total bax  (Bio-Techne corporation)

Journal: BMC Cardiovascular Disorders

Article Title: Hypoxia-inducible factor-1alpha is a critical mediator of hypoxia induced apoptosis in cardiac H9c2 and kidney epithelial HK-2 cells

doi: 10.1186/1471-2261-8-9

Figure Lengend Snippet: Flow cytometry analysis followed by Bax protein expression. H9c2 cells were transfected with pEGFP, pEGFP and pcDNA3-HA- HIF-1α or pcDNA3-Flag-DN-HIF-1α and were sorted in a flow cytometer. The EGFP positive sorted cells were subjected to hypoxia for 6 h. Total Bax expression in the same lysates was used to monitor protein loading. N = Normoxia; H = Hypoxia. The data are representative of 3 separate experiments and only 2 experiments using dominant negative Hif-1α (right panel).

Article Snippet: After blocking with TBS/5% skim milk, the membranes were incubated overnight at 4°C with primary antibodies against Hif-1α (1:500 dilution, Novus Biologicals, Littleton, CO), the active monomer of Bax (Monoclonal antibody 6A7, 0.5 μg/ml concentration, Alexis Biochemicals, San Diego, CA) or total Bax (1:1000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA), followed by secondary antibodies conjugated with horseradish peroxidase (1:10,000) (Pierce, Rockford, IL) for 1 h at room temperature.

Techniques: Flow Cytometry, Expressing, Transfection, Dominant Negative Mutation

Journal: BMC Cardiovascular Disorders

Article Title: Hypoxia-inducible factor-1alpha is a critical mediator of hypoxia induced apoptosis in cardiac H9c2 and kidney epithelial HK-2 cells

doi: 10.1186/1471-2261-8-9

Figure Lengend Snippet: HIF-1α promotes Bax activation in HK-2 cells. Cells were subjected to normoxia or hypoxia of 16 h after sorting in a flow cytometer. Active and Total Bax protein expression of the whole cell lysates of EGFP positive cells is shown of cells transfected with either pEGFP or pEGFP and pcDNA3-HA-Hif-1α or pcDNA3-DN-Hif-1α. N = Normoxia; H = Hypoxia. Data shown is representative of two separate experiments.

Article Snippet: After blocking with TBS/5% skim milk, the membranes were incubated overnight at 4°C with primary antibodies against Hif-1α (1:500 dilution, Novus Biologicals, Littleton, CO), the active monomer of Bax (Monoclonal antibody 6A7, 0.5 μg/ml concentration, Alexis Biochemicals, San Diego, CA) or total Bax (1:1000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA), followed by secondary antibodies conjugated with horseradish peroxidase (1:10,000) (Pierce, Rockford, IL) for 1 h at room temperature.

Techniques: Activation Assay, Flow Cytometry, Expressing, Transfection

Journal: PLoS ONE

Article Title: Role of Mitofusin 2 in the Renal Stress Response

doi: 10.1371/journal.pone.0031074

Figure Lengend Snippet: ( A ) Immunoblot of active Bax ( upper panel ) detected with an anti-6A7 epitope-specific antibody in MFN2 f/f proximal tubule epithelial lysates of cells transduced with no virus ( NT ), empty adenovirus ( CTL ) or Cre adenovirus ( MFN2- ) at baseline and 30 min after ATP depletion; total Bax controls are show for sample loading (lower panel) ; and ( B ) densitometric analysis of the relative increase in active 6A7-Bax content after metabolic stress.

Article Snippet: Total Bax antibody was purchased from Cell Signaling Technologies (Danvers, MA).

Techniques: Western Blot, Transduction

Journal: PLoS ONE

Article Title: Fibronectin-Integrin Signaling Is Required for L-Glutamine’s Protection against Gut Injury

doi: 10.1371/journal.pone.0050185

Figure Lengend Snippet: A) IEC-6 cells were treated with different concentrations of GLN (0, 2, 10, and 20 mM) with or without 1 h prior PD98059 treatment. Cell survival was measured via MTS assay. Results are shown as mean±SEM (n = 3). B) [T(P) 202 /Y(P) 204 ]ERK1/2 and total ERK1/2 levels were determined by Western blot analysis after basal and stressed 43°C conditions without recovery. ERK1/2 activation is shown as mean fold change relative to total ERK1/2±SEM and ratioed to 0 mM GLN (n = 3).

Article Snippet: Primary antibodies against HSP32 and HSP70 (1∶10,000 dilution) (StressGen, Victoria, BC, Canada) and FN (1∶10,000), or against total ERK1/2, [T(P) 202 /Y(P) 204 ]ERK1/2, total HSF-1, [S(P) 303 ]HSF-1, cleaved caspase-3, cleaved PARP, bax, and bcl-2 (1∶1,000) (Cell signaling, Danvers, MA) were used.

Techniques: MTS Assay, Western Blot, Activation Assay

Journal: PLoS ONE

Article Title: Fibronectin-Integrin Signaling Is Required for L-Glutamine’s Protection against Gut Injury

doi: 10.1371/journal.pone.0050185

Figure Lengend Snippet: GLN is protective in the intestine by preventing FN degradation after thermal injury, as well as by activating the protective FN-Integrin signaling pathway. GLN phosphorylates ERK1/2 via the FN-Integrin pathway leading to HSF-1 activation, which enhances HSP expression to prevent apoptosis.

Article Snippet: Primary antibodies against HSP32 and HSP70 (1∶10,000 dilution) (StressGen, Victoria, BC, Canada) and FN (1∶10,000), or against total ERK1/2, [T(P) 202 /Y(P) 204 ]ERK1/2, total HSF-1, [S(P) 303 ]HSF-1, cleaved caspase-3, cleaved PARP, bax, and bcl-2 (1∶1,000) (Cell signaling, Danvers, MA) were used.

Techniques: Activation Assay, Expressing

Journal: Developmental cell

Article Title: Endolysosomal Targeting of Mitochondria is Integral to BAX-mediated Mitochondrial Permeabilization During Apoptosis Signaling

doi: 10.1016/j.devcel.2020.05.014

Figure Lengend Snippet: (A) MCF-7_shCON, MCF-7_shRab5A or MCF-7_shRab5C cells, treated with STS (4 hrs), or TNFα/AcD (6 hrs). IF of total cellular BAX (α-BAX N-20) and of BAX in active conformation (α-BAX 6A7).

Article Snippet: Following, cells were incubated with a conformation-specific BAX antibody 6A7 (Abcam) and an antibody detecting total cellular BAX (Santa Cruz Biotechnology) at 37°C for 1 hr.

Techniques: