Structured Review

Thermo Fisher atpase
<t>SDH,</t> mABC1, PIC, ANT, and <t>ATPase</t> interact with each other in co-IP studies on rat liver mitochondrial IM. IM of mitochondria was used as a control. ( A ) Co-IP of the 30-kDa component of SDH with Abs against mABC1, as well as 30- and 70-kDa components of SDH, ANT, ATPase, and PIC. ( B ) Co-IP of the 70-kDa components of SDH with Abs against 30- and 70-kDa of SDH, mABC1, ANT, ATPase, and PIC. ( C ) Co-IP of the α-subunit of ATPase with Abs against mABC1, as well as 30- and 70-kDa components of SDH, ANT, ATPase, and PIC. The α-subunit of ATPase has a molecular mass of ≈60 kDa. An Ab against the β-subunit of ATPase coimmunoprecipitated the same proteins. ( D ) Co-IP of PIC with Abs against 30- and 70-kDa component of SDH, ATPase, PIC, and mABC1. PIC has a molecular mass of ≈30 kDa. ( E ) Co-IP of mABC1 with Abs against 70-kDa components of SDH, ATPase, mABC1, and PIC. mABC1 has a molecular mass of ≈55 kDa. Negative controls included Abs against complex I and IV of the respiratory pathway, Kir6.1, and cyclophilin D. The IM of mitochondria was prepared in 1.2% digitonin and 6% lubrol WX and was dissolved in 300 mM KPi/10% ethylene glycol/5 mM EDTA/4 mM ATP/0.5 mM DTT, pH 7.9. The columns were also washed with 2–3% (octylphenoxy)polyethoxyethanol to increase the specificity of the interactions between proteins.
Atpase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Multiprotein complex containing succinate dehydrogenase confers mitochondrial ATP-sensitive K+ channel activity"

Article Title: Multiprotein complex containing succinate dehydrogenase confers mitochondrial ATP-sensitive K+ channel activity

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0401703101

SDH, mABC1, PIC, ANT, and ATPase interact with each other in co-IP studies on rat liver mitochondrial IM. IM of mitochondria was used as a control. ( A ) Co-IP of the 30-kDa component of SDH with Abs against mABC1, as well as 30- and 70-kDa components of SDH, ANT, ATPase, and PIC. ( B ) Co-IP of the 70-kDa components of SDH with Abs against 30- and 70-kDa of SDH, mABC1, ANT, ATPase, and PIC. ( C ) Co-IP of the α-subunit of ATPase with Abs against mABC1, as well as 30- and 70-kDa components of SDH, ANT, ATPase, and PIC. The α-subunit of ATPase has a molecular mass of ≈60 kDa. An Ab against the β-subunit of ATPase coimmunoprecipitated the same proteins. ( D ) Co-IP of PIC with Abs against 30- and 70-kDa component of SDH, ATPase, PIC, and mABC1. PIC has a molecular mass of ≈30 kDa. ( E ) Co-IP of mABC1 with Abs against 70-kDa components of SDH, ATPase, mABC1, and PIC. mABC1 has a molecular mass of ≈55 kDa. Negative controls included Abs against complex I and IV of the respiratory pathway, Kir6.1, and cyclophilin D. The IM of mitochondria was prepared in 1.2% digitonin and 6% lubrol WX and was dissolved in 300 mM KPi/10% ethylene glycol/5 mM EDTA/4 mM ATP/0.5 mM DTT, pH 7.9. The columns were also washed with 2–3% (octylphenoxy)polyethoxyethanol to increase the specificity of the interactions between proteins.
Figure Legend Snippet: SDH, mABC1, PIC, ANT, and ATPase interact with each other in co-IP studies on rat liver mitochondrial IM. IM of mitochondria was used as a control. ( A ) Co-IP of the 30-kDa component of SDH with Abs against mABC1, as well as 30- and 70-kDa components of SDH, ANT, ATPase, and PIC. ( B ) Co-IP of the 70-kDa components of SDH with Abs against 30- and 70-kDa of SDH, mABC1, ANT, ATPase, and PIC. ( C ) Co-IP of the α-subunit of ATPase with Abs against mABC1, as well as 30- and 70-kDa components of SDH, ANT, ATPase, and PIC. The α-subunit of ATPase has a molecular mass of ≈60 kDa. An Ab against the β-subunit of ATPase coimmunoprecipitated the same proteins. ( D ) Co-IP of PIC with Abs against 30- and 70-kDa component of SDH, ATPase, PIC, and mABC1. PIC has a molecular mass of ≈30 kDa. ( E ) Co-IP of mABC1 with Abs against 70-kDa components of SDH, ATPase, mABC1, and PIC. mABC1 has a molecular mass of ≈55 kDa. Negative controls included Abs against complex I and IV of the respiratory pathway, Kir6.1, and cyclophilin D. The IM of mitochondria was prepared in 1.2% digitonin and 6% lubrol WX and was dissolved in 300 mM KPi/10% ethylene glycol/5 mM EDTA/4 mM ATP/0.5 mM DTT, pH 7.9. The columns were also washed with 2–3% (octylphenoxy)polyethoxyethanol to increase the specificity of the interactions between proteins.

