiwuling group (LW4).

Article Snippet: Immunohistochemistry for TNF-α, Cyt C and FADD was performed with anti-TNF-α antibody, anti-Cyt C antibody and anti-FADD antibody, respectively (Wuhan Gugebio, GB13188; Abcam, ab133504; Abcam, ab124812).

Techniques: Expressing, Mouse Assay, Immunohistochemistry

Journal: Scientific Reports

Article Title: Hypoxia-induced autophagy mediates cisplatin resistance in lung cancer cells

doi: 10.1038/srep12291

Figure Lengend Snippet: Hypoxia suppressed cisplatin-induced Bax translocation and Cyto C release. A549 and SPC-A1 cells were treated as mentioned above, then cells were immunostained by Bax or Cyto C antibody following MitoTracker staining. ( A ) Bax cellular localization. ( B ) Cyto C cellular localization. Representative images were from three independent experiments. Green: Bax or Cyto C; Red: mitochondria; Blue: nucleus; the scale bar: 10 μm.

Article Snippet: Then cells were permeabilized in PBS containing 0.5% Triton X-100 and incubated for 2 h with primary antibody rabbit anti-Bax (abcam,1:200, ab10813) or rabbit anti-Cyto C (abcam, 1:200, ab53056).

Techniques: Translocation Assay, Staining

Journal: Scientific Reports

Article Title: Hypoxia-induced autophagy mediates cisplatin resistance in lung cancer cells

doi: 10.1038/srep12291

Figure Lengend Snippet: Hypoxia blocked cisplatin-induced subcellular redistribution of pro-apoptotic proteins in lung cancer cells. A549 and SPC-A1 cells were treated as indicated and then cells were fractionated into cytosolic fraction and the mitochondrial fraction. ( A ) COX IV protein expression in the cytosolic fraction and mitochondrial fraction (lane 1: normoxia, lane 2: hypoxia). ( B ) The total protein expressions of Bax and Cyto C. ( C ) The protein expression of Bax and Cyto C in cytosol and mitochondria. ( D ) The ratio of the expression of Bax or Cyto C in the cytoplasm to the expression of Bax or Cyto C in the mitochondira. The results were shown as means ± SD of three independent experiments. ** p

Article Snippet: Then cells were permeabilized in PBS containing 0.5% Triton X-100 and incubated for 2 h with primary antibody rabbit anti-Bax (abcam,1:200, ab10813) or rabbit anti-Cyto C (abcam, 1:200, ab53056).

Techniques: Expressing

Journal: Oncology Reports

Article Title: Crosstalk between p38 MAPK and caspase-9 regulates mitochondria-mediated apoptosis induced by tetra-α-(4-carboxyphenoxy) phthalocyanine zinc photodynamic therapy in LoVo cells

doi: 10.3892/or.2017.6071

Figure Lengend Snippet: Effect of TαPcZn-PDT treatment, with or without siRNA-p38 MAPK or z-LEHD-fmk, on Bcl-2, Bax, AIF, Cyto c , Bid and caspase-3 levels in LoVo cells analyzed by immunoblot assay. In the absence or presence of siRNA- p38 MAPK (12.5 nmol/l) for 48 h or z-LEHD-fmk (10 µmol/l) for 2.5 h, LoVo cells were treated with TαPcZn (54 µmol/l), exposed to red light irradiation (53.7 J/cm 2 ) and then incubated for 3 h. The expression of (A) Bcl-2 and Bax, (B) AIF and Cyto c and (C) Bid and caspase-3 was analyzed by immunoblot assay. Values presented are representative of three independent experiments (mean ± standard deviation; **P

Article Snippet: Anti-p38 MAPK, anti-phosphorylated (p)-p38 MAPK, anti-Bcl-2, anti-Bax, anti-AIF, anti-Cyto c , anti-Bid, anti-caspase-9 and anti-caspase-3 antibodies were all purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Irradiation, Incubation, Expressing, Standard Deviation

Journal: The EMBO Journal

Article Title: HBXIP functions as a cofactor of survivin in apoptosis suppression

doi: 10.1093/emboj/cdg263

Figure Lengend Snippet: Fig. 10. The X protein of the hepatitis B virus (HBX) associates with survivin through HBXIP and suppresses caspase activation. ( A ) In vitro protein binding assays were performed using His 6 -HBX incubated with GST–HBXIP, GST–survivin or GST–CD40 (control) immobilized on glutathione–Sepharose. Bound proteins were analyzed by immunoblotting using anti-HBX antibody. ( B ) His 6 -HBX or His 6 -Traf3 (CTR) proteins, purified His 6 -HBXIP, purified survivin or various combinations of these proteins were added to 293 cell extracts, normalizing all samples for total protein added using control recombinant proteins. Cyt-c and dATP were then added, and caspase activity was measured 0.5 h later, based on hydrolysis of Ac-DEVD-AFC, and compared with samples treated with control proteins (mean ± SD; n = 3). ( C ) HEK293 cells were transiently transfected with plasmids encoding FLAG-tagged-HBX or FLAG–SIP (as a control) together with Myc-survivin or HA–HBXIP or control plasmid. Lysates were subjected to immunoprecipitation using anti-FLAG antibody, demonstrating increased association of survivin with HBX when HBXIP was co-expressed (compare the last two lanes on the right). Immunoprecipitates were analyzed by immunoblotting using anti-survivin antibody (upper panel). Lysates were also analyzed by immunoblotting using anti-HBX (middle panel) or anti-HA antibodies (lower panel), confirming protein production. ( D ) His 6 -pro-caspase-9 was incubated with GST–HBXIP(+) or GST–CD40 control protein (–), with or without His 6 -HBX and untagged survivin. GST fusion proteins were recovered on glutathione–Sepharose and bound proteins were detected by immunoblotting using anti-caspase-9, anti-survivin or anti-HBX antisera. ( E ) HepG2 cell extracts were immunodepleted using anti-survivin antisera or preimmune serum (CTR), and equivalent amounts were analyzed by immunoblotting using anti-survivin antibody (top panel). Then equivalent volumes of extracts were analyzed for caspase activity based on Ac-DEVD-AFC cleavage, where lysates were incubated with HBX (+) or control protein (–) prior to adding Cyt-c and dATP.

