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  • 79
    Biotechnology Information set8
    <t>SET8</t> expression in ccRCC tissues. SET8 immunostaining in ccRCC tissues with (A) low expression and (B) high expression. Cells with a brown-stained nucleus were considered positive. Original magnification, ×200. ccRCC, clear cell renal cell carcinoma; SET8, SET domain containing (lysine methyltransferase) 8.
    Set8, supplied by Biotechnology Information, used in various techniques. Bioz Stars score: 79/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    76
    Biotechnology Information full length dnmt1
    Correlation of increased <t>DNMT1</t> levels with MAD2 up-regulation by immunohistochemistry in human breast cancer tissues. MAD2 and DNMT1 levels were determined by immunohistochemistry in a series of 85 human breast cancer cases. The staining intensities were
    Full Length Dnmt1, supplied by Biotechnology Information, used in various techniques. Bioz Stars score: 76/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    Biotechnology Information library matched porcine stk3
    FMDV infection decreases the abundance of the endogenous <t>STK3</t> protein. PK-15 cells were seeded in 3.5 cm dish, and the monolayer cells were infected with FMDV (MOI 0.5). The cells were collected at the indicated time points (0, 4, 8, 12, 16, 18, and 20 h). The expression of STK3 mRNA was determined by qPCR assay (b). The expression of endogenous STK3 and viral VP1 proteins was determined by western blotting (a).
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    78
    Biotechnology Information human ppip5k2 kinase domain
    <t>PPIP5K2</t> KD is not a phosphotransferase: Ins P 6 inhibits Ins P 8 dephosphorylation by PPIP5K2 KD ( A , top) This graphic depicts how Ins(1,3,4) P 3 stimulates Ins(1,3,4,5,6) P 5 dephosphorylation by ITPK1 [ 47 ]. The 1-phosphate group (shown in red) that is removed from Ins(1,3,4,5,6) P 5 is not released into the bulk phase but is retained by the nucleotide and donated to Ins(1,3,4) P 3 ; this returns the enzyme to its ADP state, ready for another cycle of Ins(1,3,4,5,6) P 5 dephosphorylation. ( A , bottom) The analogous potential reaction sequence for PPIP5K. ( B ) Either no enzyme (broken line) or 0.63 μg/ml PPIP5K2 KD (solid line; dotted line) was incubated with 10 μM [ 3 H]Ins P 8 and 10 mM ADP for 10 min as indicated in the Materials and methods section. Where indicated (dotted line) incubations also contained 10 μM Ins P 6 . The reactions were quenched and neutralized and analysed by Partisphere SAX HPLC as described in the Materials and methods section. Representative HPLC data are shown.
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    79
    Biotechnology Information h2 q10
    The <t>Ly49C·H2-Q10</t> Interface
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    91
    Biotechnology Information anxa2p3
    Inhibition of <t>ANXA2P3</t> in siRNA groups 2 and 3 attenuates the expression of ANXA2, whereas overexpression of ANXA2P3 increases the expression of ANXA2 in vitro . Data are presented as the means ± standard deviation (n=3). P-values were obtained by one-way analysis of variance followed by a post hoc Tukey’s test for multiple comparisons. # P
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    79
    Biotechnology Information c glutamicum atcc13032 genome sequence
    Characterization of global transcriptome in Corynebacterium <t>glutamicum</t> ATCC21300 and <t>ATCC13032.</t> ( A ) The total number of mRNA-Seq (sequencing) reads mapped in each C. glutamicum strain library; and ( B ) differentially expressed genes (DEGs) in C. glutamicum ATCC21300. Detailed information is shown in Table S2 . CDS: coding sequencing.
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    78
    Biotechnology Information b longum ncc2705
    Quantitative analysis of arabinose, xylose, and XOS (A)XOS breakdown and metabolite production as a function of time with Bifidobacterium <t>longum</t> <t>NCC2705</t> (A and B) and Eubacterium rectale ATCC 33656 (C and D) in the presence of (A)XOS during monoculture
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    88
    Biotechnology Information slc26a6 sequence
    Expression of <t>Slc26a6</t> in the duodenal and renal tissues of rats among different groups. (A) Western analysis of duodenal tissue showed no difference in the expression of SLC26A6 among lv-Slc26a6, siRNA-Slc26a6, control, and vector groups. The data are expressed as means ± SD ( n = 4 rats/group). (B) Evaluation of the expression of renal Slc26a6. The lv-Slc26a6 group showed significantly higher expression compared with the control group, while the level reduced in the siRNA-Slc26a6 group. No significant difference was observed in the expression in the vector and control groups. The data are expressed as means ± SD ( n = 4 rats/group); * P
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    76
    Biotechnology Information human dyrk1a
    Activation Loop and Active State Stabilization Comparison of the activation segment arrangements of (A) <t>DYRK1A,</t> activation segment in orange, and (B) dual-phosphorylated ERK2, activation segment in green. The ERK2 structure is from PDB ID code 2ERK . Both DYRK1A and ERK2 have a completely ordered activation loop and glycine-rich loop, and active αC conformations. The activation loop in dual-phosphorylated active ERK2 forms an extensive hydrogen-bonding network around pT183. Phosphorylated Y185 is also stabilized through an extensive interaction network that is similar to the pY321 network formed by DYRK1A.
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    78
    Biotechnology Information dysferlin construct
    <t>Mini-dysferlin</t> C72 formation requires ∼200 μM extracellular calcium, broadly correlating with the extracellular calcium concentration required for calcium-dependent membrane repair of injured muscle cells. (A) Development of a flow cytometry membrane repair assay reveals 100–200 μM as the activating concentration of extracellular Ca 2+ required for calcium-dependent membrane repair pathways in cultured human muscle cells. (B) Treatment of primary human muscle cells with the calpain inhibitor calpeptin shows dose-dependent inhibition of cell survival, with an IC 50 of 11.8 ± 5.8 μM (a representative dose–response curve is shown; the calculated IC 50 is derived from four independent dose–response curves performed on different days, one with singlet samples at each dose, three in duplicate). C) Representative Western blot of a dose–response curve showing increasing formation of cleaved mini-dysferlin C72 with increasing concentrations of extracellular calcium. (D) Pooled densitometric quantification of levels of cleaved mini-dysferlin C72 from five calcium dose–response curves ( EC 50 of ∼ 250 μM Ca 2+ , 95% confidence interval). (E, F) In vitro digestion of dysferlin-exon 40a with 0.2 A.U. of purified calpain-1 (E) and calpain-2 (F). Mini-dysferlin C72 is indicated with black arrows.
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    79
    Biotechnology Information target genes kcne3
    <t>Kcne3</t> deletion age-dependently delays ventricular repolarization. A ) Left panel: representative surface ECGs of male 9-mo-old Kcne3 +/+ and Kcne3 −/− mice. Right panel: mean ECG parameters of male Kcne3 +/+ (5 mo old, n =8; 9 mo old, n =10)
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    79
    Biotechnology Information rom1
    Sequence analysis of <t>Rom1</t> . The position of the point mutation is indicated by red arrowheads on sequence chromatograms from the Control DBF1 mouse (top) and the M-1156 mouse (bottom). A T→C single base substitution at position 1,195 of Rom1 ( M96760 , National Center for Biotechnology Information [NCBI]) was identified in the M-1156 mouse line.
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    76
    Biotechnology Information murine perilipin 1a
    Tests for antibody specificity utilizing blocking peptides. 3T3L1 adipocytes were exposed to either 1 µM isoproterenol or 100 nM L-γ-MSH. Prior to labeling, primary antibodies were preincubated with the indicated amounts (µg) of blocking peptides corresponding to pPeri-site 5, pPeri-site 6, or pHSL-serine 660. <t>Anti-phospho-perilipin</t> 1A and anti-phospho-HSL antibodies were visualized in the red and far-red fluorescence channels, respectively. A, Results are shown for cells treated with isoproterenol for 15 minutes in which anti-pPeri-site 5 and anti-pHSLserine 660 were blocked with pPeri-site 5. Upper panels depict cell images. Lower bar graphs depict Tii Pi Pm data for pPeri-site 5 and pHSL-serine 660. B, Results are shown for cells treated with isoproterenol for 10 minutes in which anti-pPeri-site 5 and anti-pHSL-serine 660 were blocked with pHSL-serine 660. C, Results are shown cells treated with L-γ-MSH for 7 minutes in which anti-pPeri-site 6 and anti-pHSLserine563 were blocked with pPeri-site 6. For A, each bar represents a single well; for B and C, each bar represents the mean ± SD for n = 3 wells.
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    Biotechnology Information elr1
    Mapping of CRD1 residues critical for <t>ELR1</t> function by gain-of-function analyses of point mutations converting murine HveA to equine ELR1 amino acids. (A) Schematic representation of the series of point mutations constructed by substituting the indicated
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    79
    Biotechnology Information cas9 protein sequence
    Multiplex genome editing in zebrafish by <t>Cas9.</t> A mix of five gRNAs ( penta gRNAs: ddx19 gRNA, 55 pg; egfp gRNA, 15 pg; gol gRNA, 25 pg; mitfa gRNA, 60 pg; and tyr gRNA, 25 pg) were coinjected with nls-zCas9-nls RNA (150 pg) into Tg(-5.1mnx1:egfp) transgenic
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    75
    Biotechnology Information camsrb2
    Effects of overexpressing <t>CaMsrB2</t> on the Phytophthora blight disease. Leaves of CaMsrB2 -transgenic tomato were inoculated with P. capsici zoospores (5 × 10 5 zoospores mL −1 ). A, Cell death in CaMsrB2 -silenced plants. Cell death on the leaves
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    78
    Biotechnology Information human cofilin gene
    CSP comparison of <t>cofilin</t> variants. A , changes in the WT cofilin structure caused by reduction ( red ) superimposed on the three-dimensional surface model of the protein (PDB ID 1Q8G ), as in Fig. 1 C. B , overlay of CSPs > 0.1 ppm between mutant C39A and the nonreduced WT ( white ) and the changes shown in A ( red ). The overlap of both is colored pink. C , comparison of mutant C139A and nonreduced WT cofilin with the same colors as in B. D , comparison of mutant C147A and nonreduced WT cofilin with the same colors as in B .
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    79
    Biotechnology Information duck origin fadv 4
    <t>FAdV-4</t> viral DNA concentration in visceral tissues samples at different dpi ( A : oral group; B : subcutaneous group).
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    Image Search Results


