rabbit polyclonal antisera against recombinant mouse twinkle protein  (Agrisera)

Journal: Science Advances

Article Title: POLRMT regulates the switch between replication primer formation and gene expression of mammalian mtDNA

doi: 10.1126/sciadv.1600963

Figure Lengend Snippet: ( A ) Steady-state protein levels of nuclear-encoded factors of mtDNA expression analyzed by Western blotting on mitochondrial extracts from hearts of control and tissue-specific knockout mice; loading, VDAC; asterisk, cross-reacting band ; for quantification, see fig. S4A. ( B ) Quantitative RT-PCR (qRT-PCR) of transcript levels of nuclear-encoded mitochondrial proteins. Normalization, β 2 M (β 2 -microglobulin). Error bars indicate ±SEM (* P < 0.05 and *** P < 0.001; two-tailed Student’s t test; see table S1). ( C and D ) Linear glycerol density gradient fractionations of mitochondrial lysates from tissue-specific knockout and control mice followed by Western blot analysis; for quantification, see figs. S5 (A to C) and S6. Samples taken from fractions 1 to 16 are of increasing density (that is, from top to bottom of the tube after separation by ultracentrifugation; as indicated by the schematic representation of the centrifuge tube to the left). Fractions were loaded from left to right on the gels as indicated by the lane numbering; input, aliquots of unfractionated lysates. The mtDNA content of the fractions was determined by Southern blotting. ( E ) Relative TFAM and POLRMT protein distribution across the gradient from control and knockout heart mitochondria.

Article Snippet: The rabbit polyclonal antisera against recombinant mouse TWINKLE, POLRMT, and TFB2M protein that lack the mitochondrial targeting signal were generated by Agrisera and subsequently affinity-purified using the corresponding recombinant protein.

Techniques: Expressing, Western Blot, Control, Knock-Out, Quantitative RT-PCR, Two Tailed Test, Southern Blot

Journal: Science Advances

Article Title: POLRMT regulates the switch between replication primer formation and gene expression of mammalian mtDNA

doi: 10.1126/sciadv.1600963

Figure Lengend Snippet: ( A and B ) Northern blot analyses of mitochondrial mRNAs, rRNAs, and tRNAs from hearts of 4-week-old control and tissue-specific knockout mice; loading, 18 S rRNA. ( C ) Relative mitochondrial RNA abundance of mRNA and rRNA levels in hearts of 4-week-old tissue-specific knockout and control mice normalized to the upper quartile of the gene count distribution. The data analyzed are from three independent RNA-seq experiments; all RNAs have *** P ≤ 0.0001. Error bars indicate ±SEM. ( D ) In vitro transcription assay at different POLRMT levels. All reactions contained a cut plasmid template (containing the human LSP and HSP promoters giving a run-off product of 101 and 180 nt, respectively). POLRMT was added at 128, 32, 8, 2, and 0.5 nM in lanes 1 to 5, respectively; lane 6, control without POLRMT; lane 7, molecular weight marker (New England Biolabs). ( E ) Quantification of the results from (D). The experiment was performed in triplicates, and HSP transcription levels were normalized to LSP for each POLRMT concentration; bars, mean value. Error bars indicate ±SD ( n = 3).

Article Snippet: The rabbit polyclonal antisera against recombinant mouse TWINKLE, POLRMT, and TFB2M protein that lack the mitochondrial targeting signal were generated by Agrisera and subsequently affinity-purified using the corresponding recombinant protein.

Techniques: Northern Blot, Control, Knock-Out, RNA Sequencing, In Vitro, Transcription Assay, Plasmid Preparation, Molecular Weight, Marker, Concentration Assay

Journal: Science Advances

Article Title: POLRMT regulates the switch between replication primer formation and gene expression of mammalian mtDNA

doi: 10.1126/sciadv.1600963

Figure Lengend Snippet: ( A ) POLRMT steady-state protein levels in heart from wild-type (+/+) and heterozygous Polrmt knockout (+/−) mice; loading, VDAC; for quantification, see fig. S7C. ( B ) Steady-state levels of mitochondrial mRNAs, rRNAs, and tRNAs; loading, 18 S rRNA; for quantification, see fig. S8A. ( C ) Steady-state protein levels of nuclear-encoded factors of mtDNA expression analyzed by Western blotting on mitochondrial heart extracts; loading, VDAC; for quantification, see fig. S9A; asterisk, cross-reacting band . ( D ) De novo–synthesized mitochondrial transcripts from hearts of 52-week-old mice. Steady-state levels of individual mitochondrial transcripts were verified with a radiolabeled probe ( mt-Co1 ); input, Western blot analysis (POLRMT and VDAC) after labeling. ( E ) 7 S RNA levels in mouse hearts by Northern blotting on total RNA; loading, 18 S rRNA. ( F ) Quantification of mtDNA by quantitative PCR (qPCR) with mt-Co1 , mt-Nd1 , and mt-Nd5 probes on mouse heart. Signals were normalized to the 18 S signal; n = 3. Error bars indicate ±SEM. ( G ) De novo–synthesized DNA of isolated mitochondria from hearts of 12-week-old mice. The mtDNA was radioactively labeled in organello, isolated and boiled to release newly synthesized 7 S DNA before Southern blotting; input, Western blotting (POLRMT and VDAC) after labeling; for quantification, see fig. S10B.

Article Snippet: The rabbit polyclonal antisera against recombinant mouse TWINKLE, POLRMT, and TFB2M protein that lack the mitochondrial targeting signal were generated by Agrisera and subsequently affinity-purified using the corresponding recombinant protein.

Techniques: Knock-Out, Expressing, Western Blot, Synthesized, Labeling, Northern Blot, Real-time Polymerase Chain Reaction, Isolation, Southern Blot

Journal: Science Advances

Article Title: POLRMT regulates the switch between replication primer formation and gene expression of mammalian mtDNA

doi: 10.1126/sciadv.1600963

Figure Lengend Snippet: At high POLRMT levels, mitochondrial transcription initiation is activated from both the HSP and LSP resulting in mtDNA gene expression. At low POLRMT levels, only LSP is active and an RNA primer for replication of mtDNA is synthesized.

Article Snippet: The rabbit polyclonal antisera against recombinant mouse TWINKLE, POLRMT, and TFB2M protein that lack the mitochondrial targeting signal were generated by Agrisera and subsequently affinity-purified using the corresponding recombinant protein.

Techniques: Gene Expression, Synthesized