Techniques Used: Co-Immunoprecipitation Assay

Silver staining and Western blot analysis of the purified M-fraction. ( A ) Silver-stained SDS/PAGE gel of the mitochondrial IM extract and the purified M-fraction. We loaded ≈150 and 40 μg of protein in the IM and M-fraction lanes, respectively. ( B ) Western blots of the M-fraction with Abs against the 30- and 70-kDa components of SDH, PIC, α-subunit of ATPase, ANT, and mABC1. We loaded 40 μg of the M-fraction in each lane. The Ab against mABC1 also recognizes a smaller ≈30-kDa band.
Figure Legend Snippet: Silver staining and Western blot analysis of the purified M-fraction. ( A ) Silver-stained SDS/PAGE gel of the mitochondrial IM extract and the purified M-fraction. We loaded ≈150 and 40 μg of protein in the IM and M-fraction lanes, respectively. ( B ) Western blots of the M-fraction with Abs against the 30- and 70-kDa components of SDH, PIC, α-subunit of ATPase, ANT, and mABC1. We loaded 40 μg of the M-fraction in each lane. The Ab against mABC1 also recognizes a smaller ≈30-kDa band.

Techniques Used: Silver Staining, Western Blot, Purification, Staining, SDS Page

Related Articles

Clone Assay:

Article Title: Molecular Characterization of the ?-Subunit of Na+/K+ ATPase from the Euryhaline Barnacle Balanus improvisus Reveals Multiple Genes and Differential Expression of Alternative Splice Variants
Article Snippet: .. Cloning of Na+ /K+ ATPases To clone the NAK1 cDNA, 5´ and 3´ rapid amplification of cDNA ends (RACE) using the GeneRacerTM kit (Invitrogen) was performed with PCR primers based on ESTs from a previously sequenced library of B .improvisus cyprids (Alm Rosenblad et al. unpublished data). ..

Isolation:

Article Title: Multiprotein complex containing succinate dehydrogenase confers mitochondrial ATP-sensitive K+ channel activity
Article Snippet: .. After three rounds of booster immunization, the serum was isolated and total IgG was purified by using the protein-A purification system (Pierce). mAbs against SDH (both the 30- and 70-kDa components) and ATPase were purchased from Molecular Probes. .. Polyclonal Ab against ANT was purchased from Santa Cruz Biotechnology.

Spectrophotometry:

Article Title: Unchanged mitochondrial organization and compartmentation of high-energy phosphates in creatine-deficient GAMT−/− mouse hearts
Article Snippet: .. ATP kinetics of ATPases and ADP kinetics of endogenous PK were recorded using an Evolution 600 spectrophotometer (Thermo Fisher Scientific) equipped with a Peltier water-cooled cell changer (SPE 8 W, Thermo Fisher Scientific) to maintain temperature at 25°C. .. ADP production by ATPases in permeabilized cardiomyocytes was recorded in 2 ml respiration solution using a coupled assay consisting of 5 mM PEP (no. P-7002, Sigma-Aldrich), 0.3 mM NADH (no. 10128015001, Roche), 7.2 U/ml LDH (no. 61311, Sigma-Aldrich), and 20 U/ml PK (no. 10109045001, Roche).

