A-6404

antibody from Invitrogen Antibodies

Targeting: MT-CO2 CO2, COX2, MTCO2

Provider product page for A-6404

Immunocytochemistry

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Antibody data

Antibody Data
Antigen structure
References [78]
Comments [0]
Validations
Immunocytochemistry [1]
Other assay [38]

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Product number: A-6404 - Provider product page
Provider: Invitrogen Antibodies
Product name: MTCO2 Monoclonal Antibody (12C4F12)
Antibody type: Monoclonal
Antigen: Other
Description: To facilitate the study of COX structure and mitochondrial biogenesis, Invitrogen offers several subunit-specific mouse anti-OxPhos Complex IV monoclonal antibodies. The binding specificity of our anti-OxPhos Complex IV monoclonal antibody preparations allows researchers to investigate the regulation, assembly, and orientation of COX subunits in a variety of organisms. The antibodies have also proven valuable for analyzing human mitochondrial myopathies and related disorders as well as mitochondrial DNA depletion due to drug toxicity. Alexa Fluor TM 488 and Alexa Fluor TM 594 conjugates of anti- OxPhos Complex IV subunit I are also available for direct staining of these COX proteins. Cell lines can be screened for subunit expression levels for each of the OxPhos complexes by simple western blotting. These results can be combined with native gel electrophoresis or sucrose gradient centrifugation to gather additional information regarding the assembly state of the OxPhos complexes. Many of our antibodies against OxPhos subunits may also be used for immunocytochemical analysis. Image analysis of an antibody's staining pattern can reveal the relative expression and localization of a subunit. This approach has been particularly useful for studying OxPhos subunit expression in diseased muscle fibers and for screening Complex IV-deficient patients.
Antibody clone number: 12C4F12
Concentration: 1 mg/mL

MT-CO2 protein structure - A-6404 shown in red.

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Experimental details: Figure 3 Ratio of AACs protein content estimated through WB in HK2, RCC-Shaw, and CaKi-1 cells grown in serum-free medium versus complete medium conditions with respect to actin content (panel a ) or COXII content (panel b ). Data are presented as mean + SE of at least three independent experiments. * p < 0.05; nonparametric Wilcoxon two-tailed test between starved and physiological conditions. For a representative WB see Supplementary Figure S3 . The protein concentration from extraction assays was reported in Supplementary Table S2 .

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Experimental details: Figure 2 PUMA induces accumulation of GFP-LC3 puncta, which preferentially colocalize with mitochondria. A) Saos-2 TetOn PUMA cells were infected with GFP-LC3 expressing Adenovirus, left for 16 hours and then treated with doxycycline. Cells were maintained in the presence of z-VAD-fmk throughout the experiment. 24 hours later, cells were fixed and immunostained with anti-Tom20 antibody to visualize the mitochondria. B) U2OS cells were infected with GFP-LC3 expressing Adenovirus, left for 16 hours and then transfected with PUMA expression plasmid. Cells were maintained in the presence of z-VAD-fmk throughout the experiment. 24 hours later, cells were fixed and immunostained with anti-Tom20 (upper panel) or anti-GM130 (lower panel). Cells exhibiting GFP-LC3 puncta were then observed under a fluorescence microscope to compare the co-localization of GFP-LC3 puncta with mitchondria (Tom20) or Golgi (GM130). C) U2OS cells were transfected with vector control or PUMA expression plasmid and maintained in the presence of z-VAD-fmk throughout the experiment. Cells were harvested 24 or 48 hours after transfection and cell lysates were immunoblotted with the indicated antibodies to detect levels of mitochondria, Golgi and ER proteins.