    SET8 expression in ccRCC tissues. SET8 immunostaining in ccRCC tissues with (A) low expression and (B) high expression. Cells with a brown-stained nucleus were considered positive. Original magnification, ×200. ccRCC, clear cell renal cell carcinoma; SET8, SET domain containing (lysine methyltransferase) 8.

    Journal: Oncology Letters

    Article Title: miR-502-mediated histone methyltransferase SET8 expression is associated with clear cell renal cell carcinoma risk

    doi: 10.3892/ol.2017.7115

    Figure Lengend Snippet: SET8 expression in ccRCC tissues. SET8 immunostaining in ccRCC tissues with (A) low expression and (B) high expression. Cells with a brown-stained nucleus were considered positive. Original magnification, ×200. ccRCC, clear cell renal cell carcinoma; SET8, SET domain containing (lysine methyltransferase) 8.

    Article Snippet: The primers for amplification were 5′-CCTGGTCAGTGGTCAGCAAAT-3′ (sense) and 5′-CTGGGAAACACGCTCAAAATC-3′ (antisense) for rs16917496 in the 3′UTR of SET8 (National Center for Biotechnology Information database: http://www.ncbi.nlm.niih.gov/snp ).

    Techniques: Expressing, Immunostaining, Staining

    Selection of SET8 -siRNA renal carcinoma 786-O cells by western blotting. siRNA, small interfering RNA; SET8 , SET domain containing (lysine methyltransferase) 8.

    Journal: Oncology Letters

    Article Title: miR-502-mediated histone methyltransferase SET8 expression is associated with clear cell renal cell carcinoma risk

    doi: 10.3892/ol.2017.7115

    Figure Lengend Snippet: Selection of SET8 -siRNA renal carcinoma 786-O cells by western blotting. siRNA, small interfering RNA; SET8 , SET domain containing (lysine methyltransferase) 8.

    Article Snippet: The primers for amplification were 5′-CCTGGTCAGTGGTCAGCAAAT-3′ (sense) and 5′-CTGGGAAACACGCTCAAAATC-3′ (antisense) for rs16917496 in the 3′UTR of SET8 (National Center for Biotechnology Information database: http://www.ncbi.nlm.niih.gov/snp ).

    Techniques: Selection, Western Blot, Small Interfering RNA

    SET8 -knockdown inhibits renal carcinoma 786-O cell colony formation by colony formation assay. *P

    Journal: Oncology Letters

    Article Title: miR-502-mediated histone methyltransferase SET8 expression is associated with clear cell renal cell carcinoma risk

    doi: 10.3892/ol.2017.7115

    Figure Lengend Snippet: SET8 -knockdown inhibits renal carcinoma 786-O cell colony formation by colony formation assay. *P

    Article Snippet: The primers for amplification were 5′-CCTGGTCAGTGGTCAGCAAAT-3′ (sense) and 5′-CTGGGAAACACGCTCAAAATC-3′ (antisense) for rs16917496 in the 3′UTR of SET8 (National Center for Biotechnology Information database: http://www.ncbi.nlm.niih.gov/snp ).

    Techniques: Colony Assay

    SET8 -knockdown attenuates c-Myc and MMP-7 mRNA expression in renal carcinoma 786-O cells (n=3). MMP-7, matrix metalloproteinase-7; siRNA, small interfering RNA; SET8 , SET domain containing (lysine methyltransferase) 8.

    Journal: Oncology Letters

    Article Title: miR-502-mediated histone methyltransferase SET8 expression is associated with clear cell renal cell carcinoma risk

    doi: 10.3892/ol.2017.7115

    Figure Lengend Snippet: SET8 -knockdown attenuates c-Myc and MMP-7 mRNA expression in renal carcinoma 786-O cells (n=3). MMP-7, matrix metalloproteinase-7; siRNA, small interfering RNA; SET8 , SET domain containing (lysine methyltransferase) 8.

    Article Snippet: The primers for amplification were 5′-CCTGGTCAGTGGTCAGCAAAT-3′ (sense) and 5′-CTGGGAAACACGCTCAAAATC-3′ (antisense) for rs16917496 in the 3′UTR of SET8 (National Center for Biotechnology Information database: http://www.ncbi.nlm.niih.gov/snp ).

