Journal: PLoS ONE

Article Title: Post-Injury Treatment with 7,8-Dihydroxyflavone, a TrkB Receptor Agonist, Protects against Experimental Traumatic Brain Injury via PI3K/Akt Signaling

doi: 10.1371/journal.pone.0113397

Figure Lengend Snippet: Antibodies used in immunofluorescence and western blot.

Article Snippet: Phospho-Bad Ser136 , Cell signaling , 4366 , Rabbit , Monoclonal , WB 1∶1000.

Techniques: Immunofluorescence, Western Blot

Journal: PLoS ONE

Article Title: Salidroside Improves Behavioral and Histological Outcomes and Reduces Apoptosis via PI3K/Akt Signaling after Experimental Traumatic Brain Injury

doi: 10.1371/journal.pone.0045763

Figure Lengend Snippet: ( A, B, C ) Representative immunoblots and densitometric analysis showed a significant increase in p-Akt Ser473 level in the ipsilateral cortex of 20 mg/kg salidroside (SALD 20)-treated injured mice at day 1 compared with vehicle-treated injured mice. The level of p-Akt Thr308 was increased following SALD 20 treatment at day 3 compared with vehicle-treated injured mice. ( D, E, F ) Representative immunoblots and densitometric analysis showed that there were no differences in the levels of p-Bad and p-FOXO1 in the ipsilateral cortex between the SALD 20 and vehicle groups at day 1 or 3. ( G, H, I ) Representative immunoblots and densitometric analysis showed that the mitochondrial Bcl-2/Bax ratio significantly increased in SALD-20 treated injured mice but no difference was found in the total Bcl-2/Bax ratio. Values are presented as mean ± SEM; # P <0.05, ## P <0.01 versus sham controls, and * P <0.05 versus vehicle-treated injured mice (n = 6 mice/group at each time point, repeated measures two-way ANOVA for p-Akt Ser473, p-Akt Thr308, p-Bad, and p-FOXO1 levels; one-way ANOVA for Bcl-2/Bax ratio).

Article Snippet: p-Bad (Ser136) , Cell signaling , 4366 , Rabbit , Monoclonal , WB 1∶1000.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Salidroside Improves Behavioral and Histological Outcomes and Reduces Apoptosis via PI3K/Akt Signaling after Experimental Traumatic Brain Injury

doi: 10.1371/journal.pone.0045763

Figure Lengend Snippet: Representative immunoblots and densitometric analysis showed that (A, B, C, D) levels of cleaved caspase 3, p-Akt Ser473, p-Akt Thr308, (E, F, G) p-Bad and p-FOXO1 in ipsilateral hippocampal samples from injured animals at post-injury day 1 or 3 were not significantly different from those in sham-injured animals (n = 6 mice/group at each time point, repeated measures two-way ANOVA).

Article Snippet: p-Bad (Ser136) , Cell signaling , 4366 , Rabbit , Monoclonal , WB 1∶1000.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Salidroside Improves Behavioral and Histological Outcomes and Reduces Apoptosis via PI3K/Akt Signaling after Experimental Traumatic Brain Injury

doi: 10.1371/journal.pone.0045763

Figure Lengend Snippet: ( A, B, C, D, E, F ) Representative immunoblots and densitometric analysis in the cortex from sham-injured mice or mice subject to cortical impact injury, infused intracerebroventricular (icv) with saline (vehicle) or LY294002. Mice received either icv pretreatment of LY294002 followed by 20 mg/kg salidroside (SALD 20) after injury (S20Y group) or icv pretreatment of saline followed by SALD 20 after CCI (S20V group). LY294002 did not affect Akt, Bad, or FOXO1 phosphorylation in sham-operated mice, but it significantly abolished SALD 20-induced preservation of p-Akt Thr308 and p-FOXO1 levels in the ipsilateral hemisphere at 1 day post-injury. The levels of p-Akt Ser473 and p-Bad were similar between the S20V and S20LY groups. Values are presented as mean ± SEM; * P <0.05, ** P <0.01 versus sham + vehicle (ShamV), and & P <0.05 versus sham + LY294002 (ShamLY), and # P <0.05 versus S20V group (n = 6 mice/group, one-way ANOVA). ( G ) Representative cresyl violet-stained brain sections of S20V and S20LY mice at 28 days post-injury. Scale bar is 1 mm. ( H ) Quantification analysis showed that there was a significant decrease in the residual cerebral tissue ratio in the S20LY group compared with the S20S group. Values are presented as mean ± SEM; * P <0.05 versus S20V group (n = 6 mice/group, Student’s t -test).