Article Snippet: Mouse monoclonal antibodies against active caspase-9 and Cyt-c were purchased from PharMingen (San Diego, CA).

Techniques: Activation Assay, In Vitro, Protein Binding, Incubation, Purification, Recombinant, Activity Assay, Transfection, Plasmid Preparation, Immunoprecipitation

Journal: The EMBO Journal

Article Title: HBXIP functions as a cofactor of survivin in apoptosis suppression

doi: 10.1093/emboj/cdg263

Figure Lengend Snippet: Fig. 6. Combination of HBXIP and survivin inhibits recruitment of pro-caspase-9 to activated Apaf1. ( A ) GST–survivin, GST–CD40 or GST–HBXIP with or without purified survivin (untagged) was incubated with His 6 -pro-caspase-9, active His 6 -caspase-9 (lacking CARD domain) or His 6 -pro-caspase-3. GST fusion proteins were recovered using glutathione–Sepharose and bound proteins were detected by immunoblotting using anti-caspase-9 or anti-caspase-3 antisera. An equivalent amount of proteins was loaded directly onto gels as a control (‘input’). ( B ) [ 35 S]pro-caspase-9 was incubated with His 6 -Apaf1, Cyt-c and dATP, with or without GST–HBXIP and survivin. His 6 -Apaf1 and associated proteins were recovered by adsorption to Ni-resin, and bound proteins were analyzed by autoradiography (for caspase-9) or immunoblotting using anti-Apaf1 or anti-Cyt-c antibodies. ( C ) Purified His 6 -Apaf1, His 6 -pro-caspase-9, Cyt-c and dATP were incubated without (left panel) or with (right panel) purified survivin and HBXIP. Proteins were analyzed by gel-sieve chromatography using immunoblotting to detect proteins in eluted fractions. ( D ) Caspase activity in column fractions was determined by monitoring cleavage of Ac-DEVD-AFC after addition of pro-caspase-3.

Article Snippet: Mouse monoclonal antibodies against active caspase-9 and Cyt-c were purchased from PharMingen (San Diego, CA).

Techniques: Purification, Incubation, Adsorption, Autoradiography, Chromatography, Activity Assay

Journal: The EMBO Journal

Article Title: HBXIP functions as a cofactor of survivin in apoptosis suppression

doi: 10.1093/emboj/cdg263

Figure Lengend Snippet: Fig. 9. Regulation of caspase activation and apoptosis by endogenous HBXIP. ( A ) HeLa cells were transfected with siRNA targeting HBXIP or control RNA (CTR), and cells were analyzed by immunoblotting, using anti-HBXIP (upper panel), anti-survivin (middle) and anti-α- tubulin (lower) antisera, thus verifying siRNA-mediated reductions in endogenous HBXIP expression. ( B ) Extracts prepared from HeLa cells after treatment with HBXIP–siRNA or control RNA were incubated with Cyt-c and dATP, in the presence or absence of recombinant survivin (left panel) or XIAP (right panel), and caspase activity was measured based on release of AFC from Ac-DEVD-AFC substrate [mean ± SD; n = 3). ( C ) The percentage of apoptotic cells (mean + SD; n = 3) was determined by DAPI staining following culture of control RNA- or HBXIP siRNA-transfected HeLa cells with 25 µM VP-16 or STS.

Article Snippet: Mouse monoclonal antibodies against active caspase-9 and Cyt-c were purchased from PharMingen (San Diego, CA).

Techniques: Activation Assay, Transfection, Expressing, Incubation, Recombinant, Activity Assay, Staining

Journal: The EMBO Journal

Article Title: HBXIP functions as a cofactor of survivin in apoptosis suppression

doi: 10.1093/emboj/cdg263

Figure Lengend Snippet: Fig. 5. Combination of HBXIP and survivin inhibits caspase-9 activation. Recombinant pro-caspase-9 was incubated with purified His 6 -Apaf1, Cyt-c and dATP, with or without recombinant HBXIP, survivin, or the combination of these proteins or various control (CTR) proteins. ( A and B ) Caspase-9 activity was measured by cleavage of fluorogenic substrate Ac-LEHD-AFC, or ( C ) proteolytic processing of ∼50 kDa pro-caspase-9 to large (∼35 kDa) and small (∼12 kDa) subunits was analyzed by immunoblotting analysis using anti-caspase-9 antibody. Data in (B) represent mean ± SD ( n = 3), while data in (A) show representative enzyme progress curves. In (C), levels of recombinant proteins were also compared by immunoblotting using anti-HBXIP, anti-survivin and anti-His antibodies (lower three panels). Minus signs indicate addition of an equivalent amount of a control protein (GST–CD40 or His 6 -TRAF3) instead of survivin or HBXIP.

Article Snippet: Mouse monoclonal antibodies against active caspase-9 and Cyt-c were purchased from PharMingen (San Diego, CA).

Techniques: Activation Assay, Recombinant, Incubation, Purification, Activity Assay

Journal: The EMBO Journal

Article Title: HBXIP functions as a cofactor of survivin in apoptosis suppression

doi: 10.1093/emboj/cdg263

Figure Lengend Snippet: Fig. 4. HBXIP collaborates with survivin to suppress Cyt-c-induced caspase activation. Comparisons were made of effects of prior addition of purified HBXIP, survivin or the combination on caspase activity induced in cell extracts by Cyt-c ( A ), caspase-8 ( B ) or granzyme B ( C ), measuring AFC release from Ac-DEVD-AFC. Left panels show representative enzyme progress curves, indicating cumulative release of fluorophore from Ac-DEVD-AFC (RFU/µg). Right panels represent relative enzyme rates (RFU/min/µg) compared with samples treated with control protein (mean ± SD; n = 3 experiments). Several control (CTR) proteins were tested, with representative results shown, here using His 6 -TRAF3.

Article Snippet: Mouse monoclonal antibodies against active caspase-9 and Cyt-c were purchased from PharMingen (San Diego, CA).

