Journal: eLife

Article Title: Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells

doi: 10.7554/eLife.67624

Figure Lengend Snippet: ( A ) Schematic illustration of OCIAD1 domain organization. ( B ) Representative images of fixed U2OS cells stained with Mitotracker (magenta) and immunolabeled using anti-OCIAD1 antibodies (green). Lower panel is a magnification of the inset shown in the upper panel. ( C ) OCIAD1 is an integral membrane protein. Sodium carbonate extraction fractions (pH 10.5–11.5) immunoblotted with anti-OCIAD1, anti-TIM50, anti-ATP5A1, and anti-SDHA antibodies. P and S indicate pellet and soluble fractions, respectively. ( D ) OCIAD1 localizes to the inner membrane. Protease protection assay fractions immunoblotted with anti-OCIAD1, anti-prohibitin 2 (PHB2), anti-TIM50, anti-ATP5A1, and anti-SDHA antibodies. (OMM: outer mitochondrial membrane, IMM: inner mitochondrial membrane, IMS: intermembrane space). ( E ) Schematic illustration of OCIAD1 topology within the inner membrane.

Article Snippet: The sample was halved and incubated with either mouse anti-PHB2 antibodies (Proteintech, cat# 66424–1-Ig, 70 ng, ~1.8 μL) or vehicle (PBS) on ice of 90 min.

Techniques: Staining, Immunolabeling, Membrane, Extraction

Journal: eLife

Article Title: Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells

doi: 10.7554/eLife.67624

Figure Lengend Snippet: ( A ) Volcano plot showing the statistical significance (−log 10 false discovery rate [FDR] adjusted p-value; y axis) versus log 2 fold change (x axis) of proteins enriched in OCIAD1 pull-down performed on DSP-crosslinked K562 cell lysates from OCIAD1 knockdown cells and OCIAD1 knockdown cells rescued with wildtype OCIAD1. Proteins with a log 2 fold change ≥ 1.5 and an adjusted p-value < 0.05 were considered significantly enriched (n = 3 biological replicates, n.s. = non significantly enriched). ( B ) Blue-native polyacrylamide gel electrophoresis (BN-PAGE) of lauryl maltose neopentyl glycol (LMNG) detergent-solubilized mitochondrial membranes isolated from U2OS control, OCIAD1 knockdown, and OCIAD1 knockdown cells rescued with wildtype OCIAD1. The membrane was immunoblotted with anti-OCIAD1 and anti-PHB2 antibodies. ( C ) BN-PAGE of LMNG detergent-solubilized mitochondrial membranes isolated from U2OS control cells (n = 3 biological replicates) and immunoblotted with anti-OCIAD1 and anti-PHB2 antibodies. Electrophoresis was stopped before elution of the migration front to calculate the fraction of OCIAD1 that associates with PHB2 assemblies (66.91 ± 0.35%). ( D ) Mitochondria from K562 cells solubilized with LMNG and pre-incubated with anti-Phb2 antibodies (solid line) or vehicle (dotted line) were analyzed by BN-PAGE and immunoblotted with anti-OCIAD1 and anti-prohibitin two antibodies. Line scan traces represent the distribution profile of P (brown) and OCIAD1 (light blue). Figure 4—source data 1. Output of the limma statistical analysis of the OCIAD1 interactome in K562 OCIAD1 knockdown cells and K562 OCIAD1 knockdown cells rescued with wildtype OCIAD1. Figure 4—source data 2. Output of the limma statistical analysis of the whole proteome analysis by LC-MS/MS.

Article Snippet: The sample was halved and incubated with either mouse anti-PHB2 antibodies (Proteintech, cat# 66424–1-Ig, 70 ng, ~1.8 μL) or vehicle (PBS) on ice of 90 min.

Techniques: Knockdown, Polyacrylamide Gel Electrophoresis, Isolation, Control, Membrane, Electrophoresis, Migration, Incubation, Liquid Chromatography with Mass Spectroscopy