Article Snippet: p-Bad (Ser136) , Cell signaling , 4366 , Rabbit , Monoclonal , WB 1∶1000.

Techniques: Western Blot, Preserving, Staining

Journal: PLoS ONE

Article Title: Salidroside Improves Behavioral and Histological Outcomes and Reduces Apoptosis via PI3K/Akt Signaling after Experimental Traumatic Brain Injury

doi: 10.1371/journal.pone.0045763

Figure Lengend Snippet: Antibodies used in immunofluorescence and western blot.

Article Snippet: p-Bad (Ser136) , Cell signaling , 4366 , Rabbit , Monoclonal , WB 1∶1000.

Techniques: Immunofluorescence, Western Blot

Journal: PLoS ONE

Article Title: Bid Participates in Genotoxic Drug-Induced Apoptosis of HeLa Cells and Is Essential for Death Receptor Ligands' Apoptotic and Synergistic Effects

doi: 10.1371/journal.pone.0002844

Figure Lengend Snippet: A) Cells were stably transfected with plasmids encoding one of three different bid -specific shRNAs or a scrambled control shRNA as described in . For analysis of the bid knockdown, cells were subjected to Western blotting with a polyclonal Bid antibody. α-Tubulin served as a loading control. B) Control cells expressing the scrambled (Ctrl 1, Ctrl 2), or the bid specific shRNA (Bid kd 4) and clone (Bid kd 1) were incubated with Cycloheximide (CHX) (1 µg/ml) and an agonistic Fas antibody or vehicle (clone CH11) at the indicated concentrations or vehicle for 4 h. Caspase-3 like activity was measured by cleavage of the fluorigenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. # p<0.05: difference from control cells (Ctrl). C) Cell lysates were subjected to Western blotting with antibodies for the indicated proteins, as described in . D) HeLa Bid kd cells were stably transfected with a vector coding for YFP- bid -CFP (GFP-Bid); cell lysates were subjected to Western blotting with GFP and Bid antibodies. E) HeLa Bid kd cells expressing GFP-Bid were treated with CHX (1 µg/ml) and an activating Fas antibody (100 ng/ml). Controls were treated with vehicle for 4 h; caspase-3 like activity was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. # p<0.05 difference from control cells (Ctrl).

Article Snippet: The following primary antibodies were used: a rabbit polyclonal APAF-1 (Chemicon, Carrigtwohill, Ireland), a rabbit polyclonal Bad or caspase-3 (Cell Signaling, Bray, Ireland), a mouse monoclonal Bak or Bcl-2 (Santa Cruz, Heidelberg, Germany), a rabbit polyclonal Bax (Upstate, Carrigtwohill, Ireland), a rabbit polyclonal Bcl-X (BD Biosciences, Erembodgem, Belgium), a mouse monoclonal Bim or caspase-8 (Alexis Biochemicals, Blessington, Ireland), a goat polyclonal Bid (R&D Systems, Abingdon, UK), a rabbit polyclonal caspase-9 (Calbiochem, Darmstadt, Germany), a rabbit polyclonal DR4 or DR5 (Abcam, Cambridge, UK), a rabbit poyclonal GFP (Clontech, Oxford, UK), a mouse monoclonal KDEL (Stressgen, York, UK), a rabbit polyclonal cleaved PARP (NEB, Bray, Ireland), a rabbit polyclonal smac/DIABLO (R&D Systems), a mouse monoclonal XIAP (BD Biosciences), a mouse monoclonal β-actin or α-tubulin (Sigma Aldrich, Dublin, Ireland).