Techniques: Activation Assay, Purification, Activity Assay

Journal: The EMBO Journal

Article Title: HBXIP functions as a cofactor of survivin in apoptosis suppression

doi: 10.1093/emboj/cdg263

Figure Lengend Snippet: Fig. 1. Differences in caspase suppression by survivin and XIAP. Comparisons were made of the effects of purified survivin (left panels) and XIAP (middle panels) on ( A ) recombinant active caspase-3 or ( B and C ) caspase-3-like protease activity induced in cell extracts by Cyt-c. Caspase activity was continuously measured, based on cleavage of Ac-DEVD-AFC fluorigenic substrate [relative fluorescence units (RFU)/min/µg]. In (A), 100 pM recombinant active caspase-3 was incubated with recombinant survivin (left panel) or XIAP (middle panel) or with control protein. In (B), purified survivin (left panel) or XIAP (middle panel), or control protein was added to 293 cell lysates concurrently with Cyt-c and dATP. In (C), survivin (left panel) or XIAP (middle panel) was added to cell extracts after Cyt-c and dATP. Data shown in the panels on the right are normalized relative to control protein (mean ± SD; n = 3).

Article Snippet: Mouse monoclonal antibodies against active caspase-9 and Cyt-c were purchased from PharMingen (San Diego, CA).

Techniques: Purification, Recombinant, Activity Assay, Fluorescence, Incubation

Journal: The EMBO Journal

Article Title: HBXIP functions as a cofactor of survivin in apoptosis suppression

doi: 10.1093/emboj/cdg263

Figure Lengend Snippet: Fig. 8. Elevations of survivin and HBXIP associated with caspase suppression. ( A ) Protein samples from tumors (T) and non-malignant tissue (NT) of three patients with HBV-related hepatocellular carcinoma and two normal livers specimens (N1 and N2) were normalized for protein content, and analyzed by SDS–PAGE/immunoblotting using antisera specific for HBXIP, survivin or α-tubulin. ( B ) Caspase activity in the same tissue samples was measured using the fluorigenic substrate Ac-DEVD-AFC after addition of Cyt-c and dATP (left panel) or granzyme B (right panel). Results are expressed relative to caspase activity generated in the normal liver specimen, N1 (mean ± SD; n = 3).

Article Snippet: Mouse monoclonal antibodies against active caspase-9 and Cyt-c were purchased from PharMingen (San Diego, CA).

Techniques: SDS Page, Activity Assay, Generated

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Cytochrome c is rapidly reduced in the cytosol after mitochondrial outer membrane permeabilization

doi: 10.1007/s10495-010-0455-2

Figure Lengend Snippet: Representative traces of oxygen consumption ( a and b ) and oxidation changes in cyt c and cyt aa 3 ( c and d ) from control cells ( a and c ) and anisomycin treated cells ( b and d ) after release of cyt c . The arrows indicate addition of myxothiazol/antimycin

Article Snippet: This large reduction in cyt c together with a small reduction in cyt aa3 is indicative of a reduced pool of cyt c that no longer has physical access to CytOx and is no longer in redox equilibrium with cyt aa3 .

Techniques:

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Cytochrome c is rapidly reduced in the cytosol after mitochondrial outer membrane permeabilization

doi: 10.1007/s10495-010-0455-2

Figure Lengend Snippet: Effect of antimycin on the rate of reduction of cyt c ( a ) after treatment with anisomycin ( arrow ANT ). Rate of cyt c reduction pre-and post-antimycin ( b ) expressed as mean ± SD ( n = 6). ( c ) Effect of anoxia on the reduction of cyt c after anisomycin.

Article Snippet: This large reduction in cyt c together with a small reduction in cyt aa3 is indicative of a reduced pool of cyt c that no longer has physical access to CytOx and is no longer in redox equilibrium with cyt aa3 .

Techniques:

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Cytochrome c is rapidly reduced in the cytosol after mitochondrial outer membrane permeabilization

doi: 10.1007/s10495-010-0455-2

Figure Lengend Snippet: A cartoon of the electron transport chain, intermembrane space and permeabilized outer membrane showing complexes I–IV and cyt c which can either be oxidized (not carrying an electron) or reduced (carrying an electron as indicated by the black

Article Snippet: This large reduction in cyt c together with a small reduction in cyt aa3 is indicative of a reduced pool of cyt c that no longer has physical access to CytOx and is no longer in redox equilibrium with cyt aa3 .

Techniques:

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Cytochrome c is rapidly reduced in the cytosol after mitochondrial outer membrane permeabilization

doi: 10.1007/s10495-010-0455-2

Figure Lengend Snippet: Oxygen concentration ( a ) and oxidation changes ( b ) in cyt c and cyt aa 3 during anoxia and after administration of rotenone ( arrow R) from a representative study. The shaded area denotes the time when cyt c and cyt aa 3 are reduced due to hypoxia

Article Snippet: This large reduction in cyt c together with a small reduction in cyt aa3 is indicative of a reduced pool of cyt c that no longer has physical access to CytOx and is no longer in redox equilibrium with cyt aa3 .

Techniques: Concentration Assay

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Cytochrome c is rapidly reduced in the cytosol after mitochondrial outer membrane permeabilization

doi: 10.1007/s10495-010-0455-2

Figure Lengend Snippet: Total Content ( a ) and oxidation state ( b ) of mitochondrial cyt c and cyt aa 3 in control cells (control), in anisomycin cells pretreatment (baseline) and 90 min post treatment (Anisomycin). All values are mean ± SD ( n = 6). Control and Anisomycin

Article Snippet: This large reduction in cyt c together with a small reduction in cyt aa3 is indicative of a reduced pool of cyt c that no longer has physical access to CytOx and is no longer in redox equilibrium with cyt aa3 .