    Techniques: Expressing, Small Interfering RNA

    SET8 -knockdown suppresses renal carcinoma 786-O cell migration by wound healing assay. *P

    Journal: Oncology Letters

    Article Title: miR-502-mediated histone methyltransferase SET8 expression is associated with clear cell renal cell carcinoma risk

    doi: 10.3892/ol.2017.7115

    Figure Lengend Snippet: SET8 -knockdown suppresses renal carcinoma 786-O cell migration by wound healing assay. *P

    Article Snippet: The primers for amplification were 5′-CCTGGTCAGTGGTCAGCAAAT-3′ (sense) and 5′-CTGGGAAACACGCTCAAAATC-3′ (antisense) for rs16917496 in the 3′UTR of SET8 (National Center for Biotechnology Information database: http://www.ncbi.nlm.niih.gov/snp ).

    Techniques: Migration, Wound Healing Assay

    SET8 -knockdown suppresses renal carcinoma 786-O cell invasion by Transwell assay. *P

    Journal: Oncology Letters

    Article Title: miR-502-mediated histone methyltransferase SET8 expression is associated with clear cell renal cell carcinoma risk

    doi: 10.3892/ol.2017.7115

    Figure Lengend Snippet: SET8 -knockdown suppresses renal carcinoma 786-O cell invasion by Transwell assay. *P

    Article Snippet: The primers for amplification were 5′-CCTGGTCAGTGGTCAGCAAAT-3′ (sense) and 5′-CTGGGAAACACGCTCAAAATC-3′ (antisense) for rs16917496 in the 3′UTR of SET8 (National Center for Biotechnology Information database: http://www.ncbi.nlm.niih.gov/snp ).

    Techniques: Transwell Assay

    SET8 -knockdown cells were subjected to MTT assay to determine cell proliferation. *P

    Journal: Oncology Letters

    Article Title: miR-502-mediated histone methyltransferase SET8 expression is associated with clear cell renal cell carcinoma risk

    doi: 10.3892/ol.2017.7115

    Figure Lengend Snippet: SET8 -knockdown cells were subjected to MTT assay to determine cell proliferation. *P

    Article Snippet: The primers for amplification were 5′-CCTGGTCAGTGGTCAGCAAAT-3′ (sense) and 5′-CTGGGAAACACGCTCAAAATC-3′ (antisense) for rs16917496 in the 3′UTR of SET8 (National Center for Biotechnology Information database: http://www.ncbi.nlm.niih.gov/snp ).

    Techniques: MTT Assay

    Correlation of increased DNMT1 levels with MAD2 up-regulation by immunohistochemistry in human breast cancer tissues. MAD2 and DNMT1 levels were determined by immunohistochemistry in a series of 85 human breast cancer cases. The staining intensities were

    Journal:

    Article Title: Retinoblastoma Pathway Dysregulation Causes DNA Methyltransferase 1 Overexpression in Cancer via MAD2-Mediated Inhibition of the Anaphase-Promoting Complex

    doi: 10.2353/ajpath.2007.060779

    Figure Lengend Snippet: Correlation of increased DNMT1 levels with MAD2 up-regulation by immunohistochemistry in human breast cancer tissues. MAD2 and DNMT1 levels were determined by immunohistochemistry in a series of 85 human breast cancer cases. The staining intensities were

    Article Snippet: Full-length DNMT1 (nucleotides from 238 to 5088, National Center for Biotechnology Information (NCBI) RefSeq ) and N-terminal deletions were polymerase chain reaction (PCR) amplified with a Sal I site extension on the reverse primer (5′-GTCGACGCGG-TACCCTTGGCAAAGCA-3′) and the following forward primers: full-length 5′-ATGCCGGCGCGTACC-3′ and 120-amino acid N-terminal deletion 5′-ATGGCAGATGCCAACAGCC-3′.

    Techniques: Immunohistochemistry, Staining

    Correlation of MAD2 overexpression with DNMT1 dysregulation and RB hyperphosphorylation. A: U2OS, MCF-7, HMEC, MCF-10A, PREC, and HCT116 cells were synchronized in G 1 phase for 24 hours with 20 μmol/L lovastatin, and immunoblotting was performed

    Journal:

    Article Title: Retinoblastoma Pathway Dysregulation Causes DNA Methyltransferase 1 Overexpression in Cancer via MAD2-Mediated Inhibition of the Anaphase-Promoting Complex

    doi: 10.2353/ajpath.2007.060779

    Figure Lengend Snippet: Correlation of MAD2 overexpression with DNMT1 dysregulation and RB hyperphosphorylation. A: U2OS, MCF-7, HMEC, MCF-10A, PREC, and HCT116 cells were synchronized in G 1 phase for 24 hours with 20 μmol/L lovastatin, and immunoblotting was performed

    Article Snippet: Full-length DNMT1 (nucleotides from 238 to 5088, National Center for Biotechnology Information (NCBI) RefSeq ) and N-terminal deletions were polymerase chain reaction (PCR) amplified with a Sal I site extension on the reverse primer (5′-GTCGACGCGG-TACCCTTGGCAAAGCA-3′) and the following forward primers: full-length 5′-ATGCCGGCGCGTACC-3′ and 120-amino acid N-terminal deletion 5′-ATGGCAGATGCCAACAGCC-3′.

    Techniques: Over Expression

    Increased DNMT1 Levels Correlated with MAD2 Up-Regulation by Immunohistochemistry in a Series of 85 Cases of Human Breast Cancer

    Journal:

    Article Title: Retinoblastoma Pathway Dysregulation Causes DNA Methyltransferase 1 Overexpression in Cancer via MAD2-Mediated Inhibition of the Anaphase-Promoting Complex

    doi: 10.2353/ajpath.2007.060779

    Figure Lengend Snippet: Increased DNMT1 Levels Correlated with MAD2 Up-Regulation by Immunohistochemistry in a Series of 85 Cases of Human Breast Cancer

    Article Snippet: Full-length DNMT1 (nucleotides from 238 to 5088, National Center for Biotechnology Information (NCBI) RefSeq ) and N-terminal deletions were polymerase chain reaction (PCR) amplified with a Sal I site extension on the reverse primer (5′-GTCGACGCGG-TACCCTTGGCAAAGCA-3′) and the following forward primers: full-length 5′-ATGCCGGCGCGTACC-3′ and 120-amino acid N-terminal deletion 5′-ATGGCAGATGCCAACAGCC-3′.

    Techniques: Immunohistochemistry

    DNMT1-FLAG protein expression after small interferring RNA (siRNA) depletion of CDC20 and FZR1 in U2OS and MCF-7 cells. A: CDC20, FZR1, and lamin A/C (negative control) were depleted by siRNA transfection for 36 hours, and FLAG-tagged DNMT1 full-length

    Journal:

    Article Title: Retinoblastoma Pathway Dysregulation Causes DNA Methyltransferase 1 Overexpression in Cancer via MAD2-Mediated Inhibition of the Anaphase-Promoting Complex

    doi: 10.2353/ajpath.2007.060779

    Figure Lengend Snippet: DNMT1-FLAG protein expression after small interferring RNA (siRNA) depletion of CDC20 and FZR1 in U2OS and MCF-7 cells. A: CDC20, FZR1, and lamin A/C (negative control) were depleted by siRNA transfection for 36 hours, and FLAG-tagged DNMT1 full-length

    Article Snippet: Full-length DNMT1 (nucleotides from 238 to 5088, National Center for Biotechnology Information (NCBI) RefSeq ) and N-terminal deletions were polymerase chain reaction (PCR) amplified with a Sal I site extension on the reverse primer (5′-GTCGACGCGG-TACCCTTGGCAAAGCA-3′) and the following forward primers: full-length 5′-ATGCCGGCGCGTACC-3′ and 120-amino acid N-terminal deletion 5′-ATGGCAGATGCCAACAGCC-3′.