Purification:

Article Title: Multiprotein complex containing succinate dehydrogenase confers mitochondrial ATP-sensitive K+ channel activity
Article Snippet: .. After three rounds of booster immunization, the serum was isolated and total IgG was purified by using the protein-A purification system (Pierce). mAbs against SDH (both the 30- and 70-kDa components) and ATPase were purchased from Molecular Probes. .. Polyclonal Ab against ANT was purchased from Santa Cruz Biotechnology.

Polymerase Chain Reaction:

Article Title: Molecular Characterization of the ?-Subunit of Na+/K+ ATPase from the Euryhaline Barnacle Balanus improvisus Reveals Multiple Genes and Differential Expression of Alternative Splice Variants
Article Snippet: .. Cloning of Na+ /K+ ATPases To clone the NAK1 cDNA, 5´ and 3´ rapid amplification of cDNA ends (RACE) using the GeneRacerTM kit (Invitrogen) was performed with PCR primers based on ESTs from a previously sequenced library of B .improvisus cyprids (Alm Rosenblad et al. unpublished data). ..

Rapid Amplification of cDNA Ends:

Article Title: Molecular Characterization of the ?-Subunit of Na+/K+ ATPase from the Euryhaline Barnacle Balanus improvisus Reveals Multiple Genes and Differential Expression of Alternative Splice Variants
Article Snippet: .. Cloning of Na+ /K+ ATPases To clone the NAK1 cDNA, 5´ and 3´ rapid amplification of cDNA ends (RACE) using the GeneRacerTM kit (Invitrogen) was performed with PCR primers based on ESTs from a previously sequenced library of B .improvisus cyprids (Alm Rosenblad et al. unpublished data). ..