Techniques:

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Cytochrome c is rapidly reduced in the cytosol after mitochondrial outer membrane permeabilization

doi: 10.1007/s10495-010-0455-2

Figure Lengend Snippet: Comparison of mitochondrial fraction ( a ) and cytosolic fraction ( b ) of cyt c measured by Western analysis with the time course of cyt c and cyt aa 3 oxidation changes ( c ). Cells were treated with anisomycin ( arrow A ) and rotenone ( arrow R ) and an anoxia

Article Snippet: This large reduction in cyt c together with a small reduction in cyt aa3 is indicative of a reduced pool of cyt c that no longer has physical access to CytOx and is no longer in redox equilibrium with cyt aa3 .

Techniques: Western Blot

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Cytochrome c is rapidly reduced in the cytosol after mitochondrial outer membrane permeabilization

doi: 10.1007/s10495-010-0455-2

Figure Lengend Snippet: Comparison of cleaved caspase 9 (cC9) ( a ), full length Caspase (fC3) and cleaved caspase 3 (cC3) ( b ) measured by Western analysis with the time course of cyt c and cyt aa 3 oxidation changes. Cells were treated with anisomycin ( arrow A ) and rotenone (

Article Snippet: This large reduction in cyt c together with a small reduction in cyt aa3 is indicative of a reduced pool of cyt c that no longer has physical access to CytOx and is no longer in redox equilibrium with cyt aa3 .

Techniques: Western Blot

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Cytochrome c is rapidly reduced in the cytosol after mitochondrial outer membrane permeabilization

doi: 10.1007/s10495-010-0455-2

Figure Lengend Snippet: Change in attenuation spectra obtained during anoxia prior to anisomycin treatment and at saturating oxygen tension 90 min after anisomycin treatment when release of cyt c was complete. The anisomycin treated spectrum has been offset by −1mOD

Article Snippet: This large reduction in cyt c together with a small reduction in cyt aa3 is indicative of a reduced pool of cyt c that no longer has physical access to CytOx and is no longer in redox equilibrium with cyt aa3 .

Techniques:

Journal: Bioscience Reports

Article Title: 2,5-Hexanedione mediates neuronal apoptosis through suppression of NGF via PI3K/Akt signaling in the rat sciatic nerve

doi: 10.1042/BSR20181122

Figure Lengend Snippet: The effect of NGF on HD-induced mitochondrial apoptosis via the PI3K/Akt signaling pathway VSC4.1 cells were exposed to 10 mM 2,5-HD, 10 mM 2,5-HD + 50 μg/l NGF or 10 mM 2,5-HD + 50 μg/l NGF + 25 μM LY294002 for 24 h. Western blot analysis was used to detect the Akt, p-Akt, Bad and p-Bad protein expression levels. ( A and B ) Detection of expression levels of Akt and p-Akt (A) or Bad and p-Bad (B) in VSC4.1 cells, respectively. Data are presented as mean ± S.D. ( n =3). ( C ) Cyt c was examined by immunofluorescence test in each condition (scale bar = 50 μm). ( D ) TUNEL labeling with DAPI counter staining was performed to detect apoptotic cells in each condition (scale bar = 20 μm). ( E ) Quantification of the abundance ratio of TUNEL-positive cells in each condition. ( F ) Detection of caspase-3 activity in each condition. a P

Article Snippet: Immunofluorescence VSC4.1 cells were fixed through incubation with a 4% paraformaldehyde solution for 10 min, followed by 0.3% TritonX-100 solution for 5 min and 10% goat serum albumin blocking solution at room temperature for 1 h. After the appropriate preparations, the cells were incubated with the primary antibody against Cyt c (1:500, Abcam, Cambridge, U.K.) at 4°C overnight, followed by an incubation with the secondary antibody labeled with Alexa Fluor 488 (Jackson, West Grove, U.S.A.) at room temperature for 45 min. Before mounting, cells were incubated for 5-min in 5 μg/ml DAPI solution.

Techniques: Western Blot, Expressing, Immunofluorescence, TUNEL Assay, Labeling, Staining, Activity Assay

Journal: Bioscience Reports

Article Title: 2,5-Hexanedione mediates neuronal apoptosis through suppression of NGF via PI3K/Akt signaling in the rat sciatic nerve

doi: 10.1042/BSR20181122

Figure Lengend Snippet: The effect of HD on the apoptosis induction test in the rat sciatic nerve ( A ) TUNEL labeling with DAPI counter staining to detect apoptotic cells in the rat sciatic nerve (scale bar = 20 μm). ( B ) Quantification of TUNEL-positive cells. ( C ) Detection of caspase-3 activity in the rat sciatic nerve. ( D ) The expression level of Cyt c determined by Western blot analysis in the mitochondria or cytosolic fractions and their quantifications. a P

Article Snippet: Immunofluorescence VSC4.1 cells were fixed through incubation with a 4% paraformaldehyde solution for 10 min, followed by 0.3% TritonX-100 solution for 5 min and 10% goat serum albumin blocking solution at room temperature for 1 h. After the appropriate preparations, the cells were incubated with the primary antibody against Cyt c (1:500, Abcam, Cambridge, U.K.) at 4°C overnight, followed by an incubation with the secondary antibody labeled with Alexa Fluor 488 (Jackson, West Grove, U.S.A.) at room temperature for 45 min. Before mounting, cells were incubated for 5-min in 5 μg/ml DAPI solution.

Techniques: TUNEL Assay, Labeling, Staining, Activity Assay, Expressing, Western Blot

Journal: Bioscience Reports

Article Title: 2,5-Hexanedione mediates neuronal apoptosis through suppression of NGF via PI3K/Akt signaling in the rat sciatic nerve

doi: 10.1042/BSR20181122

Figure Lengend Snippet: IGF-1 inhibition of HD-induced mitochondrial apoptosis via the PI3K/Akt signaling pathway in the VSC4.1 cells VSC4.1 cells were exposed to 10 mM 2,5-HD, 10 mM 2,5-HD + 50 ng/ml IGF-1 for 24 h. Western blot analysis was used to detect Akt, p-Akt, Bad and p-Bad protein expression. ( A and B ) Detection of expression levels of Akt and p-Akt (A) or Bad and p-Bad (B), respectively. Data are presented as mean ± S.D. ( n =3). ( C ) Examination of the dimerization of Bad/Bcl-xL complex in the mitochondria using Co-IP. ( D ) Immunofluorescence test for Cyt c release in VSC4.1 cells. ( E ) The detection of caspase-3 activity. ( F ) TUNEL labeling with DAPI counter staining was performed to detect apoptotic cells in VSC4.1 cells. ( G ) Quantification of the abundance ratio of TUNEL-positive cells in each condition. a P