Techniques: Stable Transfection, Transfection, shRNA, Western Blot, Expressing, Incubation, Activity Assay, Plasmid Preparation

Journal: PLoS ONE

Article Title: Bid Participates in Genotoxic Drug-Induced Apoptosis of HeLa Cells and Is Essential for Death Receptor Ligands' Apoptotic and Synergistic Effects

doi: 10.1371/journal.pone.0002844

Figure Lengend Snippet: Control and HeLa Bid kd were pre-incubated with the pan-caspase inhibitor zVAD-fmk (100 µM) for 1 h followed by treatment for the specified times with CHX (1 µg/ml) in combination with an agonistic Fas antibody or recombinant TRAIL at the indicated concentrations. Controls were treated with vehicle. A, B) Caspase-3 like activity was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. # p<0.05 difference from control cells (Ctrl). C, D) Cells were treated as indicated and lysates were subjected to Western blotting with a polyclonal caspase-3, a monoclonal Poly-ADP-Ribose Polymerase (PARP) antibody and a monoclonal α-tubulin antibody. E, F) Cells were treated as indicated and apoptosis was assessed by flow cytometric evaluation of Annexin-V FITC conjugated binding to phosphatidylserine in non-permeabilized cells. Data are means+/−SD from n = 3 separate experiments. # p<0.05 difference from control cells (Ctrl).

Article Snippet: The following primary antibodies were used: a rabbit polyclonal APAF-1 (Chemicon, Carrigtwohill, Ireland), a rabbit polyclonal Bad or caspase-3 (Cell Signaling, Bray, Ireland), a mouse monoclonal Bak or Bcl-2 (Santa Cruz, Heidelberg, Germany), a rabbit polyclonal Bax (Upstate, Carrigtwohill, Ireland), a rabbit polyclonal Bcl-X (BD Biosciences, Erembodgem, Belgium), a mouse monoclonal Bim or caspase-8 (Alexis Biochemicals, Blessington, Ireland), a goat polyclonal Bid (R&D Systems, Abingdon, UK), a rabbit polyclonal caspase-9 (Calbiochem, Darmstadt, Germany), a rabbit polyclonal DR4 or DR5 (Abcam, Cambridge, UK), a rabbit poyclonal GFP (Clontech, Oxford, UK), a mouse monoclonal KDEL (Stressgen, York, UK), a rabbit polyclonal cleaved PARP (NEB, Bray, Ireland), a rabbit polyclonal smac/DIABLO (R&D Systems), a mouse monoclonal XIAP (BD Biosciences), a mouse monoclonal β-actin or α-tubulin (Sigma Aldrich, Dublin, Ireland).

Techniques: Incubation, Recombinant, Activity Assay, Western Blot, Binding Assay

Journal: PLoS ONE

Article Title: Bid Participates in Genotoxic Drug-Induced Apoptosis of HeLa Cells and Is Essential for Death Receptor Ligands' Apoptotic and Synergistic Effects

doi: 10.1371/journal.pone.0002844

Figure Lengend Snippet: Control and HeLa Bid kd cells were pre-incubated with the pan-caspase inhibitor zVAD-fmk (100 µM) for 1 h before treatment with the indicated concentrations of Staurosporine for the indicated times. A) Caspase-3 like activity was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. n.s. = not significant versus control (Ctrl). B) Cells were treated as indicated and lysates were subjected to Western blotting with a polyclonal caspase-3, a monoclonal Poly-ADP-Ribose Polymerase (PARP) antibody and a monoclonal α-tubulin antibody. C, D) The small molecule Bid inhibitor BI6C9 mimics the effects of the knockdown of Bid in HeLa cells. Parental HeLa cells were pre-incubated with the indicated concentrations of BI6C9 for 16 h prior to treatment with an agonistic Fas antibody, Staurosporine (STS) or vehicle, for 6 h. Apoptosis was assessed by flow cytometric evaluation of Annexin-V FITC conjugated binding to phosphatidylserine in non-permeabilized cells. Data are means+/−SD from n = 3 separate experiments. ∗ p<0.05: difference from control cultures treated with Fas antibody or Staurosporine in the absence of BI6C9.