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    Thermo Fisher v atpase
    Effects of rotenone on synaptosomal [ 3 H]-DA uptake, and changes in protein levels of CoxIV, <t>V-ATPase</t> H and <t>SV2a</t> in 3-month-old mouse striatal synaptosomes. ( a ) Greater reduction in LRRK2 R1441G mutant synaptosomal [ 3 H]-DA uptake at 100 nM rotenone as compared to similarly treated WT ( WT: 7045 ± 583 cpm; mutant: 5311 ± 405 cpm ). Data represents mean ± standard error of mean (SEM) from at least seven independent experiments. (b) Protein expression levels of CoxIV, V-ATPase H and SV2a in 3-month-old mouse striatal synaptosomes. CoxIV and V-ATPase H levels in total striatal synaptosomal lysates were significantly lower in mutant mice compared to WT controls (N = 6). Statistical significance between groups was analyzed using Student’s unpaired t-test, *p
    V Atpase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher atpase 6
    Mitochondrial FoxO3A regulates mitochondrial gene expression in metabolically stressed cancer cells. a Scheme of plasmids used. b ChIP analysis of exogenous FoxO3A recruitment at FHRE #1–2 sites on mtDNA (FHRE #1: bp 14,963–15,110; FHRE #2: bp 15,400–15,469) upon LG (0.75 mM glucose, 24 h) in HCT116 cells transfected with HA-FoxO3A-WT-FLAG. c , f ChIP analysis of endogenous FoxO3A recruitment at FHRE #1–2 sites on mtDNA in HCT116 ( c ), and HT29 and Rho 0 HT29 (negative control) ( f ) cells upon LG (24 h). d , g Mitochondrial gene regulation in HCT116 ( d ) and HT29 ( g ) cells upon LG (24 h) assessed by RT-PCR. Black bars: <t>ATPase</t> 6 and 8 genes; white bars: COX1, COX2 , and COX3 genes; gray bars: ND1, ND2, ND3, ND4, ND4L, ND5 , and ND6 genes; light gray bar: CYTOCHROME B gene. The dotted line corresponds to the expression levels detected in cells cultured in HG. e Co-immunoprecipitation analysis with the indicated antibodies of PK-treated HCT116 mitochondrial fractions upon LG (24 h). h Upper panel: scheme of gRNA location in human FoxO3A locus. Targeting sites and proto-spacer adjacent motifs (PAMs) and the deleted region are indicated. Lower panel: immunoblot analysis of HCT116-FoxO3A +/+ and HCT116-FoxO3A −/− cells with different anti-FoxO3A antibodies upon 2-DG (1 mM, 6 h) treatment. i – p HCT116-FoxO3A −/− cells were transfected with the indicated FoxO3A plasmids for 48 h and treated with 2-DG (1 mM, 6 h). i Immunoblot analysis of total and mitochondrial proteins. l , m , o , p ChIP analysis of exogenous FoxO3A recruitment at FHRE p21–p27 sites on nuclear DNA ( l , o ) and FHRE #1–2 sites on mtDNA ( m , p ). n Immunoblot analysis of nuclear and mitochondrial proteins. b , c , f , l , m , o , p Anti-IgGs were used as controls. e , h , i , n β-actin, TFAM, and LAMIN B were used as total, mitochondrial and nuclear lysate controls, respectively, as appropriate. fl. full-length FoxO3A, cl. cleaved FoxO3A, N-term. N-terminal domain, FKH-DBD forkhead DNA-binding domain, NLS nuclear localization signal, TAD transactivation domain, C-term. C-terminal domain. The presented results are representative of at least three independent experiments. Where applicable, data are presented as mean ± SEM and significance was calculated with Student’s t test; * p
    Atpase 6, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher atpase activity
    A pivotal role for UPF2 in the SMD pathway. a GST-pulldown assays of Stau1 with GST-UPF1CH and GST-UPF2s in the absence and presence of dsRNA. The dsRNA was partially labeled with 32 P to enable its detection by autoradiography (lower panels of input and precipitate). As before, proteins were detected by staining with Coomassie Brilliant Blue (CBB). The co-precipitation of dsRNA with <t>UPF1</t> is significantly enhanced in the presence of UPF2 due to strong interactions between UPF2 and Stau1 in the presence of dsRNA. b RNA-dependent <t>ATPase</t> of UPF1 in complex with UPF2 and Stau1, performed using an enzyme-coupled phosphate detection assay. The ATPase activity of UPF1 in the presence of either UPF2 or Stau1 served as controls. The data points and their error bars represent the mean values and standard deviation (s.d.) from three independent experiments. c Quantitative (q) PCRs to determine levels of known ADAR1/Stau1 targets, XIAP , MDM2 , CCNG1 , and RAD51 , upon knockdown of SMD proteins in U2OS cells. Target mRNA levels were normalized to that of the GAPDH transcript in every case. The control siRNA refers to a scrambled sequence that does not specifically target any transcript. Knockdown of UPF2 in combination with ADAR1 leads to increase in the levels of SMD target. The ADAR1/Stau1 knockdown was performed as a positive control. The data were obtained from duplicates of the indicated number of biological replicates, with error bars denoting the standard deviation (s.d.) between the biological replicates. Individual data points are represented as solid circles, while the mean of each data series is represented as a column. The differences in mRNA levels between the ADAR1 knockdown and the ADAR1/UPF2 or the ADAR1/Stau1 knockdown samples is significant, as indicated by the p values obtained from unpaired t tests. The source data for a , b are provided as a source data file
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    Effects of rotenone on synaptosomal [ 3 H]-DA uptake, and changes in protein levels of CoxIV, V-ATPase H and SV2a in 3-month-old mouse striatal synaptosomes. ( a ) Greater reduction in LRRK2 R1441G mutant synaptosomal [ 3 H]-DA uptake at 100 nM rotenone as compared to similarly treated WT ( WT: 7045 ± 583 cpm; mutant: 5311 ± 405 cpm ). Data represents mean ± standard error of mean (SEM) from at least seven independent experiments. (b) Protein expression levels of CoxIV, V-ATPase H and SV2a in 3-month-old mouse striatal synaptosomes. CoxIV and V-ATPase H levels in total striatal synaptosomal lysates were significantly lower in mutant mice compared to WT controls (N = 6). Statistical significance between groups was analyzed using Student’s unpaired t-test, *p

    Journal: Scientific Reports

    Article Title: Combined LRRK2 mutation, aging and chronic low dose oral rotenone as a model of Parkinson’s disease

    doi: 10.1038/srep40887

    Figure Lengend Snippet: Effects of rotenone on synaptosomal [ 3 H]-DA uptake, and changes in protein levels of CoxIV, V-ATPase H and SV2a in 3-month-old mouse striatal synaptosomes. ( a ) Greater reduction in LRRK2 R1441G mutant synaptosomal [ 3 H]-DA uptake at 100 nM rotenone as compared to similarly treated WT ( WT: 7045 ± 583 cpm; mutant: 5311 ± 405 cpm ). Data represents mean ± standard error of mean (SEM) from at least seven independent experiments. (b) Protein expression levels of CoxIV, V-ATPase H and SV2a in 3-month-old mouse striatal synaptosomes. CoxIV and V-ATPase H levels in total striatal synaptosomal lysates were significantly lower in mutant mice compared to WT controls (N = 6). Statistical significance between groups was analyzed using Student’s unpaired t-test, *p