    Techniques: Expressing, Negative Control, Transfection

    DNMT1-FLAG protein expression in U2OS and MCF-7 cells. A: FLAG-tagged DNMT1 full-length and 120-amino acid N-terminal deletion mutants were transfected into U2OS and MCF-7 cells, and protein levels were determined after 24 hours by anti-FLAG immunoblotting

    Journal:

    Article Title: Retinoblastoma Pathway Dysregulation Causes DNA Methyltransferase 1 Overexpression in Cancer via MAD2-Mediated Inhibition of the Anaphase-Promoting Complex

    doi: 10.2353/ajpath.2007.060779

    Figure Lengend Snippet: DNMT1-FLAG protein expression in U2OS and MCF-7 cells. A: FLAG-tagged DNMT1 full-length and 120-amino acid N-terminal deletion mutants were transfected into U2OS and MCF-7 cells, and protein levels were determined after 24 hours by anti-FLAG immunoblotting

    Article Snippet: Full-length DNMT1 (nucleotides from 238 to 5088, National Center for Biotechnology Information (NCBI) RefSeq ) and N-terminal deletions were polymerase chain reaction (PCR) amplified with a Sal I site extension on the reverse primer (5′-GTCGACGCGG-TACCCTTGGCAAAGCA-3′) and the following forward primers: full-length 5′-ATGCCGGCGCGTACC-3′ and 120-amino acid N-terminal deletion 5′-ATGGCAGATGCCAACAGCC-3′.

    Techniques: Expressing, Transfection

    MAD2 overexpression and stabilization of DNMT1-FLAG in U2OS cells. MAD2 or GFP control was co-transfected into U2OS cells with full-length DNMT1 or its 120-amino acid N-terminal deletion mutant, and protein levels were determined after 24 hours by anti-FLAG

    Journal:

    Article Title: Retinoblastoma Pathway Dysregulation Causes DNA Methyltransferase 1 Overexpression in Cancer via MAD2-Mediated Inhibition of the Anaphase-Promoting Complex

    doi: 10.2353/ajpath.2007.060779

    Figure Lengend Snippet: MAD2 overexpression and stabilization of DNMT1-FLAG in U2OS cells. MAD2 or GFP control was co-transfected into U2OS cells with full-length DNMT1 or its 120-amino acid N-terminal deletion mutant, and protein levels were determined after 24 hours by anti-FLAG

    Article Snippet: Full-length DNMT1 (nucleotides from 238 to 5088, National Center for Biotechnology Information (NCBI) RefSeq ) and N-terminal deletions were polymerase chain reaction (PCR) amplified with a Sal I site extension on the reverse primer (5′-GTCGACGCGG-TACCCTTGGCAAAGCA-3′) and the following forward primers: full-length 5′-ATGCCGGCGCGTACC-3′ and 120-amino acid N-terminal deletion 5′-ATGGCAGATGCCAACAGCC-3′.

    Techniques: Over Expression, Transfection, Mutagenesis

    FMDV infection decreases the abundance of the endogenous STK3 protein. PK-15 cells were seeded in 3.5 cm dish, and the monolayer cells were infected with FMDV (MOI 0.5). The cells were collected at the indicated time points (0, 4, 8, 12, 16, 18, and 20 h). The expression of STK3 mRNA was determined by qPCR assay (b). The expression of endogenous STK3 and viral VP1 proteins was determined by western blotting (a).

    Journal: BioMed Research International

    Article Title: The Kinase STK3 Interacts with the Viral Structural Protein VP1 and Inhibits Foot-and-Mouth Disease Virus Replication

    doi: 10.1155/2017/2481348

    Figure Lengend Snippet: FMDV infection decreases the abundance of the endogenous STK3 protein. PK-15 cells were seeded in 3.5 cm dish, and the monolayer cells were infected with FMDV (MOI 0.5). The cells were collected at the indicated time points (0, 4, 8, 12, 16, 18, and 20 h). The expression of STK3 mRNA was determined by qPCR assay (b). The expression of endogenous STK3 and viral VP1 proteins was determined by western blotting (a).

    Article Snippet: STK3 that was recovered from the library matched porcine STK3 (National Center for Biotechnology Information [NCBI] reference sequence GACC01000309.1).

    Techniques: Infection, Expressing, Real-time Polymerase Chain Reaction, Western Blot

    STK3 inhibits FMDV replication during virus infection. (a) PK-15 cells were seeded in 6-well plates, and the monolayer cells were transfected with 1, 2, and 3 μ g of a Myc-STK3 expressing plasmid and 3 μ g of an empty Myc vector. The empty Myc vector was used in the transfection process to ensure that the same amount of cells received the same amount of total plasmids. At 24 hpt, the cells were mock infected or infected with FMDV (MOI 0.5) for 12 h. The expression of viral RNA and the VP1 protein was detected by qPCR assay and western blotting, respectively. (b) PK-15 cells were seeded in 3.5 cm dish, and the monolayer cells were transfected with 150 nM of STK3 siRNA or NC siRNA for 48 h, followed by infection with equal amounts of FMDV (MOI 0.5). The cells were collected at the indicated time points (0, 6, and 12 h). Expression of STK3 mRNA and viral RNA was determined by qPCR assay. Expression of STK3 and the viral VP1 protein was detected by western blotting.

    Journal: BioMed Research International

    Article Title: The Kinase STK3 Interacts with the Viral Structural Protein VP1 and Inhibits Foot-and-Mouth Disease Virus Replication

    doi: 10.1155/2017/2481348

    Figure Lengend Snippet: STK3 inhibits FMDV replication during virus infection. (a) PK-15 cells were seeded in 6-well plates, and the monolayer cells were transfected with 1, 2, and 3 μ g of a Myc-STK3 expressing plasmid and 3 μ g of an empty Myc vector. The empty Myc vector was used in the transfection process to ensure that the same amount of cells received the same amount of total plasmids. At 24 hpt, the cells were mock infected or infected with FMDV (MOI 0.5) for 12 h. The expression of viral RNA and the VP1 protein was detected by qPCR assay and western blotting, respectively. (b) PK-15 cells were seeded in 3.5 cm dish, and the monolayer cells were transfected with 150 nM of STK3 siRNA or NC siRNA for 48 h, followed by infection with equal amounts of FMDV (MOI 0.5). The cells were collected at the indicated time points (0, 6, and 12 h). Expression of STK3 mRNA and viral RNA was determined by qPCR assay. Expression of STK3 and the viral VP1 protein was detected by western blotting.

    Article Snippet: STK3 that was recovered from the library matched porcine STK3 (National Center for Biotechnology Information [NCBI] reference sequence GACC01000309.1).