Supportive validation

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Experimental details: Figure 4 Depletion of ERAL1 leads to apoptosis prior to appreciable loss of mitochondrial protein synthesis ( A ) Counts of HEK-293T or HeLa cells were taken after siRNA treatment (3 or 4 days respectively) with an NT control or each of the three independent siRNAs targeted either to the EraL1 open reading frame (ORF1; ORF2) or to the corresponding 3'-UTR. Counts were performed on three (HeLa) or six (HEK-293T) independent repeats. *** P

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Experimental details: Fig. 3 Effect of FARS2 mutation on mitochondrial homeostasis. A. Cytochrome c oxidase (COX) histochemistry of the patient muscle showed a generalised loss of enzyme activity compared to age-matched control tissue. B. Respiratory chain enzyme activity in muscle biopsy, fibroblast and myoblast: activities of complex I, complex II, and complex IV were determined in control (blue) and patient (red) and normalised to citrate synthase. Results are based on three independent measurements and are shown as percent of the mean control value +- standard deviation C. Steady state levels of RC proteins in fibroblasts (left panel) and myoblasts (right panel) were determined by Western blotting. 10% SDS-PAGE was performed with cell lysates (30 mug) from control (C1, C2) and patient (P), except for FARS2 and SDHA in the bottom 2 panels where 80 mug mitochondrial protein was loaded per lane. Western blots were decorated with antibodies to the proteins indicated. Secondary alpha-antibodies were HRP conjugated and detection was by ECL + and ImageQuant software. D. High resolution northern blot analysis was performed on total RNA (2 mug) from control (C1, C2) and patient (P). Membranes were hybridised with radiolabelled probes for mt-tRNA Phe , mt-tRNA Val and mt-tRNA Leu(UUR) . Densitometric analyses were performed on all blots. A representative example is presented with the values relative to controls for the signals derived for each tRNA below the sample. E. De novo mitochondrial protein synt

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Experimental details: Figure 5 Mitochondrial Complex IV Remodeling after miR-181c Treatment. (A) qPCR data show that overexpression of miR-181c significantly reduces the mRNA levels of all mitochondrial complex IV genes with 3 weeks treatment. The treatment protocol has no effect on other mitochondrial genes, such as ND2 (complex I) and ATPase 8 (complex V). Content of mRNA was first normalized to 12S rRNA, a mitochondrial gene, as 12S rRNA expression did not change with miR-181c overexpresssion. Then we normalized the data to the sham group. *p

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Experimental details: Figure 2 Steady-state levels of OXPHOS components and complexes. ( a ) Cell lysate from control (C1 and C2) and patient (P) fibroblasts (40 mu g) were analysed by SDS-PAGE (12%) and immunoblotting. Subunit-specific antibodies were used against CI (NDUFA9, NDUFB8), CII (SDHA), CIII (UQCRC2), CIV (COX1, COX2) and CV (ATPB). The outer mitochondrial membrane marker, TOM20, was used as a loading control. ( b ) Mitochondrial proteins (50 mu g) isolated from patient (P) and control (C1) fibroblasts were analysed by one-dimensional BN-PAGE (4 to 16% gradient) using subunit-specific antibodies as indicated (CI (NDUFA9), CII (SDHA), CIII (UQCRC2), CIV (COX1) and CV (ATP5A)) to assess the assembly of individual OXPHOS complexes. Complex II (SDHA) was used as a loading control.

Supportive validation

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Experimental details: Fig. 2. Assessment of mitochondrial bioenergetics in neuronal tissues (brainstem, posterior columns, nerve roots and dorsal root ganglia). A quantitative western blot assay was developed to measure FXN (A), COXI and COXII (B), cytochrome c (C), ATP synthase (D) and TOM22 (E) in neuronal tissues [brainstem, posterior columns (PC), nerve roots and dorsal root ganglia (DRG)]. (A) Representative western blot of human FXN expression in YG8R mice in the four neuronal tissues used in the study. Human FXN was only detected in YG8R mice, in which the transgene was present, and not in wild type (WT; data not shown). Western blot results were quantified for each lane using Fujifilm's Multi-Gauge software. To allow for loading variation, values were normalized to the actin control. Final values were expressed as a ratio to the value of FXN expression in nerve roots. DRG and nerve roots (distal tissues) expressed less FXN than the posterior columns and brainstem (proximal tissues). Values are expressed as mean+-s.e.m.; *** P