Article Snippet: Immunofluorescence VSC4.1 cells were fixed through incubation with a 4% paraformaldehyde solution for 10 min, followed by 0.3% TritonX-100 solution for 5 min and 10% goat serum albumin blocking solution at room temperature for 1 h. After the appropriate preparations, the cells were incubated with the primary antibody against Cyt c (1:500, Abcam, Cambridge, U.K.) at 4°C overnight, followed by an incubation with the secondary antibody labeled with Alexa Fluor 488 (Jackson, West Grove, U.S.A.) at room temperature for 45 min. Before mounting, cells were incubated for 5-min in 5 μg/ml DAPI solution.

Techniques: Inhibition, Western Blot, Expressing, Co-Immunoprecipitation Assay, Immunofluorescence, Activity Assay, TUNEL Assay, Labeling, Staining

Journal: Journal of Neuroinflammation

Article Title: Targeting the NLRP3 inflammasome to attenuate spinal cord injury in mice

doi: 10.1186/s12974-017-0980-9

Figure Lengend Snippet: BAY 11-7082 or A438079 inhibits mitochondrial dysfunction. a Quantitative real-time PCR analysis of ATP synthase 3 days post injury. b Quantitative real-time PCR analysis of mitochondrial (mt) DNA 3 days post injury. c MMP level in the spinal cord tissue. d Representative western blots of cytosolic cytochrome c (Cyt C) 3 days post injury. e Quantification analysis of cytosolic Cyt C 3 days post injury. * p

Article Snippet: Moreover, the level of cytosolic Cyt C (1:1000; Abcam, Cambridge, UK) was also determined.

Techniques: Real-time Polymerase Chain Reaction, Western Blot

Journal: Molecular Cancer

Article Title: SapC-DOPS nanovesicles induce Smac- and Bax-dependent apoptosis through mitochondrial activation in neuroblastomas

doi: 10.1186/s12943-015-0336-y

Figure Lengend Snippet: SapC-DOPS treatment causes redistribution of apoptogenic proteins and Bax oligomerization in mitochondria of neuroblastoma cells. A) Immunoblots from whole cell extracts showing apoptotic protein expression changes following treatment with 50 μM SapC-DOPS. Fractions indicate fold-change estimated by densitometric analysis of proteins normalized to β-Actin corresponding to the lane. Fold change indicated for caspase-3 corresponds to the cleaved 19 kDa fragment. Right lanes: (−) refers to negative control (Schwann cells treated with SapC-DOPS) and (+) refers to positive control (SK-N-SH cells treated with 10 μM staurosporine) for 24 h. B) Relocation of Smac and Cyto c in SK-N-SH cells. Cox4 and Tubulin served as loading control for the mitochondrial and cytoplasmic fractions, respectively. C) Bax redistribution in neuroblastoma cells following SapC-DOPS treatment. D) Bax oligomerization in neuroblastoma cells. **P

Article Snippet: Anti-Bcl-2, anti-β-Actin (Abcam, Cambridge, MA), anti-Cyto c (eBioscience, San Diego, CA), anti-AIF, anti-caspase-3, anti-cleaved caspase-3 (Cell Signaling Technology, Boston, MA, anti-Survivin, Smac/Diablo, α-Tubulin (Novus biological, Littleton, CO), anti-COX-4, anti-Bax (N-20; Santa Cruz Biotechnology, La Jolla, CA), anti-Bax (polymer-recognizing A67 clone; Sigma, St.Louis, MO) and anti- cleaved PARP (Millipore, Bedford, MA).

Techniques: Western Blot, Expressing, Negative Control, Positive Control

Journal: Molecular Cancer

Article Title: SapC-DOPS nanovesicles induce Smac- and Bax-dependent apoptosis through mitochondrial activation in neuroblastomas

doi: 10.1186/s12943-015-0336-y

Figure Lengend Snippet: Apoptotic redistribution of Smac and Cyto c is independent of ROS formation, Cyclophilin D activity and Ca 2+ . A) Effect of N-acetyl cysteine (NAC)-mediated ROS inhibition on viability of SK-N-SH cells following SapC-DOPS treatment. B) Effect of pretreatment with 2 mM N-acetyl cysteine (NAC) following SapC-DOPS (50 μM) treatment on Smac and Cyto c relocation in SK-N-SH cells. C) Effect of 1 μM cyclosporine A (CsA)-pretreatment on Smac and Cyto c relocation following SapC-DOPS (50 μM) treatment in SK-N-SH cells. D) Flow cytometric measurement of Ca 2+ using Fluo-3 AM in SK-N-SH cells treated with 50 μM SapC-DOPS. Values represent geometric mean of fluorescence. Points, mean of three to five experiments; bars, SE. Western blots are representative of three independent experiments.

Article Snippet: Anti-Bcl-2, anti-β-Actin (Abcam, Cambridge, MA), anti-Cyto c (eBioscience, San Diego, CA), anti-AIF, anti-caspase-3, anti-cleaved caspase-3 (Cell Signaling Technology, Boston, MA, anti-Survivin, Smac/Diablo, α-Tubulin (Novus biological, Littleton, CO), anti-COX-4, anti-Bax (N-20; Santa Cruz Biotechnology, La Jolla, CA), anti-Bax (polymer-recognizing A67 clone; Sigma, St.Louis, MO) and anti- cleaved PARP (Millipore, Bedford, MA).

Techniques: Activity Assay, Inhibition, Flow Cytometry, Fluorescence, Western Blot

Journal: Analytical chemistry

Article Title: Characterization of Intramolecular Interactions of Cytochrome c Using Hydrogen Deuterium Exchange - Trapped Ion Mobility Spectrometry– Mass Spectrometry and Molecular Dynamics

doi: 10.1021/acs.analchem.7b00844

Figure Lengend Snippet: Left panel IMS spectra of the +6 to +11 charges states of cyt c as a function of the organic content (e.g., % methanol). Right panel : IMS spectra of the +6 to +11 charge states of cyt c as a function of the activation energy (e.g., deflector voltage).