    Techniques: Infection, Transfection, Expressing, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot

    STK3 interacts with VP1. (a) HEK-293T cells were seeded in 10-cm dish, and the monolayer cells were transfected with 8 μ g of a Myc- STK3 expressing plasmid (+), 8 μ g of a FLAG-VP1 expressing plasmid (+), 8 μ g of an empty FLAG vector (−), or 8 μ g of an empty Myc vector (−). At 24 hpt, the cells were lysed, and the lysates were immunoprecipitated with mouse anti-Myc or mouse normal IgG antibodies and subjected to western blotting. The immunoprecipitated antibody-antigen complexes and whole-cell lysates were analyzed by immunoblotting using anti-FLAG, anti-Myc, or anti- β -actin antibodies. (b) Similar transfection and immunoprecipitation experiments were performed as described above. However, the lysates were immunoprecipitated with mouse anti-FLAG or mouse normal IgG antibodies and subjected to western blotting. (c) PK-15 cells were seeded in 10 cm dish, and the monolayer cells were transfected with 10 μ g of a FLAG-VP1 expressing plasmid or 10 μ g of an empty FLAG vector. The cells were lysed at 30 hpt. Cell lysates were immunoprecipitated with goat anti-STK3 and goat IgG antibodies and subjected to western blotting. The whole-cell lysates and immunoprecipitated antibody-antigen complexes were analyzed by immunoblotting using anti-STK3, anti-FLAG, or anti- β -actin antibodies. (d) Similar infection and immunoprecipitation experiments were performed as described above. However, the lysates were immunoprecipitated with mouse anti-FLAG or mouse normal IgG antibodies and subjected to western blotting. (e) HEK293T cells were seeded in Nunc glass bottom dishes, and the monolayer cells were transfected with 1.5 μ g of a Myc-STK3 expressing plasmid, 1.5 μ g of a FLAG-VP1 expressing plasmid, or 1.5 μ g of an empty vector. At 24 hpt, the expression of Myc-STK3 and FLAG-VP1 was detected by an IFA analysis. Cells were double-immunostained for Myc-STK3 (red) and FLAG-VP1 (green); cellular nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue).

    Journal: BioMed Research International

    Article Title: The Kinase STK3 Interacts with the Viral Structural Protein VP1 and Inhibits Foot-and-Mouth Disease Virus Replication

    doi: 10.1155/2017/2481348

    Figure Lengend Snippet: STK3 interacts with VP1. (a) HEK-293T cells were seeded in 10-cm dish, and the monolayer cells were transfected with 8 μ g of a Myc- STK3 expressing plasmid (+), 8 μ g of a FLAG-VP1 expressing plasmid (+), 8 μ g of an empty FLAG vector (−), or 8 μ g of an empty Myc vector (−). At 24 hpt, the cells were lysed, and the lysates were immunoprecipitated with mouse anti-Myc or mouse normal IgG antibodies and subjected to western blotting. The immunoprecipitated antibody-antigen complexes and whole-cell lysates were analyzed by immunoblotting using anti-FLAG, anti-Myc, or anti- β -actin antibodies. (b) Similar transfection and immunoprecipitation experiments were performed as described above. However, the lysates were immunoprecipitated with mouse anti-FLAG or mouse normal IgG antibodies and subjected to western blotting. (c) PK-15 cells were seeded in 10 cm dish, and the monolayer cells were transfected with 10 μ g of a FLAG-VP1 expressing plasmid or 10 μ g of an empty FLAG vector. The cells were lysed at 30 hpt. Cell lysates were immunoprecipitated with goat anti-STK3 and goat IgG antibodies and subjected to western blotting. The whole-cell lysates and immunoprecipitated antibody-antigen complexes were analyzed by immunoblotting using anti-STK3, anti-FLAG, or anti- β -actin antibodies. (d) Similar infection and immunoprecipitation experiments were performed as described above. However, the lysates were immunoprecipitated with mouse anti-FLAG or mouse normal IgG antibodies and subjected to western blotting. (e) HEK293T cells were seeded in Nunc glass bottom dishes, and the monolayer cells were transfected with 1.5 μ g of a Myc-STK3 expressing plasmid, 1.5 μ g of a FLAG-VP1 expressing plasmid, or 1.5 μ g of an empty vector. At 24 hpt, the expression of Myc-STK3 and FLAG-VP1 was detected by an IFA analysis. Cells were double-immunostained for Myc-STK3 (red) and FLAG-VP1 (green); cellular nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue).

    Article Snippet: STK3 that was recovered from the library matched porcine STK3 (National Center for Biotechnology Information [NCBI] reference sequence GACC01000309.1).

    Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Infection, Immunofluorescence

    STK3 interacts with VP1 truncated mutants. (a) Schematics showing a series of FLAG-tagged truncated VP1 mutants. (b) HEK-293T cells were seeded in a 10-cm dish, and the monolayer cells were transfected with 8 μ g of a Myc-STK3 expressing plasmid, 8 μ g of a FLAG-VP1 expressing plasmid, 8 μ g of FLAG-VP1 mutants expressing plasmids, or 8 μ g of an empty FLAG vector. The cells were lysed at 24 hpt. The lysates were immunoprecipitated with mouse anti-FLAG or mouse IgG antibodies and subjected to western blotting. The immunoprecipitated antibody-antigen complexes and whole-cell lysates were analyzed by anti-FLAG, anti-Myc, or anti- β -actin antibodies. (c) Similar transfection and immunoprecipitation experiments were performed as described above.

    Journal: BioMed Research International

    Article Title: The Kinase STK3 Interacts with the Viral Structural Protein VP1 and Inhibits Foot-and-Mouth Disease Virus Replication

    doi: 10.1155/2017/2481348

    Figure Lengend Snippet: STK3 interacts with VP1 truncated mutants. (a) Schematics showing a series of FLAG-tagged truncated VP1 mutants. (b) HEK-293T cells were seeded in a 10-cm dish, and the monolayer cells were transfected with 8 μ g of a Myc-STK3 expressing plasmid, 8 μ g of a FLAG-VP1 expressing plasmid, 8 μ g of FLAG-VP1 mutants expressing plasmids, or 8 μ g of an empty FLAG vector. The cells were lysed at 24 hpt. The lysates were immunoprecipitated with mouse anti-FLAG or mouse IgG antibodies and subjected to western blotting. The immunoprecipitated antibody-antigen complexes and whole-cell lysates were analyzed by anti-FLAG, anti-Myc, or anti- β -actin antibodies. (c) Similar transfection and immunoprecipitation experiments were performed as described above.

    Article Snippet: STK3 that was recovered from the library matched porcine STK3 (National Center for Biotechnology Information [NCBI] reference sequence GACC01000309.1).

    Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot

    PPIP5K2 KD is not a phosphotransferase: Ins P 6 inhibits Ins P 8 dephosphorylation by PPIP5K2 KD ( A , top) This graphic depicts how Ins(1,3,4) P 3 stimulates Ins(1,3,4,5,6) P 5 dephosphorylation by ITPK1 [ 47 ]. The 1-phosphate group (shown in red) that is removed from Ins(1,3,4,5,6) P 5 is not released into the bulk phase but is retained by the nucleotide and donated to Ins(1,3,4) P 3 ; this returns the enzyme to its ADP state, ready for another cycle of Ins(1,3,4,5,6) P 5 dephosphorylation. ( A , bottom) The analogous potential reaction sequence for PPIP5K. ( B ) Either no enzyme (broken line) or 0.63 μg/ml PPIP5K2 KD (solid line; dotted line) was incubated with 10 μM [ 3 H]Ins P 8 and 10 mM ADP for 10 min as indicated in the Materials and methods section. Where indicated (dotted line) incubations also contained 10 μM Ins P 6 . The reactions were quenched and neutralized and analysed by Partisphere SAX HPLC as described in the Materials and methods section. Representative HPLC data are shown.