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Experimental details: Fig 2 Involvement of mitochondria in cooperative cytotoxic action of rosiglitazone and LA-12. (a) The release of cytochrome c into the cytoplasm (cytoplasmic fraction) of HCT116 cells pretreated (24 h) with rosiglitazone (RGZ, 50 muM) and subsequently treated (48 h) with LA-12 (0.75 muM), detected by Western blotting after cell fractionation. (b) Cleavage of caspase-9, PARP, Bax protein level (Western blotting), and (c) changes in mitochondrial membrane potential (MMP, flow cytometry) in HCT116 wt and Bax-/- cells treated as in a). (d) The level of Noxa, Bim, Puma, Bid, Bax and Bak protein (Western blotting) in HCT116 wt cells treated as in a). Results are means + S.E.M. or representatives of three independent experiments. Statistical significance: P < 0.05, * versus control, ++ versus RGZ or Omicron versus LA-12, and Delta for wt versus Bax-/- cells.

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Experimental details: Fig 7 Stable knock-down of GTPBP3 disturbs Complex I assembly and reduces the expression of Complex I assembly factors NDUFAF3 and NDUFAF4 (A) Western blot analysis of OXPHOS subunits ND1, NDUFS3 and NDUFB8 (Complex I), SDHA (Complex II), COXII and COXIV (Complex IV), and beta-subunit (Complex V) in shGTPBP3-1, shGTPBP3-2 and negative control (NC) cells. The filter was also probed with porin as a loading control. (B) Densitometric analysis of OXPHOS subunits normalized to porin and represented as % of NC. (C) Representative Blue Native-PAGE of OXPHOS complexes in shGTPBP3-1, shGTPBP3-2 and NC cells. (D) Densitometric analysis of OXPHOS Complexes normalized to Complex-II (loading control) and represented as % of NC. (E) qRT-PCR analysis of C20ORF7 , NUBPL , NDUFAF3 and NDUFAF4 mRNA expression in shGTPBP3 and NC cells. All data are the mean +- SEM of at least three independent biological replicates. Differences from NC values were found to be statistically significant at *p

Supportive validation

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Experimental details: Figure 3 Ratio of AACs protein content estimated through WB in HK2, RCC-Shaw, and CaKi-1 cells grown in serum-free medium versus complete medium conditions with respect to actin content (panel a ) or COXII content (panel b ). Data are presented as mean + SE of at least three independent experiments. * p < 0.05; nonparametric Wilcoxon two-tailed test between starved and physiological conditions. For a representative WB see Supplementary Figure S3 . The protein concentration from extraction assays was reported in Supplementary Table S2 .

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Experimental details: Figure 8 Representative immunofluorescence analysis of mitochondrial complex IV subunit II protein in AECII from lung tissue sections of newborn (NB), P15, and adult (AL) animals. Lung tissue samples from the three postnatal stages were embedded into paraffin. Thereafter, 3 mu m paraffin sections were cut with a rotation microtome and processed further for indirect double immunofluorescence.The lung sections were incubated overnight for double labelling with primary antibodies against mitochondrial complex IV subunit II and pro-SP-C, a marker for AECII ( Table 1 ). The following morning, the sections were washed and incubated with the secondary antibodies ( Table 1 ) for 2 h at room temperature. Double fluorescence samples were analyzed by confocal laser scanning microscopy (CLSM) with a Leica TCS SP5. (a, d, and g) Double immunofluorescence stainings of AECII with their marker protein pro-SP-C. (b, e, and h) IF preparations for the mitochondrial complex IV subunit II. (c, f, and i) Double IF overlay for complex IV subunit II combined with pro-SP-C. NB, newborn; P15, postnatal day 15; AL, adult. Bars represent 20 mu m.