    Article Snippet: COX IV (1:2000, Abcam #ab16056, 16 kD), NDUSF4 (1:500, Santa-Cruz #sc-100567, 18 kD), TH (1:2000, Millipore #MAB318, 58 kD), Actin (1:500, Santa-Cruz #sc-1615, 43 kD), Synaptophysin (1:2000, Cell Signaling #D35E4, 38 kD), V-ATPase (1:1000, ThermoFisher #PA5-22134, 55 kD), SV2a (1:1000, Santa-Cruz #sc-11936, 93 kD) were the primary antibodies used.

    Techniques: Mutagenesis, Expressing, Mouse Assay

    Mitochondrial FoxO3A regulates mitochondrial gene expression in metabolically stressed cancer cells. a Scheme of plasmids used. b ChIP analysis of exogenous FoxO3A recruitment at FHRE #1–2 sites on mtDNA (FHRE #1: bp 14,963–15,110; FHRE #2: bp 15,400–15,469) upon LG (0.75 mM glucose, 24 h) in HCT116 cells transfected with HA-FoxO3A-WT-FLAG. c , f ChIP analysis of endogenous FoxO3A recruitment at FHRE #1–2 sites on mtDNA in HCT116 ( c ), and HT29 and Rho 0 HT29 (negative control) ( f ) cells upon LG (24 h). d , g Mitochondrial gene regulation in HCT116 ( d ) and HT29 ( g ) cells upon LG (24 h) assessed by RT-PCR. Black bars: ATPase 6 and 8 genes; white bars: COX1, COX2 , and COX3 genes; gray bars: ND1, ND2, ND3, ND4, ND4L, ND5 , and ND6 genes; light gray bar: CYTOCHROME B gene. The dotted line corresponds to the expression levels detected in cells cultured in HG. e Co-immunoprecipitation analysis with the indicated antibodies of PK-treated HCT116 mitochondrial fractions upon LG (24 h). h Upper panel: scheme of gRNA location in human FoxO3A locus. Targeting sites and proto-spacer adjacent motifs (PAMs) and the deleted region are indicated. Lower panel: immunoblot analysis of HCT116-FoxO3A +/+ and HCT116-FoxO3A −/− cells with different anti-FoxO3A antibodies upon 2-DG (1 mM, 6 h) treatment. i – p HCT116-FoxO3A −/− cells were transfected with the indicated FoxO3A plasmids for 48 h and treated with 2-DG (1 mM, 6 h). i Immunoblot analysis of total and mitochondrial proteins. l , m , o , p ChIP analysis of exogenous FoxO3A recruitment at FHRE p21–p27 sites on nuclear DNA ( l , o ) and FHRE #1–2 sites on mtDNA ( m , p ). n Immunoblot analysis of nuclear and mitochondrial proteins. b , c , f , l , m , o , p Anti-IgGs were used as controls. e , h , i , n β-actin, TFAM, and LAMIN B were used as total, mitochondrial and nuclear lysate controls, respectively, as appropriate. fl. full-length FoxO3A, cl. cleaved FoxO3A, N-term. N-terminal domain, FKH-DBD forkhead DNA-binding domain, NLS nuclear localization signal, TAD transactivation domain, C-term. C-terminal domain. The presented results are representative of at least three independent experiments. Where applicable, data are presented as mean ± SEM and significance was calculated with Student’s t test; * p

