HG14236-ANR Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93
    Sino Biological sap18
    Binding of A671 and related compounds to <t>SAP18.</t> a Western blotting for SAP18 following pull-down of Sepharose 6B beads loaded with indicated compounds. Sepharose 6B beads without compound used as control. b Binding of A671 to recombinant SAP18 in pull-down assays. c Western blot for SAP18 expression 12 h after treatment with indicated concentrations of A670-A672 compounds. d , e Expression of SIRT3 in HEL cells treated for 24 h with indicated concentrations of A670 ( d ) or A672 ( e ). f , g Growth curves of HEL cells treated for indicated periods and concentrations of A670 ( f ) or A672 ( g ). h Pull-down assay for binding of A611 to SAP18 and SIRT3 in HEL cells. The input used as a positive control. P
    Sap18, supplied by Sino Biological, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sap18/product/Sino Biological
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sap18 - by Bioz Stars, 2021-04
    93/100 stars
      Buy from Supplier

    86
    OriGene pumilio 1 pum1 nm 014676 human tagged orf clone
    AS PIM1 Directly Phosphorylates Endogenous High-Confidence Substrates in Prostate Cancer Cells A–D) Validation of endogenous PIM1 substrates. LNCaP-AS-PIM1 thiophosphorylates <t>PUM1</t> (A), CHMP7 (B), NDRG1 (C), and KIF18A (D). Endogenous proteins were immunoprecipitated from LNCaP-WT-PIM1 and LNCaP-AS-PIM1 and analyzed by Western blot for the presence of thiophosphorylation (ThioP), or immunoprecipitated PUM1, CHMP7, NDRG1, and KIF18A. E-H) Phosphorylation site validation using WT and phosphorylation site mutant substrates. Substrates (Myc-PUM1, FLAG-CHMP7, FLAG-NDRG1, and GFP-6HIS-KIF18A; either wild type (WT) or the indicated PIM1 phosphorylation site mutation) were expressed in 293T cells with AS-PIM1 and thiophosphorylation labeling completed. Substrates were immunoprecipitated using Myc, FLAG, or GFP-magnetic beads, and Western blot performed to detect thiophosphorylated, or the immunoprecipitated substrates. Western blots are representative of two independent experiments.
    Pumilio 1 Pum1 Nm 014676 Human Tagged Orf Clone, supplied by OriGene, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pumilio 1 pum1 nm 014676 human tagged orf clone/product/OriGene
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pumilio 1 pum1 nm 014676 human tagged orf clone - by Bioz Stars, 2021-04
    86/100 stars
      Buy from Supplier

    86
    Sino Biological chmp7 cdna orf clone human c dykddddk tag
    AS PIM1 Directly Phosphorylates Endogenous High-Confidence Substrates in Prostate Cancer Cells A–D) Validation of endogenous PIM1 substrates. LNCaP-AS-PIM1 thiophosphorylates PUM1 (A), <t>CHMP7</t> (B), NDRG1 (C), and KIF18A (D). Endogenous proteins were immunoprecipitated from LNCaP-WT-PIM1 and LNCaP-AS-PIM1 and analyzed by Western blot for the presence of thiophosphorylation (ThioP), or immunoprecipitated PUM1, CHMP7, NDRG1, and KIF18A. E-H) Phosphorylation site validation using WT and phosphorylation site mutant substrates. Substrates (Myc-PUM1, FLAG-CHMP7, FLAG-NDRG1, and GFP-6HIS-KIF18A; either wild type (WT) or the indicated PIM1 phosphorylation site mutation) were expressed in 293T cells with AS-PIM1 and thiophosphorylation labeling completed. Substrates were immunoprecipitated using Myc, FLAG, or GFP-magnetic beads, and Western blot performed to detect thiophosphorylated, or the immunoprecipitated substrates. Western blots are representative of two independent experiments.
    Chmp7 Cdna Orf Clone Human C Dykddddk Tag, supplied by Sino Biological, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/chmp7 cdna orf clone human c dykddddk tag/product/Sino Biological
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    chmp7 cdna orf clone human c dykddddk tag - by Bioz Stars, 2021-04
    86/100 stars
      Buy from Supplier