    Journal: Bioscience Reports

    Article Title: The kinetic properties of a human PPIP5K reveal that its kinase activities are protected against the consequences of a deteriorating cellular bioenergetic environment

    doi: 10.1042/BSR20120115

    Figure Lengend Snippet: PPIP5K2 KD is not a phosphotransferase: Ins P 6 inhibits Ins P 8 dephosphorylation by PPIP5K2 KD ( A , top) This graphic depicts how Ins(1,3,4) P 3 stimulates Ins(1,3,4,5,6) P 5 dephosphorylation by ITPK1 [ 47 ]. The 1-phosphate group (shown in red) that is removed from Ins(1,3,4,5,6) P 5 is not released into the bulk phase but is retained by the nucleotide and donated to Ins(1,3,4) P 3 ; this returns the enzyme to its ADP state, ready for another cycle of Ins(1,3,4,5,6) P 5 dephosphorylation. ( A , bottom) The analogous potential reaction sequence for PPIP5K. ( B ) Either no enzyme (broken line) or 0.63 μg/ml PPIP5K2 KD (solid line; dotted line) was incubated with 10 μM [ 3 H]Ins P 8 and 10 mM ADP for 10 min as indicated in the Materials and methods section. Where indicated (dotted line) incubations also contained 10 μM Ins P 6 . The reactions were quenched and neutralized and analysed by Partisphere SAX HPLC as described in the Materials and methods section. Representative HPLC data are shown.

    Article Snippet: The human PPIP5K2 kinase domain (residues 1–366) [hPPIP5K2KD , NCBI (National Center for Biotechnology Information) accession number NP_056031.2] and its single-site mutants were expressed in Escherichia coli and purified as previously described [ ].

    Techniques: De-Phosphorylation Assay, Sequencing, Incubation, High Performance Liquid Chromatography

    ATPase activity of PPIP5K2 KD ( A ) and ( B ) Ortho phosphate released from the hydrolysis of ATP was measured using a Malachite Green detection method [ 55 ]. The 27–270 μg/ml PPIP5K2 KD was incubated at 37°C for 120 min in 50 μl reactions containing 20 mM Tris/HCl, pH 7.5, 10 mM ATP (unless otherwise indicated), 100 mM KCl, 0.1 mM EDTA and 5 mM MgCl 2 . ( A ) Stimulation of PPIP5K2 KD ATPase activity by Ins P 3 , Ins P 4 , Ins P 5 , Ins P 6 and 5-Ins P 7 (concentrations as indicated, [ATP]=5 mM). ( B ) Effect of ATP concentration (0.2, 1 or 5 mM) on basal (absence of Ins P ) and Ins P -stimulated (25 μM) ATPase activity. Results are presented as the means±S.D. ( n =3). ( C ) and ( D ) provide representative HPLC data indicating that 15 μM Ins P 5 (dotted lines) does not inhibit phosphorylation of either Ins P 6 or 5- Ins P 7 , respectively. In ( C ), 20 μg/ml PPIP5K2 KD was incubated for 15 min with reaction buffer (see the Materials and methods section) containing 15 μM [ 3 H]Ins P 6 , 5 mM ATP and 0. 5 mM ADP. In ( D ), 0.41 μg/ml PPIP5K2 KD was incubated for 5 min with reaction buffer (see the Materials and methods section) containing 1 μM 5-[ 3 H]Ins P 7 , 5 mM ATP and 0.5 mM ADP. The reactions were quenched and neutralized and analysed by Partisphere SAX HPLC as described in the Materials and methods section.

    Journal: Bioscience Reports

    Article Title: The kinetic properties of a human PPIP5K reveal that its kinase activities are protected against the consequences of a deteriorating cellular bioenergetic environment

    doi: 10.1042/BSR20120115

    Figure Lengend Snippet: ATPase activity of PPIP5K2 KD ( A ) and ( B ) Ortho phosphate released from the hydrolysis of ATP was measured using a Malachite Green detection method [ 55 ]. The 27–270 μg/ml PPIP5K2 KD was incubated at 37°C for 120 min in 50 μl reactions containing 20 mM Tris/HCl, pH 7.5, 10 mM ATP (unless otherwise indicated), 100 mM KCl, 0.1 mM EDTA and 5 mM MgCl 2 . ( A ) Stimulation of PPIP5K2 KD ATPase activity by Ins P 3 , Ins P 4 , Ins P 5 , Ins P 6 and 5-Ins P 7 (concentrations as indicated, [ATP]=5 mM). ( B ) Effect of ATP concentration (0.2, 1 or 5 mM) on basal (absence of Ins P ) and Ins P -stimulated (25 μM) ATPase activity. Results are presented as the means±S.D. ( n =3). ( C ) and ( D ) provide representative HPLC data indicating that 15 μM Ins P 5 (dotted lines) does not inhibit phosphorylation of either Ins P 6 or 5- Ins P 7 , respectively. In ( C ), 20 μg/ml PPIP5K2 KD was incubated for 15 min with reaction buffer (see the Materials and methods section) containing 15 μM [ 3 H]Ins P 6 , 5 mM ATP and 0. 5 mM ADP. In ( D ), 0.41 μg/ml PPIP5K2 KD was incubated for 5 min with reaction buffer (see the Materials and methods section) containing 1 μM 5-[ 3 H]Ins P 7 , 5 mM ATP and 0.5 mM ADP. The reactions were quenched and neutralized and analysed by Partisphere SAX HPLC as described in the Materials and methods section.

    Article Snippet: The human PPIP5K2 kinase domain (residues 1–366) [hPPIP5K2KD , NCBI (National Center for Biotechnology Information) accession number NP_056031.2] and its single-site mutants were expressed in Escherichia coli and purified as previously described [ ].

    Techniques: Activity Assay, Incubation, Concentration Assay, High Performance Liquid Chromatography

    Effect of ATP/ADP and AMP upon the equilibrium condition for PPIP5K2 KD ( A ) and ( B ) show a time course of Ins P 6 and 5-Ins P 7 phosphorylation, respectively. In ( A ), 20 μg/ml enzyme was incubated with reaction buffer (see the Materials and methods section) containing 15 μM Ins P 6 , 5 mM ATP and 0.5 mM ADP. In ( B ), 0.33 μg/ml enzyme was incubated with reaction buffer (see the Materials and methods section) containing 1 μM 5-Ins P 7 and 5 mM ATP. Assays were quenched and neutralized and analysed by Partisphere SAX HPLC as described in the Materials and methods section. ( C ) and ( D ) show the equilibrium point for the phosphorylation of 5-Ins P 7 to Ins P 8 in which 0.33 μg/ml enzyme was incubated as described in the Materials and methods section under initial conditions of 1 μM 5-Ins P 7 and either 1 (squares) or 5 (diamonds) mM ATP; either the ATP/ADP ratio ( C ) or the ATP/AMP ratio ( D ) was varied as indicated. Assays were conducted for 60 min. The reactions were then quenched and neutralized and analysed by Partisphere SAX HPLC as described in the Materials and methods section.

    Journal: Bioscience Reports

    Article Title: The kinetic properties of a human PPIP5K reveal that its kinase activities are protected against the consequences of a deteriorating cellular bioenergetic environment

    doi: 10.1042/BSR20120115

    Figure Lengend Snippet: Effect of ATP/ADP and AMP upon the equilibrium condition for PPIP5K2 KD ( A ) and ( B ) show a time course of Ins P 6 and 5-Ins P 7 phosphorylation, respectively. In ( A ), 20 μg/ml enzyme was incubated with reaction buffer (see the Materials and methods section) containing 15 μM Ins P 6 , 5 mM ATP and 0.5 mM ADP. In ( B ), 0.33 μg/ml enzyme was incubated with reaction buffer (see the Materials and methods section) containing 1 μM 5-Ins P 7 and 5 mM ATP. Assays were quenched and neutralized and analysed by Partisphere SAX HPLC as described in the Materials and methods section. ( C ) and ( D ) show the equilibrium point for the phosphorylation of 5-Ins P 7 to Ins P 8 in which 0.33 μg/ml enzyme was incubated as described in the Materials and methods section under initial conditions of 1 μM 5-Ins P 7 and either 1 (squares) or 5 (diamonds) mM ATP; either the ATP/ADP ratio ( C ) or the ATP/AMP ratio ( D ) was varied as indicated. Assays were conducted for 60 min. The reactions were then quenched and neutralized and analysed by Partisphere SAX HPLC as described in the Materials and methods section.