    Journal: Cell Death & Disease

    Article Title: Uncoupling FoxO3A mitochondrial and nuclear functions in cancer cells undergoing metabolic stress and chemotherapy

    doi: 10.1038/s41419-018-0336-0

    Figure Lengend Snippet: Mitochondrial FoxO3A regulates mitochondrial gene expression in metabolically stressed cancer cells. a Scheme of plasmids used. b ChIP analysis of exogenous FoxO3A recruitment at FHRE #1–2 sites on mtDNA (FHRE #1: bp 14,963–15,110; FHRE #2: bp 15,400–15,469) upon LG (0.75 mM glucose, 24 h) in HCT116 cells transfected with HA-FoxO3A-WT-FLAG. c , f ChIP analysis of endogenous FoxO3A recruitment at FHRE #1–2 sites on mtDNA in HCT116 ( c ), and HT29 and Rho 0 HT29 (negative control) ( f ) cells upon LG (24 h). d , g Mitochondrial gene regulation in HCT116 ( d ) and HT29 ( g ) cells upon LG (24 h) assessed by RT-PCR. Black bars: ATPase 6 and 8 genes; white bars: COX1, COX2 , and COX3 genes; gray bars: ND1, ND2, ND3, ND4, ND4L, ND5 , and ND6 genes; light gray bar: CYTOCHROME B gene. The dotted line corresponds to the expression levels detected in cells cultured in HG. e Co-immunoprecipitation analysis with the indicated antibodies of PK-treated HCT116 mitochondrial fractions upon LG (24 h). h Upper panel: scheme of gRNA location in human FoxO3A locus. Targeting sites and proto-spacer adjacent motifs (PAMs) and the deleted region are indicated. Lower panel: immunoblot analysis of HCT116-FoxO3A +/+ and HCT116-FoxO3A −/− cells with different anti-FoxO3A antibodies upon 2-DG (1 mM, 6 h) treatment. i – p HCT116-FoxO3A −/− cells were transfected with the indicated FoxO3A plasmids for 48 h and treated with 2-DG (1 mM, 6 h). i Immunoblot analysis of total and mitochondrial proteins. l , m , o , p ChIP analysis of exogenous FoxO3A recruitment at FHRE p21–p27 sites on nuclear DNA ( l , o ) and FHRE #1–2 sites on mtDNA ( m , p ). n Immunoblot analysis of nuclear and mitochondrial proteins. b , c , f , l , m , o , p Anti-IgGs were used as controls. e , h , i , n β-actin, TFAM, and LAMIN B were used as total, mitochondrial and nuclear lysate controls, respectively, as appropriate. fl. full-length FoxO3A, cl. cleaved FoxO3A, N-term. N-terminal domain, FKH-DBD forkhead DNA-binding domain, NLS nuclear localization signal, TAD transactivation domain, C-term. C-terminal domain. The presented results are representative of at least three independent experiments. Where applicable, data are presented as mean ± SEM and significance was calculated with Student’s t test; * p

    Article Snippet: FoxO3A (CST #BK99199S), FoxO3A (CST #2497), COX4 3E11 (CST #4850), BCL2 50E3 (CST #2870), PDH C54G1 (CST #3205), FLAG M2 (Sigma #F1804), HA-Tag C29F4 (CST #3724), β-actin (CST #3700), AMPK-α Thr 172 (CST #2531), AMPK-α (CST #2532), p44/42 MAP2K1-ERK1/2 Thr202/Tyr204 (CST #9106), p44/42 MA2PK1-ERK1/2 (CST #9102), p-Acetyl-CoA Ser 79 (CST #3661), TFAM D5C8 (CST #8076), HSP60 D307 (CST #4870), IgG (CST #2729S), mtRNAPOL H300 (Santa Cruz #SC-67350), SIRT3 D22A3 (CST #5490), LAMIN B1 (CST #12586S) (Cell Signaling Technologies, Danvers, MA) PARP1 (p85 fragment, G7341, Promega, Madison, WI), ATPase 6 (#PA5-37129, Thermo Fisher Scientific), COXI (#A6403, Molecular probes, Carlsbad, CA), ND6 (#A31857, Molecular probes) were used as primary antibodies.