    88
    Sino Biological mrpp3
    Mitochondrial pre-RNA processing defects upon UPR mt a, qPCR of mitochondrial pre-RNA at tRNA Met and tRNA Lys RNAseP processing sites upon induction of UPR mt with GTPP (6h). Error bars, averages ±s.d. (n=3 biological replicates). b , RNA-seq for analysis of mitochondrial pre-RNA processing defects based on number of reads crossing the tRNA/mRNA gene junction. Slope of coverage in the tRNA gene adjacent to the cut site is used as a measure of processing. c-d , Normalized RNA-seq coverage across tRNA/mRNA gene borders for tRNA Met ( c ) and tRNA Lys ( d ) with average of slopes (±s.d.) from b indicated in the inset (n=3 biological replicates, two-tailed p-values *p≤0.05, ***p≤0.001). e , Quantitative western blot analysis of <t>MRPP3</t> levels upon treatment of cells with DMSO, GTPP, CDDO, or GTPP + CDDO co-treatment for 6 hours. f , Mitochondrial pre-RNA accumulation upon co-treatment with GTPP and CDDO (as in a ). Data are average values ±s.d. (n=3 biological replicates). For gel source data, see Supplementary Figure 1 .
    Mrpp3, supplied by Sino Biological, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mrpp3/product/Sino Biological
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mrpp3 - by Bioz Stars, 2021-04
    88/100 stars
      Buy from Supplier

    Image Search Results


    Binding of A671 and related compounds to SAP18. a Western blotting for SAP18 following pull-down of Sepharose 6B beads loaded with indicated compounds. Sepharose 6B beads without compound used as control. b Binding of A671 to recombinant SAP18 in pull-down assays. c Western blot for SAP18 expression 12 h after treatment with indicated concentrations of A670-A672 compounds. d , e Expression of SIRT3 in HEL cells treated for 24 h with indicated concentrations of A670 ( d ) or A672 ( e ). f , g Growth curves of HEL cells treated for indicated periods and concentrations of A670 ( f ) or A672 ( g ). h Pull-down assay for binding of A611 to SAP18 and SIRT3 in HEL cells. The input used as a positive control. P

    Journal: Communications Biology

    Article Title: A C21-steroidal derivative suppresses T-cell lymphoma in mice by inhibiting SIRT3 via SAP18-SIN3

    doi: 10.1038/s42003-020-01458-3

    Figure Lengend Snippet: Binding of A671 and related compounds to SAP18. a Western blotting for SAP18 following pull-down of Sepharose 6B beads loaded with indicated compounds. Sepharose 6B beads without compound used as control. b Binding of A671 to recombinant SAP18 in pull-down assays. c Western blot for SAP18 expression 12 h after treatment with indicated concentrations of A670-A672 compounds. d , e Expression of SIRT3 in HEL cells treated for 24 h with indicated concentrations of A670 ( d ) or A672 ( e ). f , g Growth curves of HEL cells treated for indicated periods and concentrations of A670 ( f ) or A672 ( g ). h Pull-down assay for binding of A611 to SAP18 and SIRT3 in HEL cells. The input used as a positive control. P

    Article Snippet: Similarly, SAP18 (pCMV3-SAP18-GFPSpark®; Sino Biological, China) overexpression studies conducted using Lipofectamine 3000 and hygromycin (250 µg/ml) used for cell selection.

    Techniques: Binding Assay, Western Blot, Recombinant, Expressing, Pull Down Assay, Positive Control

    Correlation between SAP18 and SIRT3 transcription in BCALL patients predicts drug response. a A model for the effect of A671 on SAP18/SIN3 and SIRT3, and its impact on leukemic cell survival. A671 binds and stabilizes SAP18, leading to activation of the SIN3 complex and transcriptional repression of SIRT3 expression, which culminates in apoptotic cell death. b A significant negative correlation ( P = 0.0001 by two tailed student t -test) of SIRT3 and SAP18 in patients with BCALL, and correlation ( r ) of 0.4. (Spearman’s rank analysis). c , d Overall survival rates for high SIRT3 and SAP18 expression in BCALL patients determined by Log-rank (Mantel-Cox) test.

