Journal: Journal of neurochemistry

Article Title: Prohibitin is a positive modulator of mitochondrial function in PC12 cells under oxidative stress

doi: 10.1111/jnc.14472

Figure Lengend Snippet: (A) Levels of PHB1 and SLP-2 were measured in whole cell lysates by immunoblot. HSP60 was used as a loading control. (B) Cardiolipin content was measured in mitochondria isolated from shSCR and shPHB PC12 cells. (C) Cardiolipin binding proteins were pulled down from mitochondrial lysates using cardiolipin coated beads and separated by SDS-PAGE for immunoblotting. Uncoated beads were used to control for non-specific binding and unbound lysate was used as an input control. *p<0.05, n=3 separate experiments. (D) The data are shown as mean±SEM.

Article Snippet: Cardlolipin Binding Assay Mitochondrial cardiolipin-binding proteins were pulled down using cardiolipin coated beads (P-BCLP, Echelon Biosciences, Salt Lake City, UT).

Techniques: Western Blot, Isolation, Binding Assay, SDS Page

Journal: Cell reports

Article Title: Mitoregulin: A lncRNA-Encoded Microprotein that Supports Mitochondrial Supercomplexes and Respiratory Efficiency

doi: 10.1016/j.celrep.2018.06.002

Figure Lengend Snippet: (A and B) Wild-type (A) and GFP-tagged (B) human Mtln were expressed in neonatal rat cardiomyocytes, and co-localization with MitoTracker red was evaluated. Representative photomicrographs are shown. Scale bars, 10 μm. (C) Mitochondrial pellets were isolated from wild-type (WT) or Mtln-knockout (KO) C2C12 myoblast cells, and western blot was performed on various fractions. (D) Mitochondrial pellets harvested from WT or Mtln-KO skeletal muscle tissues were treated with increasing digitonin concentrations to release OMMs, and pellet and supernatant fraction fractions were subjected to western blot analysis. Cox4 and Vdac1 are known IMM and OMM proteins, respectively. Gapdh is a cytosolic protein known to associate with mitochondria in some cases. (E) Mitochondrial pellets harvested from WT skeletal muscle tissues were resuspended in isotonic, hypotonic, or isotonic plus triton buffers in the absence or presence of proteinase K and subjected to western blot analysis. Proteins with known localization to various mitochondrial compartments (e.g., matrix, IMM, and intermembrane space [IMS]) were evaluated as controls. (F) Western blot analysis performed on WT and Mtln-KO cardiac tissue lysates subjected to pull-down assay using cardiolipin (CL)-coated or control beads. Subunit c, a known cardiolipin-binding protein, serves as the positive control. (G) Lipid-strip binding assay performed using synthetic Mtln protein followed by anti-Mtln immunoblot.

Article Snippet: Cardiolipin-bound agarose beads and control beads (Echelon) were incubated with 500 μg of mouse heart lysates for 2 h at 4°C, followed by 5 washes in 10 mM HEPES, 150 mM NaCl and 0.25% (v/v) NP-40.

Techniques: Isolation, Knock-Out, Western Blot, Pull Down Assay, Binding Assay, Positive Control, Stripping Membranes

Journal: Cell reports

Article Title: Mitoregulin: A lncRNA-Encoded Microprotein that Supports Mitochondrial Supercomplexes and Respiratory Efficiency

doi: 10.1016/j.celrep.2018.06.002

Figure Lengend Snippet:

Article Snippet: Cardiolipin-bound agarose beads and control beads (Echelon) were incubated with 500 μg of mouse heart lysates for 2 h at 4°C, followed by 5 washes in 10 mM HEPES, 150 mM NaCl and 0.25% (v/v) NP-40.

Techniques: Recombinant, Isolation, Glutathione Assay, Knock-Out, Negative Control, Multiplex Assay, CRISPR, Plasmid Preparation, Software