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Experimental details: Figure 2. RBFA binds to helix 45 of 12S rRNA but has a distinct function from that of ERAL1. ( A ) The graph represents the human mtDNA sequence with locations and numbers of the CLIP tags indicated. Tags were generated as described in the Experimental Procedures. ( B ) The location of the greatest number of CLIP tags from three independent experiments is depicted spanning helix 45 and protruding into helix 44. The two dimethylated adenines in helix 45 are indicated in light grey. The terminal C-residue represents the near 3'-terminus of the 12S mt-rRNA, corresponding to nt1597 of mtDNA. ( C ) Lysates (30 mug) from HeLa, 143B.206 parental and Rho 0 cells were analyzed by western blot to compare the relative expression levels of RBFA, components of the OXPHOS complexes (COX2, NDUFB8 and SDHA) and members of the mitoribosome (mS29, mS40, mL62 and uL3m,). Cytosolic RP-S6 is also shown. ( D ) Cell growth of each cell line was determined after 3 days treatment of six RBFA-targeted siRNAs (33 nM) (lanes 1-6) compared with control (NT lane 7). Inset: western blot of cell lysates (25 ug) after treatment with RBFA siRNA 2 and 6 to assess the level of depletion with beta-actin as the loading control. ( E ) Top panel: HEK293 cells were grown for 72 h in the presence of either si-NT (lanes 1 and 3) or si-RBFA (lanes 2 and 4). ERAL1-FLAG expression was induced 4 h after siRNA transfection (lanes 3 and 4). Lower panel: HEK293 cells were grown for 72 h in the presence of either si-NT (lane

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Experimental details: Figure 3. Depletion of RBFA has no appreciable effect on mitochondrial protein synthesis. ( A ) HEK293 cells were treated with RBFA (lanes 2 and 3) or non-targeting (NT, lanes 1 and 4) siRNA for 3 days and extracts (25 ug) were subjected to western blotting to compare OXPHOS (COX2, NDUFB8 and SDHA) or mitoribosomal (mS40, mS29 and uL3m) protein levels. ( B ) Following similar siRNA treatment (NT or RBFA-2) for 3 (lanes 1 and 2) or 6 (lanes 3 and 4) days, cells were metabolically labelled ( 35 S-met/cys) and extracts (3 days--30 ug; 6 days--50 ug) separated by 15% denaturing PAGE. Migration of the 13 mtDNA-encoded polypeptides is indicated and loading confirmed by Coomassie blue (CBB) staining.

Supportive validation

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Experimental details: Figure 4. Depletion of RBFA causes a decrease in 12S rRNA modification. ( A ) Schematic presenting sequence conservation of helix 45 (dashed line) and the primer extension assay used to measure the modification levels at adenine residues A 936 / A 937 in human 12S rRNA. If the modification is present, the primer extends four residues, and in its absence the primer extends a further five residues. ( B ) To determine the modification status of 12S rRNA bound to ERAL1-FLAG (lane 2, 3-day induction) or RBFA-FLAG (lane 3, 3 days), these proteins were immunoprecipitated from HEK293 cells and the bound RNA was extracted. Samples were subjected to primer extension and denaturing PAGE. (Right panel) Cells were depleted of ERAL1 (3 days) followed by 4 days of siRNA treatment; si-NT (lane 7) or si-RBFA (lane 8). RNA was extracted and primer extension performed. Primer alone (lane 1, 4), extension on unmethylated template (lane 5) and wild-type cells (lane 6) were controls performed in parallel. ( C ) Western blot (50 ug of cell extracts) determined the steady-state levels of ERAL1, TFB1M and COXII in cells depleted of RBFA. SDHA and beta-actin were used as loading controls. ( D ) HEK293 cells were grown for 5 days in the presence of NT or RBFA siRNA with induction of mS27-FLAG (IP SSU) or mL62-FLAG (IP LSU) on day 3. (Left panel) Cell extracts were subjected to immunoprecipitation and bound 12S rRNA was assessed by primer extension alongside controls (primer alone, lane 1; unmethylate