    Journal: Communications Biology

    Article Title: A C21-steroidal derivative suppresses T-cell lymphoma in mice by inhibiting SIRT3 via SAP18-SIN3

    doi: 10.1038/s42003-020-01458-3

    Figure Lengend Snippet: Correlation between SAP18 and SIRT3 transcription in BCALL patients predicts drug response. a A model for the effect of A671 on SAP18/SIN3 and SIRT3, and its impact on leukemic cell survival. A671 binds and stabilizes SAP18, leading to activation of the SIN3 complex and transcriptional repression of SIRT3 expression, which culminates in apoptotic cell death. b A significant negative correlation ( P = 0.0001 by two tailed student t -test) of SIRT3 and SAP18 in patients with BCALL, and correlation ( r ) of 0.4. (Spearman’s rank analysis). c , d Overall survival rates for high SIRT3 and SAP18 expression in BCALL patients determined by Log-rank (Mantel-Cox) test.

    Article Snippet: Similarly, SAP18 (pCMV3-SAP18-GFPSpark®; Sino Biological, China) overexpression studies conducted using Lipofectamine 3000 and hygromycin (250 µg/ml) used for cell selection.

    Techniques: Activation Assay, Expressing, Two Tailed Test

    A671 binds SAP18 within the SIN3 suppressor complex to transcriptionally repress SIRT3 expression. a , b Efficient depletion of SAP18 in HEL cells using shRNA as determined by western blotting ( a ) and Q-RT-PCR ( b ). Untreated or scrambled transfected vector (Vector) cells used as control. c Depletion of SAP18 in HEL cells (Sh-SAP18) induces SIRT3 transcription. d Marginal effect of Sh-SAP18 on cell proliferation. e Survival rate of Sh-SAP18 and controls cells treated for 24 h with indicated concentrations of A671. f SIN3A transcription factor (TF) binding sites within the Sirtuin gene family promoters. DNA sequencing data derived from SIN3A chromatin immunoprecipitation (ChIP) for two cell types (GM12878, B-lymphocyte lymphoblastoid, and H1-hESC, human embryonic stem cells) obtained through the ENCODE database (for details see supplementary Fig. 8 ). Of the Sirtuin genes, SIN3A observed to bind within the promoters of all but SIRT7 . The greatest binding observed for SIRT3 was ~108% higher than SIRT1 in GM12878 cells and ~40% higher than SIRT2 in the H1-hESCs. g Expression of SAP18 in HEL cells treated with actinomycin D (10 μM) alone or in combination with A671 (0.5 μM) for 12 (top) or 24 h (bottom). GAPDH used as a loading control. h A three-dimensional view of the predicted interaction of A671 with SAP18. i Position of chemical interactions with the indicated amino acids within SAP18. j Predicted affinity properties of A671 to SAP18 and SIRT3. P

    Journal: Communications Biology

    Article Title: A C21-steroidal derivative suppresses T-cell lymphoma in mice by inhibiting SIRT3 via SAP18-SIN3