Article Snippet: The following primary antibodies were used: a rabbit polyclonal APAF-1 (Chemicon, Carrigtwohill, Ireland), a rabbit polyclonal Bad or caspase-3 (Cell Signaling, Bray, Ireland), a mouse monoclonal Bak or Bcl-2 (Santa Cruz, Heidelberg, Germany), a rabbit polyclonal Bax (Upstate, Carrigtwohill, Ireland), a rabbit polyclonal Bcl-X (BD Biosciences, Erembodgem, Belgium), a mouse monoclonal Bim or caspase-8 (Alexis Biochemicals, Blessington, Ireland), a goat polyclonal Bid (R&D Systems, Abingdon, UK), a rabbit polyclonal caspase-9 (Calbiochem, Darmstadt, Germany), a rabbit polyclonal DR4 or DR5 (Abcam, Cambridge, UK), a rabbit poyclonal GFP (Clontech, Oxford, UK), a mouse monoclonal KDEL (Stressgen, York, UK), a rabbit polyclonal cleaved PARP (NEB, Bray, Ireland), a rabbit polyclonal smac/DIABLO (R&D Systems), a mouse monoclonal XIAP (BD Biosciences), a mouse monoclonal β-actin or α-tubulin (Sigma Aldrich, Dublin, Ireland).

Techniques: Incubation, Activity Assay, Western Blot, Binding Assay

Journal: PLoS ONE

Article Title: Bid Participates in Genotoxic Drug-Induced Apoptosis of HeLa Cells and Is Essential for Death Receptor Ligands' Apoptotic and Synergistic Effects

doi: 10.1371/journal.pone.0002844

Figure Lengend Snippet: Control cells and HeLa Bid kd cells were pre-incubated with the pan-caspase inhibitor zVAD-fmk (100 µM) where specified for 1 h before treatment with the ER stressors Tunicamycin, Brefeldin A, and Thapsigargin at the indicated concentrations for the indicated times. A) Cell lysates were subjected to Western blotting with a monoclonal KDEL antibody, which detects Grp94 and Grp78, and a monoclonal α-tubulin antibody. B, C, D) Caspase-3 like activity was measured by cleavage of the fluorigenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. n.s. = not significant versus control (Ctrl). E) Cells were treated as indicated and lysates were subjected to Western blotting with a polyclonal caspase-3 antibody, a monoclonal Poly-ADP-Ribose Polymerase (PARP) antibody and a monoclonal α-tubulin antibody. F) Apoptosis was assessed by flow cytometric evaluation of Annexin-V FITC conjugated binding to phosphatidylserine in non-permeabilized cells. Data are means+/−SD from n = 3 separate experiments. n.s. = not significant versus control (Ctrl).

Article Snippet: The following primary antibodies were used: a rabbit polyclonal APAF-1 (Chemicon, Carrigtwohill, Ireland), a rabbit polyclonal Bad or caspase-3 (Cell Signaling, Bray, Ireland), a mouse monoclonal Bak or Bcl-2 (Santa Cruz, Heidelberg, Germany), a rabbit polyclonal Bax (Upstate, Carrigtwohill, Ireland), a rabbit polyclonal Bcl-X (BD Biosciences, Erembodgem, Belgium), a mouse monoclonal Bim or caspase-8 (Alexis Biochemicals, Blessington, Ireland), a goat polyclonal Bid (R&D Systems, Abingdon, UK), a rabbit polyclonal caspase-9 (Calbiochem, Darmstadt, Germany), a rabbit polyclonal DR4 or DR5 (Abcam, Cambridge, UK), a rabbit poyclonal GFP (Clontech, Oxford, UK), a mouse monoclonal KDEL (Stressgen, York, UK), a rabbit polyclonal cleaved PARP (NEB, Bray, Ireland), a rabbit polyclonal smac/DIABLO (R&D Systems), a mouse monoclonal XIAP (BD Biosciences), a mouse monoclonal β-actin or α-tubulin (Sigma Aldrich, Dublin, Ireland).