Journal: Cell reports

Article Title: Mitoregulin: A lncRNA-Encoded Microprotein that Supports Mitochondrial Supercomplexes and Respiratory Efficiency

doi: 10.1016/j.celrep.2018.06.002

Figure Lengend Snippet: (A and B) Wild-type (A) and GFP-tagged (B) human Mtln were expressed in neonatal rat cardiomyocytes, and co-localization with MitoTracker red was evaluated. Representative photomicrographs are shown. Scale bars, 10 μm. (C) Mitochondrial pellets were isolated from wild-type (WT) or Mtln-knockout (KO) C2C12 myoblast cells, and western blot was performed on various fractions. (D) Mitochondrial pellets harvested from WT or Mtln-KO skeletal muscle tissues were treated with increasing digitonin concentrations to release OMMs, and pellet and supernatant fraction fractions were subjected to western blot analysis. Cox4 and Vdac1 are known IMM and OMM proteins, respectively. Gapdh is a cytosolic protein known to associate with mitochondria in some cases. (E) Mitochondrial pellets harvested from WT skeletal muscle tissues were resuspended in isotonic, hypotonic, or isotonic plus triton buffers in the absence or presence of proteinase K and subjected to western blot analysis. Proteins with known localization to various mitochondrial compartments (e.g., matrix, IMM, and intermembrane space [IMS]) were evaluated as controls. (F) Western blot analysis performed on WT and Mtln-KO cardiac tissue lysates subjected to pull-down assay using cardiolipin (CL)-coated or control beads. Subunit c, a known cardiolipin-binding protein, serves as the positive control. (G) Lipid-strip binding assay performed using synthetic Mtln protein followed by anti-Mtln immunoblot.

Article Snippet: Cardiolipin Beads , Echelon Biosciences , P-BCLP.

Techniques: Isolation, Knock-Out, Western Blot, Pull Down Assay, Binding Assay, Positive Control, Stripping Membranes

Journal: Cell reports

Article Title: Mitoregulin: A lncRNA-Encoded Microprotein that Supports Mitochondrial Supercomplexes and Respiratory Efficiency

doi: 10.1016/j.celrep.2018.06.002

Figure Lengend Snippet:

Article Snippet: Cardiolipin Beads , Echelon Biosciences , P-BCLP.

Techniques: Recombinant, Isolation, Glutathione Assay, Knock-Out, Negative Control, Multiplex Assay, CRISPR, Plasmid Preparation, Software

Journal: PLoS ONE

Article Title: Cytochrome c Negatively Regulates NLRP3 Inflammasomes

doi: 10.1371/journal.pone.0167636

Figure Lengend Snippet: (A) Immunoblots of cell lysates and either NLRP3 or sham immunoprecipitates prepared from LPS-primed THP-1 macrophage cells, stimulated with ATP or not, to detect an interaction between endogenous NLRP3 and cytochrome c. The ratios between the NLRP3 and cytochrome c levels in the immunoprecipitates were quantitated using Image J. (B) Immunoblots of cell lysates and either control or Myc immunoprecipitates to map the portion of NLRP3 important for the interaction with cytochrome c. Myc-tagged NLRP3 constructs were transfected into HEK 293T cells. The immunoprecipitates were washed, incubated with cytochrome c (50 ng), washed again, fractionated by SDS-PAGE, and immunoblotted. (C) Immunoblots of cell lysates and Myc-LRR domain immunoprecipitates incubated with cytochrome c, or not. A construct expressing a myc tagged NLRP3 LRR domain was transfected into HEK 293T cells. The Myc and control immunoprecipitates were incubated with cytochrome c (50 ng), washed, and immunoblotted. (D) Schematic of the constructs used in the above experiments (B & C). (E) Immunoblots of cell lysates and cardiolipin bead pull-downs to assess whether cytochrome c interferes with the interaction between cardiolipin and NLRP3. BSA (50 ng) or purified cytochrome c (50 ng) was added to lysates prepared from HEK 293T cells expressing NLRP3-Flag. Following a 30 minute incubation cardiolipin conjugated beads were added to the lysates for an additional 30 minutes. The caridolipin beads were washed; and the bound NLRP3 eluted in SDS-sample buffer, size fractionated by SDS PAGE, and quantitated by immunoblotting. The amount of NLRP3 in the cardiolipin pulldowns was normalized to the BSA control. The above experiments were respectively performed twice. (F) Cell lysates from cell treated with ATP, or not, were fractionated into cytosolic and mitochondrial fractions and the indicated proteins were immunoblotted. NLRP3 immunoprecipitates were prepared using the cytosolic fraction. An interaction between endogenous NLRP3 and cytochrome c was assess by immunoblotting. The ratios between the NLRP3 and cytochrome c levels in the immunoprecipitates were quantitated using Image J.

Article Snippet: Afterwards cardiolipin-coated agarose beads (Echelon) were added for an additional 30 minutes to pull down cardiolipin binding proteins.

Techniques: Western Blot, Construct, Transfection, Incubation, SDS Page, Expressing, Purification