    doi: 10.1038/s42003-020-01458-3

    Figure Lengend Snippet: A671 binds SAP18 within the SIN3 suppressor complex to transcriptionally repress SIRT3 expression. a , b Efficient depletion of SAP18 in HEL cells using shRNA as determined by western blotting ( a ) and Q-RT-PCR ( b ). Untreated or scrambled transfected vector (Vector) cells used as control. c Depletion of SAP18 in HEL cells (Sh-SAP18) induces SIRT3 transcription. d Marginal effect of Sh-SAP18 on cell proliferation. e Survival rate of Sh-SAP18 and controls cells treated for 24 h with indicated concentrations of A671. f SIN3A transcription factor (TF) binding sites within the Sirtuin gene family promoters. DNA sequencing data derived from SIN3A chromatin immunoprecipitation (ChIP) for two cell types (GM12878, B-lymphocyte lymphoblastoid, and H1-hESC, human embryonic stem cells) obtained through the ENCODE database (for details see supplementary Fig. 8 ). Of the Sirtuin genes, SIN3A observed to bind within the promoters of all but SIRT7 . The greatest binding observed for SIRT3 was ~108% higher than SIRT1 in GM12878 cells and ~40% higher than SIRT2 in the H1-hESCs. g Expression of SAP18 in HEL cells treated with actinomycin D (10 μM) alone or in combination with A671 (0.5 μM) for 12 (top) or 24 h (bottom). GAPDH used as a loading control. h A three-dimensional view of the predicted interaction of A671 with SAP18. i Position of chemical interactions with the indicated amino acids within SAP18. j Predicted affinity properties of A671 to SAP18 and SIRT3. P

    Article Snippet: Similarly, SAP18 (pCMV3-SAP18-GFPSpark®; Sino Biological, China) overexpression studies conducted using Lipofectamine 3000 and hygromycin (250 µg/ml) used for cell selection.

    Techniques: Expressing, shRNA, Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Binding Assay, DNA Sequencing, Derivative Assay, Chromatin Immunoprecipitation

    A671 is an HDAC type III inhibitor (HDACi-III) that attenuates SIRT3 by binding and activating SAP18. a HEL cells incubated for 30 min with the indicated concentrations of A671 followed by HDAC type I and IIA/IIB activity assays (Promega). b Trichostatin A used as a positive control. c HEL cells were treated with A671, followed by HDAC type III activity analysis. d Nicotinamide was used as a positive control. e – j Q-RT-PCR analysis of EL4 cells for the indicated genes following 12 h treatment with A671, DMSO, or medium. k , l Induction of SAP18 mRNA ( k ) and protein ( l ) levels by A671 in a dose-dependent manner. m , A671 treatment diminished SIRT3 transcription in a dose-dependent manner as determined by Q-RT-PCR. P

    Journal: Communications Biology

    Article Title: A C21-steroidal derivative suppresses T-cell lymphoma in mice by inhibiting SIRT3 via SAP18-SIN3

    doi: 10.1038/s42003-020-01458-3

    Figure Lengend Snippet: A671 is an HDAC type III inhibitor (HDACi-III) that attenuates SIRT3 by binding and activating SAP18. a HEL cells incubated for 30 min with the indicated concentrations of A671 followed by HDAC type I and IIA/IIB activity assays (Promega). b Trichostatin A used as a positive control. c HEL cells were treated with A671, followed by HDAC type III activity analysis. d Nicotinamide was used as a positive control. e – j Q-RT-PCR analysis of EL4 cells for the indicated genes following 12 h treatment with A671, DMSO, or medium. k , l Induction of SAP18 mRNA ( k ) and protein ( l ) levels by A671 in a dose-dependent manner. m , A671 treatment diminished SIRT3 transcription in a dose-dependent manner as determined by Q-RT-PCR. P

    Article Snippet: Similarly, SAP18 (pCMV3-SAP18-GFPSpark®; Sino Biological, China) overexpression studies conducted using Lipofectamine 3000 and hygromycin (250 µg/ml) used for cell selection.

    Techniques: Binding Assay, Incubation, Activity Assay, Positive Control, Reverse Transcription Polymerase Chain Reaction

    AS PIM1 Directly Phosphorylates Endogenous High-Confidence Substrates in Prostate Cancer Cells A–D) Validation of endogenous PIM1 substrates. LNCaP-AS-PIM1 thiophosphorylates PUM1 (A), CHMP7 (B), NDRG1 (C), and KIF18A (D). Endogenous proteins were immunoprecipitated from LNCaP-WT-PIM1 and LNCaP-AS-PIM1 and analyzed by Western blot for the presence of thiophosphorylation (ThioP), or immunoprecipitated PUM1, CHMP7, NDRG1, and KIF18A. E-H) Phosphorylation site validation using WT and phosphorylation site mutant substrates. Substrates (Myc-PUM1, FLAG-CHMP7, FLAG-NDRG1, and GFP-6HIS-KIF18A; either wild type (WT) or the indicated PIM1 phosphorylation site mutation) were expressed in 293T cells with AS-PIM1 and thiophosphorylation labeling completed. Substrates were immunoprecipitated using Myc, FLAG, or GFP-magnetic beads, and Western blot performed to detect thiophosphorylated, or the immunoprecipitated substrates. Western blots are representative of two independent experiments.