    Techniques: Expressing, Metabolic Labelling, Chromatin Immunoprecipitation, Transfection, Negative Control, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Immunoprecipitation, Binding Assay

    A pivotal role for UPF2 in the SMD pathway. a GST-pulldown assays of Stau1 with GST-UPF1CH and GST-UPF2s in the absence and presence of dsRNA. The dsRNA was partially labeled with 32 P to enable its detection by autoradiography (lower panels of input and precipitate). As before, proteins were detected by staining with Coomassie Brilliant Blue (CBB). The co-precipitation of dsRNA with UPF1 is significantly enhanced in the presence of UPF2 due to strong interactions between UPF2 and Stau1 in the presence of dsRNA. b RNA-dependent ATPase of UPF1 in complex with UPF2 and Stau1, performed using an enzyme-coupled phosphate detection assay. The ATPase activity of UPF1 in the presence of either UPF2 or Stau1 served as controls. The data points and their error bars represent the mean values and standard deviation (s.d.) from three independent experiments. c Quantitative (q) PCRs to determine levels of known ADAR1/Stau1 targets, XIAP , MDM2 , CCNG1 , and RAD51 , upon knockdown of SMD proteins in U2OS cells. Target mRNA levels were normalized to that of the GAPDH transcript in every case. The control siRNA refers to a scrambled sequence that does not specifically target any transcript. Knockdown of UPF2 in combination with ADAR1 leads to increase in the levels of SMD target. The ADAR1/Stau1 knockdown was performed as a positive control. The data were obtained from duplicates of the indicated number of biological replicates, with error bars denoting the standard deviation (s.d.) between the biological replicates. Individual data points are represented as solid circles, while the mean of each data series is represented as a column. The differences in mRNA levels between the ADAR1 knockdown and the ADAR1/UPF2 or the ADAR1/Stau1 knockdown samples is significant, as indicated by the p values obtained from unpaired t tests. The source data for a , b are provided as a source data file

    Journal: Nature Communications

    Article Title: Insights into the assembly and architecture of a Staufen-mediated mRNA decay (SMD)-competent mRNP

    doi: 10.1038/s41467-019-13080-x

    Figure Lengend Snippet: A pivotal role for UPF2 in the SMD pathway. a GST-pulldown assays of Stau1 with GST-UPF1CH and GST-UPF2s in the absence and presence of dsRNA. The dsRNA was partially labeled with 32 P to enable its detection by autoradiography (lower panels of input and precipitate). As before, proteins were detected by staining with Coomassie Brilliant Blue (CBB). The co-precipitation of dsRNA with UPF1 is significantly enhanced in the presence of UPF2 due to strong interactions between UPF2 and Stau1 in the presence of dsRNA. b RNA-dependent ATPase of UPF1 in complex with UPF2 and Stau1, performed using an enzyme-coupled phosphate detection assay. The ATPase activity of UPF1 in the presence of either UPF2 or Stau1 served as controls. The data points and their error bars represent the mean values and standard deviation (s.d.) from three independent experiments. c Quantitative (q) PCRs to determine levels of known ADAR1/Stau1 targets, XIAP , MDM2 , CCNG1 , and RAD51 , upon knockdown of SMD proteins in U2OS cells. Target mRNA levels were normalized to that of the GAPDH transcript in every case. The control siRNA refers to a scrambled sequence that does not specifically target any transcript. Knockdown of UPF2 in combination with ADAR1 leads to increase in the levels of SMD target. The ADAR1/Stau1 knockdown was performed as a positive control. The data were obtained from duplicates of the indicated number of biological replicates, with error bars denoting the standard deviation (s.d.) between the biological replicates. Individual data points are represented as solid circles, while the mean of each data series is represented as a column. The differences in mRNA levels between the ADAR1 knockdown and the ADAR1/UPF2 or the ADAR1/Stau1 knockdown samples is significant, as indicated by the p values obtained from unpaired t tests. The source data for a , b are provided as a source data file

    Article Snippet: ATPase assay The ATPase activity of UPF1 in the presence of its binding partners was determined by quantifying the amount of inorganic phosphate released upon ATP hydrolysis using a coupled colorimetric assay (EnzCheck Phosphate Kit, Thermo Fisher Scientific) .