    Article Snippet: The human PPIP5K2 kinase domain (residues 1–366) [hPPIP5K2KD , NCBI (National Center for Biotechnology Information) accession number NP_056031.2] and its single-site mutants were expressed in Escherichia coli and purified as previously described [ ].

    Techniques: Incubation, High Performance Liquid Chromatography

    The effects of R213A and K248A mutations upon ATPase activity of PPIP5K2 KD ( A ) Structure of a portion of the wild-type PPIP5K2 KD catalytic centre [ 20 ] showing water molecules bridging the interaction between the ATP analogue AMPPNP (adenosine 5′-[β,γ-imido]triphosphate) and R213 and K248. ( B ) The effect of single-site mutants of PPIP5K2 KD on basal and Ins P 6 -stimulated (25 μM) ATPase activity. ATPase activity was measured as in Figure 8 .

    Journal: Bioscience Reports

    Article Title: The kinetic properties of a human PPIP5K reveal that its kinase activities are protected against the consequences of a deteriorating cellular bioenergetic environment

    doi: 10.1042/BSR20120115

    Figure Lengend Snippet: The effects of R213A and K248A mutations upon ATPase activity of PPIP5K2 KD ( A ) Structure of a portion of the wild-type PPIP5K2 KD catalytic centre [ 20 ] showing water molecules bridging the interaction between the ATP analogue AMPPNP (adenosine 5′-[β,γ-imido]triphosphate) and R213 and K248. ( B ) The effect of single-site mutants of PPIP5K2 KD on basal and Ins P 6 -stimulated (25 μM) ATPase activity. ATPase activity was measured as in Figure 8 .

    Article Snippet: The human PPIP5K2 kinase domain (residues 1–366) [hPPIP5K2KD , NCBI (National Center for Biotechnology Information) accession number NP_056031.2] and its single-site mutants were expressed in Escherichia coli and purified as previously described [ ].

    Techniques: Activity Assay

    Phosphorylation reactions of PPIP5K2 KD and substrate saturation plots for ATP and ADP ( A ) Either no enzyme (broken line) or 27 μg/ml PPIP5K2 KD (solid line) was incubated with 10 μM [ 3 H]Ins P 6 , and 11 μM ATP for 20 min as indicated in the Materials and methods section. The reactions were quenched and neutralized and analysed by Partisphere SAX HPLC as described in the Materials and methods section. Representative HPLC data are shown. ( B ) The initial velocity of Ins P 6 phosphorylation was determined for forward reactions under pseudo first-order rate conditions [ 38 ] in which the concentration of the designated inositol phosphate was fixed at saturating levels (10 μM; Table 1 ), and the concentration of nucleotide was varied as indicated. Each individual data point was analysed by HPLC as described under the Materials and methods section. The Michaelis–Menten equation was fitted to the data using non-linear regression (GraphPad Prism v5.03). Results are presented as the means±S.E.M. ( C ) Either no enzyme (broken line) or 0.011 μg/ml PPIP5K2 KD (solid line) was incubated with 100 nM 5-[ 3 H]Ins P 7 and 10 mM ATP for 7 min as indicated in the Materials and methods section. The reactions were quenched and neutralized and analysed by Partisphere SAX HPLC as described in the Materials and methods section. Representative HPLC data are shown. ( D ) The initial velocity of 5-Ins P 7 phosphorylation was determined as described in the legend to panel ( B ) for Ins P 6 .

    Journal: Bioscience Reports

    Article Title: The kinetic properties of a human PPIP5K reveal that its kinase activities are protected against the consequences of a deteriorating cellular bioenergetic environment

    doi: 10.1042/BSR20120115

    Figure Lengend Snippet: Phosphorylation reactions of PPIP5K2 KD and substrate saturation plots for ATP and ADP ( A ) Either no enzyme (broken line) or 27 μg/ml PPIP5K2 KD (solid line) was incubated with 10 μM [ 3 H]Ins P 6 , and 11 μM ATP for 20 min as indicated in the Materials and methods section. The reactions were quenched and neutralized and analysed by Partisphere SAX HPLC as described in the Materials and methods section. Representative HPLC data are shown. ( B ) The initial velocity of Ins P 6 phosphorylation was determined for forward reactions under pseudo first-order rate conditions [ 38 ] in which the concentration of the designated inositol phosphate was fixed at saturating levels (10 μM; Table 1 ), and the concentration of nucleotide was varied as indicated. Each individual data point was analysed by HPLC as described under the Materials and methods section. The Michaelis–Menten equation was fitted to the data using non-linear regression (GraphPad Prism v5.03). Results are presented as the means±S.E.M. ( C ) Either no enzyme (broken line) or 0.011 μg/ml PPIP5K2 KD (solid line) was incubated with 100 nM 5-[ 3 H]Ins P 7 and 10 mM ATP for 7 min as indicated in the Materials and methods section. The reactions were quenched and neutralized and analysed by Partisphere SAX HPLC as described in the Materials and methods section. Representative HPLC data are shown. ( D ) The initial velocity of 5-Ins P 7 phosphorylation was determined as described in the legend to panel ( B ) for Ins P 6 .

    Article Snippet: The human PPIP5K2 kinase domain (residues 1–366) [hPPIP5K2KD , NCBI (National Center for Biotechnology Information) accession number NP_056031.2] and its single-site mutants were expressed in Escherichia coli and purified as previously described [ ].

    Techniques: Incubation, High Performance Liquid Chromatography, Concentration Assay

    Dephosphorylation reactions of PPIP5K2 KD and substrate saturation plots for ATP and ADP ( A ) Either no enzyme (broken line) or 0.56 μg/ml PPIP5K2 KD (solid line) was incubated with 10 μM [ 3 H]Ins P 8 and 5 mM ADP for 10 min as indicated in the Materials and methods section. The reactions were quenched and neutralized and analysed by Partisphere SAX HPLC as described in the Materials and methods section. Representative HPLC data are shown. ( B ) The initial velocity of Ins P 8 dephosphorylation was determined for reverse reactions under pseudo first-order rate conditions [ 38 ] in which the concentration of the designated inositol phosphate was fixed at saturating levels (10 μM; Table 1 ), and the concentration of nucleotide was varied as indicated. Each individual data point was analysed by HPLC as described under the Materials and methods section. The Michaelis–Menten equation was fitted to the data using non-linear regression (GraphPad Prism v5.03). Results are presented as the means±S.E.M. ( C ) Either no enzyme (broken line) or 27 μg/ml PPIP5K2 KD (solid line) was incubated with 10 μM 1-[ 3 H]Ins P 7 and 28 μM ADP for 45 min as indicated in the Materials and methods section. The reactions were quenched and neutralized and analysed by Partisphere SAX HPLC as described in the Materials and methods section. Representative HPLC data are shown. ( D ) The initial velocity of 1-Ins P 7 dephosphorylation was determined as described in the legend to panel ( B ) for Ins P 8 .