Article Snippet: Horse heart cyt c (C2506) was purchased from Sigma-Aldrich (St. Louis, MO).

Techniques: Activation Assay

Journal: Analytical chemistry

Article Title: Characterization of Intramolecular Interactions of Cytochrome c Using Hydrogen Deuterium Exchange - Trapped Ion Mobility Spectrometry– Mass Spectrometry and Molecular Dynamics

doi: 10.1021/acs.analchem.7b00844

Figure Lengend Snippet: Candidate structures proposed for the kinetic intermediates IMS bands of cyt c . Conformations G and H for the +7 charge state (dark red background); and C, D, and E (dark green background), for the +8, and +9 charge states, were obtained after CIA.

Article Snippet: Horse heart cyt c (C2506) was purchased from Sigma-Aldrich (St. Louis, MO).

Techniques:

Journal: Analytical chemistry

Article Title: Characterization of Intramolecular Interactions of Cytochrome c Using Hydrogen Deuterium Exchange - Trapped Ion Mobility Spectrometry– Mass Spectrometry and Molecular Dynamics

doi: 10.1021/acs.analchem.7b00844

Figure Lengend Snippet: Left panel typical mass spectra of cyt c as a function of the starting solvent conditions. Right panel : overall CCS profiles (black lines) obtained by summation of the intensity-normalized IMS resolved data (color lines). The results obtained in the positive and negative ion mode are represented on the top and bottom part, respectively.

Article Snippet: Horse heart cyt c (C2506) was purchased from Sigma-Aldrich (St. Louis, MO).

Techniques:

Journal: Analytical chemistry

Article Title: Characterization of Intramolecular Interactions of Cytochrome c Using Hydrogen Deuterium Exchange - Trapped Ion Mobility Spectrometry– Mass Spectrometry and Molecular Dynamics

doi: 10.1021/acs.analchem.7b00844

Figure Lengend Snippet: Bottom : IMS profiles obtained with CIA (blue line), and without CIA (green line). Top : High resolution IMS profiles for the +12 to +21 charge states of cyt c .

Article Snippet: Horse heart cyt c (C2506) was purchased from Sigma-Aldrich (St. Louis, MO).

Techniques:

Journal: Journal of Biophotonics

Article Title: Ultrasensitive label-free photothermal imaging, spectral identification, and quantification of cytochrome c in mitochondria, live cells, and solutions

doi: 10.1002/jbio.201000012

Figure Lengend Snippet: KB-3 cells chromophores: (A) Phase-contrast image of a KB-3 cell fragment on substrate (Axioskop 2, Zeiss, 60×, NA, 1.3); (B) fluorescent imaging of cellular mitochondria labeled with MitoTracker Red; (C) fluorescent imaging of cyt c labeled with Oregon Green conjugated with anti–cytochrome- c antibody; (D) label-free PT images of the same cell (60× objective); excitation wavelength 538 nm, laser fluence 100 mJ/cm 2 , and 5 ns delay between excitation and probe pulses.

Article Snippet: For cyt c staining, cells were treated with anti-cyt c antibody according to manufacturer’s instructions (Pharmingen, CA, USA).

Techniques: Imaging, Labeling

Journal: Journal of Biophotonics

Article Title: Ultrasensitive label-free photothermal imaging, spectral identification, and quantification of cytochrome c in mitochondria, live cells, and solutions

doi: 10.1002/jbio.201000012

Figure Lengend Snippet: Conventional absorption spectra of (A) pure forms for cyt c (II) and cyt c (III) and cyt c released from mitochondria (a sample #1, corrected for scattering) (2 μM), (B) isolated mitochondria suspension and sample of cyt c released from mitochondria under various conditions (see the text for sample types #1–#6 description; and (inset) rat hepatocyte cells in suspension in concentration 5 × 10 6 cell/mL (Spectrophotometer Cary 5000, Varian, Inc).

Article Snippet: For cyt c staining, cells were treated with anti-cyt c antibody according to manufacturer’s instructions (Pharmingen, CA, USA).

Techniques: Isolation, Concentration Assay, Spectrophotometry

Journal: Journal of Biophotonics

Article Title: Ultrasensitive label-free photothermal imaging, spectral identification, and quantification of cytochrome c in mitochondria, live cells, and solutions

doi: 10.1002/jbio.201000012

Figure Lengend Snippet: cyt c cyt c released from mitochondria (solutions of sample type #1), dash-dotted line, dotted confidence interval. The data for the calibration plots were collected in three days, the repeatability relative standard deviation 0.02.

Article Snippet: For cyt c staining, cells were treated with anti-cyt c antibody according to manufacturer’s instructions (Pharmingen, CA, USA).

Techniques: Standard Deviation

Journal: EMBO Reports

Article Title: VDAC2 is required for truncated BID-induced mitochondrial apoptosis by recruiting BAK to the mitochondria

doi: 10.1038/embor.2009.219

Figure Lengend Snippet: VDAC2 dependence of tBID-induced cyto c -GFP release and cell death in permeabilized and intact MEFs. ( A ) Fluorescence time-lapse imaging of cyto c -GFP-expressing (green) and mtDsRed-expressing (red), permeabilized wt and V2 −/− MEFs. tBID (37 nM) caused release of cyto c -GFP (green to red shift in the overlaid images) only in wt MEFs. At the end of the experiment, 600 μg/ml digitonin was added to discharge the entire mitochondrial pool. Graphs showing the means of tBID-induced cyto c -GFP release kinetics of the entire cell population in the imaging field (15–20 cells; 4–5 measurements). ( B ) tBID adenovirus (16 h of infection) induced cell death. Means±s.e. ( n =3). ( C ) Immunoblot of cell lysates confirming the rescue of VDAC2 expression in VDAC2-transfected V2 −/− MEFs. ( D ) Fluorescence time-lapse imaging of control and rescued V2 −/− MEFs transfected with cyto c -GFP. Addition of 37 nM tBID caused progressive release of cyto c -GFP (shown in greyscale). Graphs showing the mean response of the entire cell population in the imaging field (15–20 cells; 4–5 measurements). The tBID-induced fluorescence change was normalized to the effect of digitonin. Ad-tBID, tBID adenovirus; cyto c , cytochrome c ; GFP, green fluorescent protein; MEF, mouse embryonic fibroblast; tBID, truncated BID; V2 −/− , VDAC2 −/− ; VDAC, voltage-dependent anion channel; wt, wild type.