    Journal: bioRxiv

    Article Title: Identification of PIM1 substrates reveals a role for NDRG1 in prostate cancer cellular migration and invasion

    doi: 10.1101/2020.01.21.913962

    Figure Lengend Snippet: AS PIM1 Directly Phosphorylates Endogenous High-Confidence Substrates in Prostate Cancer Cells A–D) Validation of endogenous PIM1 substrates. LNCaP-AS-PIM1 thiophosphorylates PUM1 (A), CHMP7 (B), NDRG1 (C), and KIF18A (D). Endogenous proteins were immunoprecipitated from LNCaP-WT-PIM1 and LNCaP-AS-PIM1 and analyzed by Western blot for the presence of thiophosphorylation (ThioP), or immunoprecipitated PUM1, CHMP7, NDRG1, and KIF18A. E-H) Phosphorylation site validation using WT and phosphorylation site mutant substrates. Substrates (Myc-PUM1, FLAG-CHMP7, FLAG-NDRG1, and GFP-6HIS-KIF18A; either wild type (WT) or the indicated PIM1 phosphorylation site mutation) were expressed in 293T cells with AS-PIM1 and thiophosphorylation labeling completed. Substrates were immunoprecipitated using Myc, FLAG, or GFP-magnetic beads, and Western blot performed to detect thiophosphorylated, or the immunoprecipitated substrates. Western blots are representative of two independent experiments.

    Article Snippet: Plasmids, Primers, and Antibodies Constructs utilized are as follows: pCDNA 3.1 (+)-AR-FLAG , NDRG1 (HG14119-CF, Sino Biologicals), CHMP7 (HG14273-CF, Sino Biologicals), PUM1 (RC201219, Origene), KIF18A (Addgene plasmid # 23002), pCDNA 3.1 (+)-WT PIM1 , pCDNA 3.1 (+)-KD (K67M) , pLB(N)CX-WT PIM1 , pLB(N)CX-KD PIM1 . pCDNA 3.1 (+)- AS (L120G) PIM1 and pLB(N)CX-AS PIM1 (L120G) was generated using site-directed mutagenesis.

    Techniques: Immunoprecipitation, Western Blot, Mutagenesis, Labeling, Magnetic Beads

    AS PIM1 Directly Phosphorylates Endogenous High-Confidence Substrates in Prostate Cancer Cells A–D) Validation of endogenous PIM1 substrates. LNCaP-AS-PIM1 thiophosphorylates PUM1 (A), CHMP7 (B), NDRG1 (C), and KIF18A (D). Endogenous proteins were immunoprecipitated from LNCaP-WT-PIM1 and LNCaP-AS-PIM1 and analyzed by Western blot for the presence of thiophosphorylation (ThioP), or immunoprecipitated PUM1, CHMP7, NDRG1, and KIF18A. E-H) Phosphorylation site validation using WT and phosphorylation site mutant substrates. Substrates (Myc-PUM1, FLAG-CHMP7, FLAG-NDRG1, and GFP-6HIS-KIF18A; either wild type (WT) or the indicated PIM1 phosphorylation site mutation) were expressed in 293T cells with AS-PIM1 and thiophosphorylation labeling completed. Substrates were immunoprecipitated using Myc, FLAG, or GFP-magnetic beads, and Western blot performed to detect thiophosphorylated, or the immunoprecipitated substrates. Western blots are representative of two independent experiments.