Journal: eLife

Article Title: Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells

doi: 10.7554/eLife.67624

Figure Lengend Snippet: ( A ) Schematic of predicted OCIAD1 and OCIAD2 topologies. ( B ) Representative images of fixed U2OS cells stained with Mitotracker (magenta) and immunolabeled for OCIAD2 (green). The bottom panel is a magnification of the inset shown in the upper panel. ( C ) OCIAD2 is an integral membrane protein. Sodium carbonate extraction fractions (pH 10.5–11.5) immunoblotted with anti-OCIAD1, anti-OCIAD2, anti-TIM50, anti-ATP5A1, and anti-SDHA antibodies. P and S indicate pellet and soluble fractions, respectively. This panel, without the OCIAD2 blot, was shown in . ( E ) OCIAD2 localizes to the inner membrane. Protease protection assay fractions immunoblotted with anti-OCIAD1, anti-OCIAD2, anti-prohibitin 2 (PHB2), anti-TIM50, anti-ATP5A1, and anti-SDHA antibodies (OMM: outer mitochondrial membrane, IMM: inner mitochondrial membrane, IMS: intermembrane space). This panel, without the OCIAD2 blot, was shown in . ( F ) Blue-native polyacrylamide gel electrophoresis (BN-PAGE) of lauryl maltose neopentyl glycol (LMNG) detergent-solubilized mitochondrial membranes isolated from U2OS cells and immunoblotted with anti-OCIAD2 and anti-prohibitin antibodies.

Article Snippet: The sample was halved and incubated with either mouse anti-PHB2 antibodies (Proteintech, cat# 66424–1-Ig, 70 ng, ~1.8 μL) or vehicle (PBS) on ice of 90 min.

Techniques: Staining, Immunolabeling, Membrane, Extraction, Polyacrylamide Gel Electrophoresis, Isolation

Journal: eLife

Article Title: Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells

doi: 10.7554/eLife.67624

Figure Lengend Snippet: ( A ) 2D-native/SDS-PAGE analysis of mitochondrial membranes isolated from K562 control and OCIAD1 knockdown cells and immunoblotted with CYC1 and PHB2 antibodies. CIII 2 assemblies from OCIAD1 knockdown cells contained immature CYC1 of higher molecular weight. PHB2 staining served as an internal molecular size reference. Light blue horizontal lines represent the size of putative precursor (p), intermediate (i), and mature (m) CYC1. White vertical lines represent the different high-order CIII 2 assemblies. ( B ) Extracted MS2 fragment ion chromatograms (XIC) for three diagnostic CYC1 peptides detected by diaPASEF mass spectrometry in blue-native polyacrylamide gel electrophoresis (BN-PAGE) gel slices excised from control cells, OCIAD1 knockdown cells, and OCIAD1 knockdown cells rescued with wildtype OCIAD1. Individual peptides displayed highly correlated fragment ion co-elution profiles strongly supportive of peptide identification. The TPQAVALSSK ++ peptide (bottom panel), located at the N-terminus of the CYC1 hydrophobic sorting sequence, was only identified in CIII 2 assemblies from OCIAD1 knockdown cells. Conversely, the SDLELHPPSYPWSHR +++ peptide (middle panel), which uniquely identifies the N-terminus of mature CYC1 but is not present in the tryptic digest of the CYC1 precursor, was reliably detected in CIII 2 assemblies from control and OCIAD1 knockdown cells rescued with wildtype OCIAD1, but not from OCIAD1 knockdown cells. An internal peptide (LFDYFPKPYPNSEAAR +++ , top panel) common to all CYC1 species (precursor, intermediate, mature) was detected in all cell lines, albeit at lower levels in OCIAD1 cells as expected.

Article Snippet: The sample was halved and incubated with either mouse anti-PHB2 antibodies (Proteintech, cat# 66424–1-Ig, 70 ng, ~1.8 μL) or vehicle (PBS) on ice of 90 min.

Techniques: SDS Page, Isolation, Control, Knockdown, Molecular Weight, Staining, Diagnostic Assay, Data-independent acquisition, Mass Spectrometry, Polyacrylamide Gel Electrophoresis, Co-Elution Assay, Sequencing

Journal: eLife

Article Title: Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells

doi: 10.7554/eLife.67624

Figure Lengend Snippet:

Article Snippet: The sample was halved and incubated with either mouse anti-PHB2 antibodies (Proteintech, cat# 66424–1-Ig, 70 ng, ~1.8 μL) or vehicle (PBS) on ice of 90 min.

Techniques: Control, Transduction, shRNA, Knockdown, Software

Journal: iScience

Article Title: CHIP-dependent regulation of the actin cytoskeleton is linked to neuronal cell membrane integrity

doi: 10.1016/j.isci.2021.102878

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-S100A11 , Proteintech , 10237-1-ap; RRID: AB_2183478.

Techniques: Transduction, Recombinant, Data-independent acquisition, CRISPR, Control, Expressing, Stable Transfection, Clone Assay, Software