Techniques: Incubation, Western Blot, Activity Assay, Binding Assay

Journal: PLoS ONE

Article Title: Bid Participates in Genotoxic Drug-Induced Apoptosis of HeLa Cells and Is Essential for Death Receptor Ligands' Apoptotic and Synergistic Effects

doi: 10.1371/journal.pone.0002844

Figure Lengend Snippet: A) HeLa Control and HeLa Bid kd cells were pre-incubated with the pan-caspase inhibitor zVAD-fmk for 1 h where specified; cells were subsequently treated with Tunicamycin or vehicle for the indicated times. Cell lysates were subjected to Western blotting with a polyclonal DR4, a polyclonal DR5, and a monoclonal β-actin antibody. B, C) Control cells and HeLa Bid kd cells were pre-incubated for 16 h with the indicated concentrations of Tunicamycin, Thapsigargin or vehicle before treatment with TRAIL (10 ng/ml). Caspase-3 like activity was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. ∗ p<0.05: difference from Tunicamycin, Thapsigargin or TRAIL individual treatments. # p<0.05: difference from control cells (Ctrl). D) Control cells and HeLa Bid kd cells were pre-incubated with Tunicamycin (0.3 µM) or vehicle for the indicated times followed by treatment with TRAIL (10 ng/ml) for the indicated times. Cell lysates were subjected to Western blotting with a polyclonal Bid, a polyclonal caspase-3, a polyclonal Poly-ADP-Ribose Polymerase (PARP) antibody and a monoclonal α-tubulin antibody. E) Control cells and HeLa Bid kd cells were pre-incubated with Tunicamycin (0.3 µM) for 16 h followed by treatment with recombinant TRAIL (10 ng/ml) for the indicated times. Apoptosis was assessed by flow cytometric evaluation of Annexin-V FITC conjugated binding to phosphatidylserine in non-permeabilized cells. Data are means+/−SD from n = 3 separate experiments. ∗ p<0.05: difference from Tunicamycin or TRAIL individual treatments. # p<0.05: difference from control cells (Ctrl). F) Control cells and HeLa Bid kd cells were pre-incubated with a DR5 blocking peptide (50 ng/ml) for 1 h followed by pre-treatment with Tunicamycin (0.3 µM) or vehicle for 16 h. Subsequently, cells were treated with recombinant TRAIL for 3 h. Caspase-3 like activity was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. ∗ p<0.05: difference from Tunicamycin or TRAIL individual treatments. # p<0.05: difference from control cells (Ctrl).

Article Snippet: The following primary antibodies were used: a rabbit polyclonal APAF-1 (Chemicon, Carrigtwohill, Ireland), a rabbit polyclonal Bad or caspase-3 (Cell Signaling, Bray, Ireland), a mouse monoclonal Bak or Bcl-2 (Santa Cruz, Heidelberg, Germany), a rabbit polyclonal Bax (Upstate, Carrigtwohill, Ireland), a rabbit polyclonal Bcl-X (BD Biosciences, Erembodgem, Belgium), a mouse monoclonal Bim or caspase-8 (Alexis Biochemicals, Blessington, Ireland), a goat polyclonal Bid (R&D Systems, Abingdon, UK), a rabbit polyclonal caspase-9 (Calbiochem, Darmstadt, Germany), a rabbit polyclonal DR4 or DR5 (Abcam, Cambridge, UK), a rabbit poyclonal GFP (Clontech, Oxford, UK), a mouse monoclonal KDEL (Stressgen, York, UK), a rabbit polyclonal cleaved PARP (NEB, Bray, Ireland), a rabbit polyclonal smac/DIABLO (R&D Systems), a mouse monoclonal XIAP (BD Biosciences), a mouse monoclonal β-actin or α-tubulin (Sigma Aldrich, Dublin, Ireland).