    Journal: bioRxiv

    Article Title: Identification of PIM1 substrates reveals a role for NDRG1 in prostate cancer cellular migration and invasion

    doi: 10.1101/2020.01.21.913962

    Figure Lengend Snippet: AS PIM1 Directly Phosphorylates Endogenous High-Confidence Substrates in Prostate Cancer Cells A–D) Validation of endogenous PIM1 substrates. LNCaP-AS-PIM1 thiophosphorylates PUM1 (A), CHMP7 (B), NDRG1 (C), and KIF18A (D). Endogenous proteins were immunoprecipitated from LNCaP-WT-PIM1 and LNCaP-AS-PIM1 and analyzed by Western blot for the presence of thiophosphorylation (ThioP), or immunoprecipitated PUM1, CHMP7, NDRG1, and KIF18A. E-H) Phosphorylation site validation using WT and phosphorylation site mutant substrates. Substrates (Myc-PUM1, FLAG-CHMP7, FLAG-NDRG1, and GFP-6HIS-KIF18A; either wild type (WT) or the indicated PIM1 phosphorylation site mutation) were expressed in 293T cells with AS-PIM1 and thiophosphorylation labeling completed. Substrates were immunoprecipitated using Myc, FLAG, or GFP-magnetic beads, and Western blot performed to detect thiophosphorylated, or the immunoprecipitated substrates. Western blots are representative of two independent experiments.

    Article Snippet: Plasmids, Primers, and Antibodies Constructs utilized are as follows: pCDNA 3.1 (+)-AR-FLAG , NDRG1 (HG14119-CF, Sino Biologicals), CHMP7 (HG14273-CF, Sino Biologicals), PUM1 (RC201219, Origene), KIF18A (Addgene plasmid # 23002), pCDNA 3.1 (+)-WT PIM1 , pCDNA 3.1 (+)-KD (K67M) , pLB(N)CX-WT PIM1 , pLB(N)CX-KD PIM1 . pCDNA 3.1 (+)- AS (L120G) PIM1 and pLB(N)CX-AS PIM1 (L120G) was generated using site-directed mutagenesis.

    Techniques: Immunoprecipitation, Western Blot, Mutagenesis, Labeling, Magnetic Beads

    Mitochondrial pre-RNA processing defects upon UPR mt a, qPCR of mitochondrial pre-RNA at tRNA Met and tRNA Lys RNAseP processing sites upon induction of UPR mt with GTPP (6h). Error bars, averages ±s.d. (n=3 biological replicates). b , RNA-seq for analysis of mitochondrial pre-RNA processing defects based on number of reads crossing the tRNA/mRNA gene junction. Slope of coverage in the tRNA gene adjacent to the cut site is used as a measure of processing. c-d , Normalized RNA-seq coverage across tRNA/mRNA gene borders for tRNA Met ( c ) and tRNA Lys ( d ) with average of slopes (±s.d.) from b indicated in the inset (n=3 biological replicates, two-tailed p-values *p≤0.05, ***p≤0.001). e , Quantitative western blot analysis of MRPP3 levels upon treatment of cells with DMSO, GTPP, CDDO, or GTPP + CDDO co-treatment for 6 hours. f , Mitochondrial pre-RNA accumulation upon co-treatment with GTPP and CDDO (as in a ). Data are average values ±s.d. (n=3 biological replicates). For gel source data, see Supplementary Figure 1 .

    Journal: Nature

    Article Title: Mitochondrial unfolded protein response controls matrix pre-RNA processing and translation

