Journal: PLoS ONE

Article Title: Putative Structural and Functional Coupling of the Mitochondrial BK Ca Channel to the Respiratory Chain

doi: 10.1371/journal.pone.0068125

Figure Lengend Snippet: A. Detection of mitoBK Ca channel regulatory β4 subunit mRNA in astrocytoma cells. The BK Ca subunit β4 mRNA was detected at a size of 405 bp. No products were obtained for the BK Ca subunits β1, β2 and β3. GAPDH served as a positive control and was detected at a size of 496 bp. The negative control without reverse transcriptase (−RT) and samples without cDNA (−A) had no signals. The results presented are representative of seven independent experiments. B. Immunoblot of astrocytoma mitochondria, astrocytoma cell homogenate and brain homogenate fractions labeled with the anti-BK Ca channel β4 subunit antibody. A control antigen (BK Ca β4+ peptide) was used as a positive control for the specificity of the antibody. An anti-cytochrome c oxidase subunit IV antibody (COX IV) was used as a mitochondrial marker (n = 3). C. Immuno-gold electron microscopy localization of the BK Ca channel β4 regulatory subunit in mitochondria of cultured human astrocytoma cells. The β4 subunit molecules were labeled using 10 nm colloidal-gold particles (arrows). D. High-power confocal image of cultured astrocytoma cells immunolabeled to detect OxPhos (red) and β4-GFP-transfected cells (green). The superimposition of the two signals revealed the mitochondrial localization of BK Ca β4 in human astrocytoma cells (yellow). The DNA-binding dye DAPI was used to stain the cell nuclei (blue). For details concerning the astrocytoma cells, see the .

Article Snippet: The membranes were exposed to polyclonal antibodies that recognize BK Ca channel β subunit 4 (anti-β4, 1∶200, Alomone Labs) and cytochrome c oxidase subunit IV (COX IV, 1∶1000, Cell Signaling).

Techniques: Positive Control, Negative Control, Western Blot, Labeling, Marker, Electron Microscopy, Cell Culture, Immunolabeling, Transfection, Binding Assay, Staining

Journal: PLoS ONE

Article Title: Putative Structural and Functional Coupling of the Mitochondrial BK Ca Channel to the Respiratory Chain

doi: 10.1371/journal.pone.0068125

Figure Lengend Snippet: Two-dimensional separation was performed as described in the , and the PVDF membrane was first immunoblotted for the BK Ca channel β4 subunit (below, Coomassie staining panel). Next, the PVDF membrane was immunoblotted for the subunits of individual respiratory chain complexes (below the BK Ca β4 panel). The BN-PAGE was calibrated based on the location of mitochondrial respiratory chain complexes that were isolated from rat heart mitochondria (above the panel for the blue native PAGE of mitochondria from astrocytoma cells). In the native astrocytoma lysate, mitochondria BK Ca β4 co-localized with subunit I of cytochrome c oxidase. M, the monomeric form of cytochrome c oxidase; D, the dimeric form of cytochrome c oxidase; Sc 1 and Sc 2 , complexes with higher molecular weights containing cytochrome c oxidase. A typical immunoblot from three separate experiments is shown.

Article Snippet: The membranes were exposed to polyclonal antibodies that recognize BK Ca channel β subunit 4 (anti-β4, 1∶200, Alomone Labs) and cytochrome c oxidase subunit IV (COX IV, 1∶1000, Cell Signaling).

Techniques: Staining, Isolation, Blue Native PAGE, Western Blot

Journal: PLoS ONE

Article Title: Putative Structural and Functional Coupling of the Mitochondrial BK Ca Channel to the Respiratory Chain

doi: 10.1371/journal.pone.0068125

Figure Lengend Snippet: A. Detection of mitoBK Ca channel regulatory β4 subunit mRNA in astrocytoma cells. The BK Ca subunit β4 mRNA was detected at a size of 405 bp. No products were obtained for the BK Ca subunits β1, β2 and β3. GAPDH served as a positive control and was detected at a size of 496 bp. The negative control without reverse transcriptase (−RT) and samples without cDNA (−A) had no signals. The results presented are representative of seven independent experiments. B. Immunoblot of astrocytoma mitochondria, astrocytoma cell homogenate and brain homogenate fractions labeled with the anti-BK Ca channel β4 subunit antibody. A control antigen (BK Ca β4+ peptide) was used as a positive control for the specificity of the antibody. An anti-cytochrome c oxidase subunit IV antibody (COX IV) was used as a mitochondrial marker (n = 3). C. Immuno-gold electron microscopy localization of the BK Ca channel β4 regulatory subunit in mitochondria of cultured human astrocytoma cells. The β4 subunit molecules were labeled using 10 nm colloidal-gold particles (arrows). D. High-power confocal image of cultured astrocytoma cells immunolabeled to detect OxPhos (red) and β4-GFP-transfected cells (green). The superimposition of the two signals revealed the mitochondrial localization of BK Ca β4 in human astrocytoma cells (yellow). The DNA-binding dye DAPI was used to stain the cell nuclei (blue). For details concerning the astrocytoma cells, see the .

Article Snippet: The immunoreactions consisted of sequential incubations with a rabbit polyclonal anti-BK Ca channel β subunit 4 antibody (anti-β4, 1∶20, Alomone Labs) followed by species-specific donkey secondary antibodies coupled to 10 nm gold particles (Electron Microscopy Sciences).

Techniques: Positive Control, Negative Control, Western Blot, Labeling, Marker, Electron Microscopy, Cell Culture, Immunolabeling, Transfection, Binding Assay, Staining

Journal: PLoS ONE

Article Title: Putative Structural and Functional Coupling of the Mitochondrial BK Ca Channel to the Respiratory Chain

doi: 10.1371/journal.pone.0068125

Figure Lengend Snippet: Two-dimensional separation was performed as described in the , and the PVDF membrane was first immunoblotted for the BK Ca channel β4 subunit (below, Coomassie staining panel). Next, the PVDF membrane was immunoblotted for the subunits of individual respiratory chain complexes (below the BK Ca β4 panel). The BN-PAGE was calibrated based on the location of mitochondrial respiratory chain complexes that were isolated from rat heart mitochondria (above the panel for the blue native PAGE of mitochondria from astrocytoma cells). In the native astrocytoma lysate, mitochondria BK Ca β4 co-localized with subunit I of cytochrome c oxidase. M, the monomeric form of cytochrome c oxidase; D, the dimeric form of cytochrome c oxidase; Sc 1 and Sc 2 , complexes with higher molecular weights containing cytochrome c oxidase. A typical immunoblot from three separate experiments is shown.

Article Snippet: The immunoreactions consisted of sequential incubations with a rabbit polyclonal anti-BK Ca channel β subunit 4 antibody (anti-β4, 1∶20, Alomone Labs) followed by species-specific donkey secondary antibodies coupled to 10 nm gold particles (Electron Microscopy Sciences).

Techniques: Staining, Isolation, Blue Native PAGE, Western Blot