Journal: Cell Death and Differentiation

Article Title: Prohibitin levels regulate OMA1 activity and turnover in neurons

doi: 10.1038/s41418-019-0469-4

Figure Lengend Snippet: a Representative western blot of OPA1 long (L-OPA1) and short (S-OPA1) isoforms in N2a cells transiently overexpressing PHB1/2 (PHB) or empty vector. Cells were treated with 10 µM CCCP or vehicle (DMSO). β-actin was used as a loading control. b The ratio of L-OPA1 to S-OPA1 was quantified from n = 4 experiments. *p < 0.05, Student’s t test. c Cytochrome c content in mitochondrial pellets (P) or released into supernatant (S) measured by western blots of mitochondria isolated from N2a cells overexpressing PHB or vector control incubated for 30 min with 100 nM tBID at 30 °C. TIM23 served as a mitochondrial protein loading control. d Quantification of cytochrome c localization in tBID treated mitochondria relative to total cytochrome c detected, n = 5 experiments. *p < 0.05 S relative to P fraction, Student’s t test. e Schematic representation of C-terminal OMA1-Flag constructed and proposed activating cleavage site (dashed line) and resulting OMA1 isoforms. f OMA1 activation visualized by western blot of lysates prepared from N2a cells transiently expressing a C-terminal OMA1-Flag and treated with 10 µM CCCP or vehicle (DMSO). Blots were probed with antibodies to OPA1, Flag, and β-actin as a loading control. g Western blot of whole cell lysates from N2a cells overexpressing a C-terminal OMA1-Flag construct plus PHB1/2, siRNA targeting PHB1, or scrambled (Scr) control siRNA. Blots were probed with specific antibodies to Flag, PHB1, PHB2, and β-actin as a loading control. h Quantification of relative OMA1-Flag band intensity normalized by β-actin from n = 4 experiments. *p < 0.05, **p < 0.01 relative to Scr controls, Student’s t test.

Article Snippet: The N2a mouse neuroblastoma cell line (ATCC ® CCL-131TM) was cultured in DMEM with 10% fetal bovine serum at 37 °C in a 5% CO 2 incubator.

Techniques: Western Blot, Plasmid Preparation, Control, Isolation, Incubation, Construct, Activation Assay, Expressing

Journal: Cell Death and Differentiation

Article Title: Prohibitin levels regulate OMA1 activity and turnover in neurons

doi: 10.1038/s41418-019-0469-4

Figure Lengend Snippet: a Western blot of lysates from OMA1 KO MEFs transiently transfected with an internally Flag-tagged OMA1 construct and PHB1/2 or vector control probed with specific antibodies to Flag, PHB1, and β-actin as a loading control. b OMA1-Flag bands were quantified relative to β-actin from n = 5 experiments. *p < 0.05, **p < 0.01, Student’s t test. c Western blot of N2a cells transfected with internal Flag-OMA1 treated with 10 µM CCCP for 30 min or vehicle (DMSO). d OMA1-Flag bands were quantified relative to β-actin from n = 5 experiments. ***p < 0.001, Student’s t test. e Western blot of N2a cells transfected with empty vector or full length (FL) or deletion (Δ245–305) internal OMA1-Flag constructs. Hyphen indicates an empty lane to control for background signal. Blots were probed with specific antibodies to Flag and β-actin as a loading control. f Schematic representation of internally Flag-tagged OMA1 constructs with cleavages sites for the MTS and inhibitory C-terminus (activating).

Article Snippet: The N2a mouse neuroblastoma cell line (ATCC ® CCL-131TM) was cultured in DMEM with 10% fetal bovine serum at 37 °C in a 5% CO 2 incubator.

Techniques: Western Blot, Transfection, Construct, Plasmid Preparation, Control

Journal: Cell Death and Differentiation

Article Title: Prohibitin levels regulate OMA1 activity and turnover in neurons

doi: 10.1038/s41418-019-0469-4

Figure Lengend Snippet: a Membrane potential was measured in N2a cells transiently expressing empty vector, PHB1/2, scrambled siRNA control, or siRNA targeting PHB1 using 50 nM TMRM fluorescence (left panels). Background fluorescence was measured after treatment with 10 µM CCCP for 30 min (right panels). Scale bar = 25 µm. b Quantification of average TMRM fluorescence minus background fluorescence after CCCP. n = 5 vect, n = 12 PHB, n = 5 Scr, and n = 12 siPHB wells. c Western blots of whole cell lysates from N2a cells expressing empty vector, PHB1/2, scrambled siRNA control, or siRNA targeting PHB1. Blots were probed with specific antibodies to Yme1L and β-actin as a loading control. d Quantification of relative Yme1L band intensity normalized by β-actin from n = 4 independent experiments. e Western blots of whole cell lysates from N2a cells transfected with internally Flag-tagged OMA1 plus siRNA targeting Yme1L or scrambled siRNA control. Blots were probed with specific antibodies to Flag, Yme1L, PHB1, and β-actin as a loading control.

Article Snippet: The N2a mouse neuroblastoma cell line (ATCC ® CCL-131TM) was cultured in DMEM with 10% fetal bovine serum at 37 °C in a 5% CO 2 incubator.

Techniques: Membrane, Expressing, Plasmid Preparation, Control, Fluorescence, Western Blot, Transfection

Journal: Cell Death and Differentiation

Article Title: Prohibitin levels regulate OMA1 activity and turnover in neurons

doi: 10.1038/s41418-019-0469-4

Figure Lengend Snippet: a Schematic representation of full length (FL) and Δ144–163 deletion mutant internally Flag-tagged OMA1 constructs. b Western blot of CL-binding proteins pulled down using CL-conjugated beads from crude mitochondria isolated from N2a cells expressing either full length (FL) or CL-binding domain deletion (Δ144–163) internally Flag-tagged OMA1 constructs. Blots were probed with specific antibodies to Flag, PHB1 as a positive CL-binding control, and HSP60 as a non-CL-binding control. c 2D BN-PAGE of digitonin-treated mitochondria from N2a cells expressing internally Flag-tagged OMA1. Blot probed with specific antibodies to OPA1, Flag, and PHB1. OPA1, Flag, and PHB immunoreactivity align in high-molecular-weight complexes (dashed box).

Article Snippet: The N2a mouse neuroblastoma cell line (ATCC ® CCL-131TM) was cultured in DMEM with 10% fetal bovine serum at 37 °C in a 5% CO 2 incubator.

Techniques: Mutagenesis, Construct, Western Blot, Binding Assay, Isolation, Expressing, Control, High Molecular Weight