Techniques: Incubation, Western Blot, Activity Assay, Recombinant, Binding Assay, Blocking Assay

Journal: PLoS ONE

Article Title: Bid Participates in Genotoxic Drug-Induced Apoptosis of HeLa Cells and Is Essential for Death Receptor Ligands' Apoptotic and Synergistic Effects

doi: 10.1371/journal.pone.0002844

Figure Lengend Snippet: HeLa control and HeLa Bid kd cells were treated with the indicated concentrations of Etoposide, Oxaliplatin and Doxorubicin, or vehicle, for 24 h; cells were pre-incubated with the pan-caspase inhibitor zVAD-fmk (100 µM) for 1 h where specified. A, B, C) Caspase-3 like activity was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. # p<0.05 difference from control cells (Ctrl). D) Cells were treated as indicated and lysates were subjected to Western blotting with a polyclonal Bid, a polyclonal caspase-3, a polyclonal Poly-ADP-Ribose Polymerase (PARP) antibody and a monoclonal α-tubulin antibody. E) Apoptosis was assessed by flow cytometric evaluation of Annexin-V FITC conjugated binding to phosphatidylserine in non-permeabilized cells. Data are means+/−SD from n = 3 separate experiments. # p<0.05 difference from control cells (Ctrl).

Article Snippet: The following primary antibodies were used: a rabbit polyclonal APAF-1 (Chemicon, Carrigtwohill, Ireland), a rabbit polyclonal Bad or caspase-3 (Cell Signaling, Bray, Ireland), a mouse monoclonal Bak or Bcl-2 (Santa Cruz, Heidelberg, Germany), a rabbit polyclonal Bax (Upstate, Carrigtwohill, Ireland), a rabbit polyclonal Bcl-X (BD Biosciences, Erembodgem, Belgium), a mouse monoclonal Bim or caspase-8 (Alexis Biochemicals, Blessington, Ireland), a goat polyclonal Bid (R&D Systems, Abingdon, UK), a rabbit polyclonal caspase-9 (Calbiochem, Darmstadt, Germany), a rabbit polyclonal DR4 or DR5 (Abcam, Cambridge, UK), a rabbit poyclonal GFP (Clontech, Oxford, UK), a mouse monoclonal KDEL (Stressgen, York, UK), a rabbit polyclonal cleaved PARP (NEB, Bray, Ireland), a rabbit polyclonal smac/DIABLO (R&D Systems), a mouse monoclonal XIAP (BD Biosciences), a mouse monoclonal β-actin or α-tubulin (Sigma Aldrich, Dublin, Ireland).

Techniques: Incubation, Activity Assay, Western Blot, Binding Assay

Journal: PLoS ONE

Article Title: Bid Participates in Genotoxic Drug-Induced Apoptosis of HeLa Cells and Is Essential for Death Receptor Ligands' Apoptotic and Synergistic Effects

doi: 10.1371/journal.pone.0002844

Figure Lengend Snippet: A, B) HeLa Control and HeLa Bid kd cells were treated with Etoposide (10 µM) or Oxaliplatin (30 µg/ml) for the indicated times; the pan-caspase inhibitor zVAD (100 µM) was added to the cells 1 h prior to treatment where specified; cell lysates were subjected to Western blotting with a polyclonal DR4, a polyclonal DR5, and a monoclonal β-actin antibody. C, D) HeLa control and HeLa Bid kd cells were pre-incubated with Etoposide (10 µM), Oxaliplatin (30 µg/ml), or vehicle for 16 h followed by treatment with TRAIL (10 ng/ml) for 3 h. Caspase-3 like activity was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. ∗ p<0.05: difference from Etoposide, Oxaliplatin or TRAIL individual treatments. # p<0.05 difference from control cells (Ctrl).

Article Snippet: The following primary antibodies were used: a rabbit polyclonal APAF-1 (Chemicon, Carrigtwohill, Ireland), a rabbit polyclonal Bad or caspase-3 (Cell Signaling, Bray, Ireland), a mouse monoclonal Bak or Bcl-2 (Santa Cruz, Heidelberg, Germany), a rabbit polyclonal Bax (Upstate, Carrigtwohill, Ireland), a rabbit polyclonal Bcl-X (BD Biosciences, Erembodgem, Belgium), a mouse monoclonal Bim or caspase-8 (Alexis Biochemicals, Blessington, Ireland), a goat polyclonal Bid (R&D Systems, Abingdon, UK), a rabbit polyclonal caspase-9 (Calbiochem, Darmstadt, Germany), a rabbit polyclonal DR4 or DR5 (Abcam, Cambridge, UK), a rabbit poyclonal GFP (Clontech, Oxford, UK), a mouse monoclonal KDEL (Stressgen, York, UK), a rabbit polyclonal cleaved PARP (NEB, Bray, Ireland), a rabbit polyclonal smac/DIABLO (R&D Systems), a mouse monoclonal XIAP (BD Biosciences), a mouse monoclonal β-actin or α-tubulin (Sigma Aldrich, Dublin, Ireland).

Techniques: Western Blot, Incubation, Activity Assay

Journal: PLoS ONE

Article Title: N-Cadherin Mediates Neuronal Cell Survival through Bim Down-Regulation

doi: 10.1371/journal.pone.0033206

Figure Lengend Snippet: Serum-starved GT1-7 cells were seeded at low density on PL or N-cad and grown for the indicated time (A) or for 4 hours (C) in serum-free medium. In B-D, cells were subjected to a calcium switch protocol. As a positive control, cells were treated with 10 ng/ml of PMA or DMSO for 1 hour. Equivalent amount of proteins were immunobloted with antibodies to Bcl-2 and phospho-Bad. Relative densities of Bcl-2 and phospho-Bad were normalized with either α-tubulin, or β-actin. Results are the mean ± SD of three independent experiments.

Article Snippet: The following antibodies were used for western blotting : rabbit anti-Akt (1∶1000; Cell Signaling Technology, Beverly, MA), rabbit anti-phospho-Akt (1∶1000; Cell Signaling Technology), rabbit anti-Erk1/2 (1/10000; Upstate Biotechnology, Inc. Lake Placid, NY), mouse anti-phospho-Erk1/2 (1/10000; Sigma), mouse anti-Bcl-2 (1/500; Transduction Laboratories, Becton Dickinson Europe, Le Pont de Claix, France), rabbit anti-Bcl-xL (1/1000; Cell Signaling Technology), rabbit anti-Bim (1/1000; Pharmingen, San Diego, CA), rabbit anti-phospho-Bad (1/1000; Cell Signaling Technology), rabbit anti-PARP (1/1000; Cell signalling Technology), mouse anti-β-actin (1/20000; clone AC-15; Sigma), mouse anti-α-tubulin (1∶20000, clone E7, Developmental Studies Hybridoma Bank).

Techniques: Positive Control

Journal: PLoS ONE

Article Title: c-MET Protects Breast Cancer Cells from Apoptosis Induced by Sodium Butyrate

doi: 10.1371/journal.pone.0030143

Figure Lengend Snippet: (A) to (D) showed the effect of NaBu on breast cancer cell line MDA-MB-231(A and B) and MCF-7 (C and D). Majority of MDA-MB-231(B) and MCF-7(D) cancer cells were removed by the treatment of NaBu. Compared with 16% of viable MCF cells, the survival rate of MDA-MB-231 was significantly increased to 30%(E). Immunoblotting result of BAD, BAX, Bcl-2 and Cytochrome C expression indicated that MDA-MB-231 cells were resistant to the treatment of NaBu by neutralizing its apoptosis effect compared with MCF-7 cells (F). *P<0.05.

Article Snippet: After blocking in a powdered nonfat milk solution (5% in PBS) with 0.05% Tween-20, the blot was incubated with a polyclonal rabbit anti- human Bcl-2-associated death promoter (BAD) antibody (Cell Signaling, Danvers, MA),a rabbit anti- human BAX antibody (Cell Signaling), a rabbit anti- human Bcl-2 antibody (Cell Signaling), a rabbit anti- human Cytochrome C antibody (Cell Signaling), a rabbit anti-human CD133 antibody (Cell Signaling) and a rabbit anti-human beta– actin antibody (Cell Signaling) at 1∶1000 dilutions in 5% blocking solution over night at 4°C.

Techniques: Western Blot, Expressing