    doi: 10.1038/nature18302

    Figure Lengend Snippet: Mitochondrial pre-RNA processing defects upon UPR mt a, qPCR of mitochondrial pre-RNA at tRNA Met and tRNA Lys RNAseP processing sites upon induction of UPR mt with GTPP (6h). Error bars, averages ±s.d. (n=3 biological replicates). b , RNA-seq for analysis of mitochondrial pre-RNA processing defects based on number of reads crossing the tRNA/mRNA gene junction. Slope of coverage in the tRNA gene adjacent to the cut site is used as a measure of processing. c-d , Normalized RNA-seq coverage across tRNA/mRNA gene borders for tRNA Met ( c ) and tRNA Lys ( d ) with average of slopes (±s.d.) from b indicated in the inset (n=3 biological replicates, two-tailed p-values *p≤0.05, ***p≤0.001). e , Quantitative western blot analysis of MRPP3 levels upon treatment of cells with DMSO, GTPP, CDDO, or GTPP + CDDO co-treatment for 6 hours. f , Mitochondrial pre-RNA accumulation upon co-treatment with GTPP and CDDO (as in a ). Data are average values ±s.d. (n=3 biological replicates). For gel source data, see Supplementary Figure 1 .

    Article Snippet: Cell line generation Human cDNA for MRPP3 was purchased from Sino Biological (HG14131-G) and transferred into a pHAGE lentiviral vector.

    Techniques: Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Two Tailed Test, Western Blot

    Rescue of UPR mt -induced mitochondrial pre-RNA processing by MRPP3 overexpression a , Western Blot analysis of MRPP3 levels upon DMSO or GTPP treatment in the context of wild-type or MRPP3-overexpressing (o/e) cells. Quantifications of the MRPP3 bands are shown in blue (quantified with Fiji of digitally acquired images). b , Quantitative PCR analysis of non-processed mitochondrial pre-RNA levels at the tRNA Met and tRNA Lys cut sites in wild-type cells or cells overexpressing MRPP3. Shown are mean values ±s.d. (n=3 biological replicates). c , Quantitative PCR analysis of non-processed mitochondrial pre-RNA levels at the tRNA Met and tRNA Lys cut sites in wild-type cells or cells overexpressing MRPP3 upon GTPP treatment. Shown are mean values ±s.d. (n=3 biological replicates).

    Journal: Nature

    Article Title: Mitochondrial unfolded protein response controls matrix pre-RNA processing and translation

    doi: 10.1038/nature18302

    Figure Lengend Snippet: Rescue of UPR mt -induced mitochondrial pre-RNA processing by MRPP3 overexpression a , Western Blot analysis of MRPP3 levels upon DMSO or GTPP treatment in the context of wild-type or MRPP3-overexpressing (o/e) cells. Quantifications of the MRPP3 bands are shown in blue (quantified with Fiji of digitally acquired images). b , Quantitative PCR analysis of non-processed mitochondrial pre-RNA levels at the tRNA Met and tRNA Lys cut sites in wild-type cells or cells overexpressing MRPP3. Shown are mean values ±s.d. (n=3 biological replicates). c , Quantitative PCR analysis of non-processed mitochondrial pre-RNA levels at the tRNA Met and tRNA Lys cut sites in wild-type cells or cells overexpressing MRPP3 upon GTPP treatment. Shown are mean values ±s.d. (n=3 biological replicates).

    Article Snippet: Cell line generation Human cDNA for MRPP3 was purchased from Sino Biological (HG14131-G) and transferred into a pHAGE lentiviral vector.

    Techniques: Over Expression, Western Blot, Real-time Polymerase Chain Reaction

    Reversibility of UPR mt -induced mitochondrial pre-RNA processing and translation defects a , Coomassie gel staining as a loading control of the same experiment as in Fig. 4e . b , Mitochondrial pre-RNA processing was measured by qPCR in cells subjected to GTTP pulse-chase wash-out for 1-4h. Data are averages of fold changes versus untreated ±s.d., two-tailed p-values *p≤0.05, **p≤0.01, ***p≤0.001, n=3 biological replicates. c , Analysis of mitochondrial translation in wild-type or MRPP3 overexpressing cells with or without GTPP treatment. Newly synthesized proteins were labeled with 35 S and analyzed by phospho-imager. d , Immunoblot of TFB1M expression with or without 6h GTPP treatment (left). Quantification of control normalized TFB1M levels from immunoblots of two independent experiments.