Article Snippet: Primary antibodies were anti-cyto c (Pharmingen, San Jose, CA, USA), anti-BID (R & D Systems, Minneapolis, MN, USA), anti-BAK NT (Upstate, Bedford, MA, USA), anti-BAX N20 (Santa Cruz, Santa Cruz, CA, USA) and anti-VDAC2 (Abcam, Cambridge, MA, USA).

Techniques: Fluorescence, Imaging, Expressing, Infection, Transfection

Journal: EMBO Reports

Article Title: VDAC2 is required for truncated BID-induced mitochondrial apoptosis by recruiting BAK to the mitochondria

doi: 10.1038/embor.2009.219

Figure Lengend Snippet: BAX-dependent tBID-induced ΔΨ m loss and cyto c release in permeabilized BAK −/− and V2 −/− MEFs. ( A ) Immunoblot showing the presence of BAX in membrane and cytosol (upper left panels) and BAK in membrane (upper right panel) of permeabilized wt, BAK −/− and BAX −/− MEFs. Membranes from the permeabilized, washed cells were immunoblotted for BAX and BAK (lower two panels). ( B ) ΔΨ m was monitored for permeabilized and washed wt, BAK −/− and BAX −/− MEFs treated with 37 nM recombinant tBID. Immunoblot of rapidly separated supernatants (cytosol) showed the release of cyto c . ( C ) ΔΨ m was monitored for permeabilized V2 −/− MEFs treated either with 50 nM oligomeric BAX only (light grey trace) or with 50 nM BAX+37 nM tBID (dark grey trace). The supernatants (cytosol) were rapidly separated at different time points (indicated by arrows) and immunoblotted for cyto c . ( D ) Cumulative data showing BAX and BAX+tBID induced depolarization of V2 −/− MEFs. Cyto c , cytochrome c ; FCCP, carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone; MEF, mouse embryonic fibroblast; tBID, truncated BID; V2 −/− , VDAC2 −/− ; wt, wild type.

Article Snippet: Primary antibodies were anti-cyto c (Pharmingen, San Jose, CA, USA), anti-BID (R & D Systems, Minneapolis, MN, USA), anti-BAK NT (Upstate, Bedford, MA, USA), anti-BAX N20 (Santa Cruz, Santa Cruz, CA, USA) and anti-VDAC2 (Abcam, Cambridge, MA, USA).

Techniques: Recombinant

Journal: Journal of Translational Medicine

Article Title: Autologous Transplantation of Adipose-Derived Mesenchymal Stem Cells Markedly Reduced Acute Ischemia-Reperfusion Lung Injury in a Rodent Model

doi: 10.1186/1479-5876-9-118

Figure Lengend Snippet: Proposed mechanisms underlying the improvement in pulmonary functions after adipose-derived mesenchymal stem cell (ADMSC) treatment in a rodent model of pulmonary ischemia-reperfusion (IR) injury . vWF :von Willebrand factor, ET-1: Endothelin-1; TNF-α: Tumor necrosis factor-α; MMP-9: Matrix metalloproteinase-9; NF-κB: Nuclear factor kappa B; Cx43: Connexin43; IL-10: Interleukin-10; eNOS: Endothelial nitric oxide synthase; ICAM-1: Intercellular Adhesion Molecule-1; VCAM-1: Vascular cell adhesion molecule; HO-1: Heme oxygenase-1; NQO1: NAD(P)H quinone oxidoreductase; GR: Glutathione reductase; GPx: Glutathione peroxidase; Cyt C: Cytochrome C; RVSBP: Right ventricular systolic blood pressure; Sat O 2 : Oxygen saturation.

Article Snippet: The membranes were incubated with monoclonal antibodies against vascular cell adhesion molecule (VCAM)-1 (1: 100, Abcam, Cambridge, MA, USA), intercellular adhesion molecule (ICAM)-1 (1: 2000, Abcam, Cambridge, MA, USA), NAD(P)H quinone oxidoreductase (NQO)-1 (1: 1000, Abcam, Cambridge, MA, USA), connexin43 (Cx43) (1: 2000, Chemicon, Billerica, MA, USA), cytochrom C (Cyt C) (1: 2000, BD, San Jose, CA, USA) and heme oxygense (HO)-1 (1: 250, Abcam, Cambridge, MA, USA), and polyclonal antibodies against TNF-α (1: 1000, Cell Signaling, Danvers, MA, USA) and NFκB (1: 250, Abcam, Cambridge, MA, USA).

Techniques: Derivative Assay

Journal: Oncogene

Article Title: Differential role of FL-BID and t-BID during verotoxin-1-induced apoptosis in Burkitt’s lymphoma cells

doi: 10.1038/s41388-018-0123-5

Figure Lengend Snippet: Both FL-BID and caspase-8 activation are involved in CYT C and SMAC/DIABLO release from mitochondria during VT-1-induced apoptosis. a Cytosolic and mitochondrial extracts from Ramos cells preincubated (1 h) with or without the caspase-8 inhibitor Z-IETD-FMK (left) or from Ramos shCTRL and Ramos shBID cells (right), all treated or not with VT-1 for 6 h, were submitted to western blot analysis for detection of CYT C, SMAC/DIABLO, LDH and cyclophilin D (used as cytosolic and mitochondrial markers, respectively). Results shown are representative of at least three independent experiments. b CYT C (upper graphics) and SMAC/DIABLO (lower graphics) specific bands in cytosolic and mitochondrial fractions were quantified by densitometry, normalized to LDH or cyclophilin D levels and reported to the total amount (cytosolic + mitochondrial) of CYT C and SMAC/DIABLO. Values (means ± sd) are from four independent experiments. Statistical significance is as follows: ** p

Article Snippet: As seen in Fig. , this resulted in a large increase of both CYT C and SMAC/DIABLO release from mitochondria which did not depend on whether BID could be cleaved into t-BID (BID-WT, 75 ± 19% of CYT C and 50 ± 7% of SMAC/DIABLO release) or not (BID-D59A, 70 ± 14% of CYT C and 49 ± 6% of SMAC/DIABLO release) as compared to Ramos shBID cells transduced with an empty vector (26 ± 9 and 31 ± 6%, respectively).