    Techniques: Labeling, Autoradiography, Staining, Detection Assay, Activity Assay, Standard Deviation, Sequencing, Positive Control

    Interactions of Stau1 with UPF1 and UPF2. a Schematic representation of the domain arrangements of human UPF1, UPF2, and Stau1. Globular domains are shown as rectangles and flexible linkers and low-complexity regions are denoted as lines. The helicase core of UPF1 consisting of the two RecA domains is colored yellow and the cis -inhibitory CH domain is in green. The three MIF4G domains of UPF2 are shown in shades of blue, while the C-terminal UPF1-binding region of UPF2 is shown as a hatched box. The four dsRNA-binding domains (dsRBDs) and the tubulin-binding domain (TBD) of Stau1 are in purple. The Staufen-swapping motif (SSM, hatched box) together with dsRBD5 comprises the Stau1 dimerization module (dd). The constructs of UPF1 and UPF2 used in this study are indicated. b RNA-dependent ATPase activity of UPF1 in the presence of UPF2 or Stau1, performed using an enzyme-coupled phosphate detection assay. The data points and their error bars represent the mean values and standard deviation (s.d.) from three independent experiments. c GST-pulldown assays of Stau1 and UPF2 with GST-UPF1CH as a bait. GST serves as a negative control. The top and bottom panels indicate input and precipitate in this and all other GST-pulldown experiments. Binding of Stau1 to UPF1CH is ~3-fold weaker than the binding of UPF2 to UPF1CH. d GST-pulldown assays of UPF1 and Stau1 in the absence and presence of UPF2. GST-UPF1 and UPF1CH are used as baits while GST serves as a negative control. The presence of UPF2 strengthens the interaction of Stau1 with UPF1 and UPF1CH by ~3-fold. e GST-pulldown assays to determine the Stau1-binding region of UPF2. Binding of GST-Stau1 to different UPF2 constructs spanning the entire protein identified the UPF2-MIF4G3 domain as the Stau1-binding site. The corresponding negative controls using GST as bait is shown in Supplementary Fig. 1c . The asterisk (*) indicates contaminants in d , e . The source data for this figure are provided as source data files

    Journal: Nature Communications

    Article Title: Insights into the assembly and architecture of a Staufen-mediated mRNA decay (SMD)-competent mRNP

    doi: 10.1038/s41467-019-13080-x

    Figure Lengend Snippet: Interactions of Stau1 with UPF1 and UPF2. a Schematic representation of the domain arrangements of human UPF1, UPF2, and Stau1. Globular domains are shown as rectangles and flexible linkers and low-complexity regions are denoted as lines. The helicase core of UPF1 consisting of the two RecA domains is colored yellow and the cis -inhibitory CH domain is in green. The three MIF4G domains of UPF2 are shown in shades of blue, while the C-terminal UPF1-binding region of UPF2 is shown as a hatched box. The four dsRNA-binding domains (dsRBDs) and the tubulin-binding domain (TBD) of Stau1 are in purple. The Staufen-swapping motif (SSM, hatched box) together with dsRBD5 comprises the Stau1 dimerization module (dd). The constructs of UPF1 and UPF2 used in this study are indicated. b RNA-dependent ATPase activity of UPF1 in the presence of UPF2 or Stau1, performed using an enzyme-coupled phosphate detection assay. The data points and their error bars represent the mean values and standard deviation (s.d.) from three independent experiments. c GST-pulldown assays of Stau1 and UPF2 with GST-UPF1CH as a bait. GST serves as a negative control. The top and bottom panels indicate input and precipitate in this and all other GST-pulldown experiments. Binding of Stau1 to UPF1CH is ~3-fold weaker than the binding of UPF2 to UPF1CH. d GST-pulldown assays of UPF1 and Stau1 in the absence and presence of UPF2. GST-UPF1 and UPF1CH are used as baits while GST serves as a negative control. The presence of UPF2 strengthens the interaction of Stau1 with UPF1 and UPF1CH by ~3-fold. e GST-pulldown assays to determine the Stau1-binding region of UPF2. Binding of GST-Stau1 to different UPF2 constructs spanning the entire protein identified the UPF2-MIF4G3 domain as the Stau1-binding site. The corresponding negative controls using GST as bait is shown in Supplementary Fig. 1c . The asterisk (*) indicates contaminants in d , e . The source data for this figure are provided as source data files

    Article Snippet: ATPase assay The ATPase activity of UPF1 in the presence of its binding partners was determined by quantifying the amount of inorganic phosphate released upon ATP hydrolysis using a coupled colorimetric assay (EnzCheck Phosphate Kit, Thermo Fisher Scientific) .

    Techniques: Binding Assay, Construct, Activity Assay, Detection Assay, Standard Deviation, Negative Control