    Journal: Nature

    Article Title: Mitochondrial unfolded protein response controls matrix pre-RNA processing and translation

    doi: 10.1038/nature18302

    Figure Lengend Snippet: Reversibility of UPR mt -induced mitochondrial pre-RNA processing and translation defects a , Coomassie gel staining as a loading control of the same experiment as in Fig. 4e . b , Mitochondrial pre-RNA processing was measured by qPCR in cells subjected to GTTP pulse-chase wash-out for 1-4h. Data are averages of fold changes versus untreated ±s.d., two-tailed p-values *p≤0.05, **p≤0.01, ***p≤0.001, n=3 biological replicates. c , Analysis of mitochondrial translation in wild-type or MRPP3 overexpressing cells with or without GTPP treatment. Newly synthesized proteins were labeled with 35 S and analyzed by phospho-imager. d , Immunoblot of TFB1M expression with or without 6h GTPP treatment (left). Quantification of control normalized TFB1M levels from immunoblots of two independent experiments.

    Article Snippet: Cell line generation Human cDNA for MRPP3 was purchased from Sino Biological (HG14131-G) and transferred into a pHAGE lentiviral vector.

    Techniques: Staining, Real-time Polymerase Chain Reaction, Pulse Chase, Two Tailed Test, Synthesized, Labeling, Expressing, Western Blot

    Mitochondrial pre-RNA processing defects upon UPR mt a , Primer design for monitoring pre-RNA processing. Primer pairs1 3 and 2 4 will only produce PCR products for uncleaved mitochondrial pre-RNAs and allows quantitation of non-processed pre-RNAs. Primer pair 2 3 will monitor total levels for normalization. b , Quantitative PCR of MRPP3 mRNA levels upon knockdown with siRNA targeting a scrambled sequence or MRPP3. Shown are averages ±s.d. (n=3 biological replicates). c , qPCR of mitochondrial pre-RNA at tRNA Met and tRNA Lys RNAseP processing sites upon depletion of MRPP3 by RNAi. Error bars, ±s.d. (n=3 biological replicates). d , Quantitative PCR monitoring levels of non-processed pre-RNA upon treatment of cells with GTPP or the uncoupler CCCP in biological duplicate; repl., replicate e , LON protein levels as determined by quantitative proteomics ( Fig.2 ) in biological duplicate. Shown are scaled signal to noise values observed (i.e. relative abundance). f . Western blot analysis of LON levels upon control or 10μM GTPP treatment (6 h).

    Journal: Nature

    Article Title: Mitochondrial unfolded protein response controls matrix pre-RNA processing and translation

    doi: 10.1038/nature18302

    Figure Lengend Snippet: Mitochondrial pre-RNA processing defects upon UPR mt a , Primer design for monitoring pre-RNA processing. Primer pairs1 3 and 2 4 will only produce PCR products for uncleaved mitochondrial pre-RNAs and allows quantitation of non-processed pre-RNAs. Primer pair 2 3 will monitor total levels for normalization. b , Quantitative PCR of MRPP3 mRNA levels upon knockdown with siRNA targeting a scrambled sequence or MRPP3. Shown are averages ±s.d. (n=3 biological replicates). c , qPCR of mitochondrial pre-RNA at tRNA Met and tRNA Lys RNAseP processing sites upon depletion of MRPP3 by RNAi. Error bars, ±s.d. (n=3 biological replicates). d , Quantitative PCR monitoring levels of non-processed pre-RNA upon treatment of cells with GTPP or the uncoupler CCCP in biological duplicate; repl., replicate e , LON protein levels as determined by quantitative proteomics ( Fig.2 ) in biological duplicate. Shown are scaled signal to noise values observed (i.e. relative abundance). f . Western blot analysis of LON levels upon control or 10μM GTPP treatment (6 h).

    Article Snippet: Cell line generation Human cDNA for MRPP3 was purchased from Sino Biological (HG14131-G) and transferred into a pHAGE lentiviral vector.

    Techniques: Polymerase Chain Reaction, Quantitation Assay, Real-time Polymerase Chain Reaction, Sequencing, Western Blot