Techniques: Activation Assay, Western Blot

Journal: Free Radical Biology & Medicine

Article Title: KRIT1 loss-of-function induces a chronic Nrf2-mediated adaptive homeostasis that sensitizes cells to oxidative stress: Implication for Cerebral Cavernous Malformation disease

doi: 10.1016/j.freeradbiomed.2017.11.014

Figure Lengend Snippet: The increased susceptibility to apoptotic cell death induced by KRIT1 loss-of-function occurs through the intrinsic, mitochondria-mediated pathway. Wild type (K +/+ ), KRIT1 -/- (K -/- ), and KRIT1 -/- re-expressing KRIT1 (K9/6) MEF cells were grown to confluence under standard conditions. Total cell extracts ( a ) or mitochondria and cytosolic fractions ( b ) were then obtained, and proteins involved in the intrinsic, mitochondria-dependent apoptotic pathway, including the anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins ( a ), and Cytochrome c (Cyt c) and Caspase-3 (Casp-3) ( b ), were analyzed by Western blotting as described in Materials and Methods. Cox IV and α-tubulin were used as internal loading controls for WB normalization of mitochondria and total/cytoplasmic proteins, respectively. Pro-Casp-3, intact protein; Casp-3, active fragment. A representative immunoblot is shown for each experiment. Histograms alongside their respective Western blots represent the mean (± SD) of the densitometric quantification of three independent experiments. **p ≤ 0.01 and ***p ≤ 0.001 versus K9/6 cells. Notice that the basal levels of Bcl-2 and Bax proteins in K -/- cells were significantly down-regulated and up-regulated, respectively, as compared with K +/+ and K9/6 cells ( a ). In addition, a significant increase in Cyt c release into the cytosol and activation of Casp-3 was also evident in K -/- cells ( b ).

Article Snippet: Primary antibodies used in the present study included the following: mouse anti-Methylglyoxal-AGE (Arg-Pyrimidine) mAb (clone 6B) (BioLogo, Hamburg, Germany); rabbit anti-Nrf2 pAb (Bioss Antibodies, Sial, Rome, Italy); rabbit anti-Nrf2 pAb (C-20) (Santa Cruz Biotechnology); rabbit anti-Nrf2 pAb (ab31163, Abcam); rabbit anti-Nrf2 mAb [EP1808Y] (AB62352, Abcam); rabbit anti-c-Jun pAb (H-79) (Santa Cruz Biotechnology); mouse anti-phospho-c-Jun mAb (KM-1) (Santa Cruz Biotechnology); mouse anti-Heme Oxygenase 1 mAb [HO-1-1] (Abcam); rabbit anti-Bax pAb (N20) (Santa Cruz Biotechnology); rat anti-Glyoxalase 1 mAb (6F10) (Santa Cruz Biotechnology); mouse anti-p-JNK mAb (G-7) (Santa Cruz Biotechnology); rabbit anti Caspase-3 pAb (9662, Cell Signaling Technology); rabbit anti phospho-Erk5 (Thr218/Tyr220) pAb (3371, Cell Signaling Technology); mouse anti-Bcl-2 mAb (DAKO, Milan, Italy); mouse anti-Cytochrome c (Cyt c) mAb (BD Pharmingen, Milan, Italy); mouse anti-Cyt c oxidase subunit IV (Cox IV) mAb (Molecular Probes, Monza, Italy); goat anti-mouse KLF4 pAb (AF3158, R & D Systems); goat anti-human KLF4 pAb (AF3640, R & D Systems); mouse anti-β-actin mAb (C4) (Santa Cruz Biotechnology); rabbit anti-lamin β1 pAb (H-90) (Santa Cruz Biotechnology); mouse anti-α-tubulin mAb (Sigma-Aldrich).

Techniques: Expressing, Western Blot, Activation Assay

Journal: Plant Physiology

Article Title: Programmed Cell Death during Pollination-Induced Petal Senescence in Petunia 1

doi:

Figure Lengend Snippet: Abundance of Cyt c in cellular fractions at flower opening and following compatible pollination. Immunoblot of a 12% SDS-PAGE gel was probed with a monoclonal anti-Cyt c antibody. A, Crude petal extract; S, 1,500 g supernatant (extract devoid of nuclei and chloroplasts); C, 16,000 g supernatant (cytosol); M, 16,000 g pellet (mitochondria); and P, 1,500 g pellet (nucleus and chloroplast). Bar graph indicates comparative densitometry data of the Cyt c signal with the highest signal in mitochondria as 1.

Article Snippet: A monoclonal anti- Cyt c antibody from Pharmingen (San Diego) was used for immunoblot detection.

Techniques: SDS Page

Journal: Plant Physiology

Article Title: Programmed Cell Death during Pollination-Induced Petal Senescence in Petunia 1

doi:

Figure Lengend Snippet: Distribution of Cyt c during pollination-induced petal senescence. Immunoblot of a 12% SDS-PAGE gel probed with a monoclonal anti-Cyt c antibody (top). The bottom panel shows Ponceau staining of the same gel as a loading control. Bovine Cyt c (40 ng, lane C) was used as a positive control (15 kD). m, Rainbow marker; 0, 24, and 30, HACP.

Article Snippet: A monoclonal anti- Cyt c antibody from Pharmingen (San Diego) was used for immunoblot detection.

Techniques: SDS Page, Staining, Positive Control, Marker