    Journal: Bioscience Reports

    Article Title: The kinetic properties of a human PPIP5K reveal that its kinase activities are protected against the consequences of a deteriorating cellular bioenergetic environment

    doi: 10.1042/BSR20120115

    Figure Lengend Snippet: Dephosphorylation reactions of PPIP5K2 KD and substrate saturation plots for ATP and ADP ( A ) Either no enzyme (broken line) or 0.56 μg/ml PPIP5K2 KD (solid line) was incubated with 10 μM [ 3 H]Ins P 8 and 5 mM ADP for 10 min as indicated in the Materials and methods section. The reactions were quenched and neutralized and analysed by Partisphere SAX HPLC as described in the Materials and methods section. Representative HPLC data are shown. ( B ) The initial velocity of Ins P 8 dephosphorylation was determined for reverse reactions under pseudo first-order rate conditions [ 38 ] in which the concentration of the designated inositol phosphate was fixed at saturating levels (10 μM; Table 1 ), and the concentration of nucleotide was varied as indicated. Each individual data point was analysed by HPLC as described under the Materials and methods section. The Michaelis–Menten equation was fitted to the data using non-linear regression (GraphPad Prism v5.03). Results are presented as the means±S.E.M. ( C ) Either no enzyme (broken line) or 27 μg/ml PPIP5K2 KD (solid line) was incubated with 10 μM 1-[ 3 H]Ins P 7 and 28 μM ADP for 45 min as indicated in the Materials and methods section. The reactions were quenched and neutralized and analysed by Partisphere SAX HPLC as described in the Materials and methods section. Representative HPLC data are shown. ( D ) The initial velocity of 1-Ins P 7 dephosphorylation was determined as described in the legend to panel ( B ) for Ins P 8 .

    Article Snippet: The human PPIP5K2 kinase domain (residues 1–366) [hPPIP5K2KD , NCBI (National Center for Biotechnology Information) accession number NP_056031.2] and its single-site mutants were expressed in Escherichia coli and purified as previously described [ ].

    Techniques: De-Phosphorylation Assay, Incubation, High Performance Liquid Chromatography, Concentration Assay

    Ins P 6 and InsS 6 inhibit Ins P 8 dephosphorylation by PPIP5K2 KD ; a luciferase-based ATP assay A total of 0.3 μg/ml PPIP5K2 KD was incubated at 37°C with 1.4× reaction buffer (ATP Determination Kit, Molecular Probes), 650 μM ADP and 250 nM Ins P 8 for 15 min as described in the Materials and methods section. InsS 6 and Ins P 6 were included in the assays as indicated. Then 16 μl of either an ATP standard solution or reaction solution was added into 100 μl Standard Reaction Solution (ATP Determination Kit, Molecular Probes) and luminescence was recorded after 15 s using a BioTek Synergy 2. Data are presented as the means±S.E.M. ( n ≥3). A dose–response curve was fit using GraphPad Prism v5.03. The IC 50 values for InsS 6 and Ins P 6 were 0.48 and 0.32 μM, respectively.

    Journal: Bioscience Reports

    Article Title: The kinetic properties of a human PPIP5K reveal that its kinase activities are protected against the consequences of a deteriorating cellular bioenergetic environment

    doi: 10.1042/BSR20120115

    Figure Lengend Snippet: Ins P 6 and InsS 6 inhibit Ins P 8 dephosphorylation by PPIP5K2 KD ; a luciferase-based ATP assay A total of 0.3 μg/ml PPIP5K2 KD was incubated at 37°C with 1.4× reaction buffer (ATP Determination Kit, Molecular Probes), 650 μM ADP and 250 nM Ins P 8 for 15 min as described in the Materials and methods section. InsS 6 and Ins P 6 were included in the assays as indicated. Then 16 μl of either an ATP standard solution or reaction solution was added into 100 μl Standard Reaction Solution (ATP Determination Kit, Molecular Probes) and luminescence was recorded after 15 s using a BioTek Synergy 2. Data are presented as the means±S.E.M. ( n ≥3). A dose–response curve was fit using GraphPad Prism v5.03. The IC 50 values for InsS 6 and Ins P 6 were 0.48 and 0.32 μM, respectively.

    Article Snippet: The human PPIP5K2 kinase domain (residues 1–366) [hPPIP5K2KD , NCBI (National Center for Biotechnology Information) accession number NP_056031.2] and its single-site mutants were expressed in Escherichia coli and purified as previously described [ ].

    Techniques: De-Phosphorylation Assay, Luciferase, ATP Assay, Incubation

    Pathways of enzymatic synthesis of PP -Ins P s Figure modified from Shears 2009 [ 5 ]. The IP6Ks (myoinositol hexakisphosphate kinase; Kcs1 in yeast [ 48 ]), of which there are three isoforms in mammals, IP6K1, IP6K2 and IP6K3 [ 26 , 48 , 52 ], phosphorylate Ins P 6 and 1-Ins P 7 at the 5 position [ 41 , 53 ]. The PPIP5Ks (Vip1 in yeast [ 21 ]), of which there are two isoforms in mammals, PPIP5K1 and PPIP5K2 [ 18 , 19 ], phosphorylate Ins P 6 and 5-Ins P 7 at the 1 position [ 20 , 41 ]. Thus, the concerted actions of the IP6Ks and the PPIP5Ks leads to two routes of synthesis to Ins P 8 , which are designated pathway I and pathway II after Padmanabhan et al. [ 17 ]. The diphosphate groups that are added by these kinases are hydrolysed by a family of PP -Ins P phosphohydrolases (DIPPs) [ 44 , 45 ].

    Journal: Bioscience Reports

    Article Title: The kinetic properties of a human PPIP5K reveal that its kinase activities are protected against the consequences of a deteriorating cellular bioenergetic environment

    doi: 10.1042/BSR20120115

    Figure Lengend Snippet: Pathways of enzymatic synthesis of PP -Ins P s Figure modified from Shears 2009 [ 5 ]. The IP6Ks (myoinositol hexakisphosphate kinase; Kcs1 in yeast [ 48 ]), of which there are three isoforms in mammals, IP6K1, IP6K2 and IP6K3 [ 26 , 48 , 52 ], phosphorylate Ins P 6 and 1-Ins P 7 at the 5 position [ 41 , 53 ]. The PPIP5Ks (Vip1 in yeast [ 21 ]), of which there are two isoforms in mammals, PPIP5K1 and PPIP5K2 [ 18 , 19 ], phosphorylate Ins P 6 and 5-Ins P 7 at the 1 position [ 20 , 41 ]. Thus, the concerted actions of the IP6Ks and the PPIP5Ks leads to two routes of synthesis to Ins P 8 , which are designated pathway I and pathway II after Padmanabhan et al. [ 17 ]. The diphosphate groups that are added by these kinases are hydrolysed by a family of PP -Ins P phosphohydrolases (DIPPs) [ 44 , 45 ].

    Article Snippet: The human PPIP5K2 kinase domain (residues 1–366) [hPPIP5K2KD , NCBI (National Center for Biotechnology Information) accession number NP_056031.2] and its single-site mutants were expressed in Escherichia coli and purified as previously described [ ].

    Techniques: Modification

    The Ly49C·H2-Q10 Interface

    Journal:

    Article Title: Recognition of the Major Histocompatibility Complex (MHC) Class Ib Molecule H2-Q10 by the Natural Killer Cell Receptor Ly49C

    doi: 10.1074/jbc.M116.737130

    Figure Lengend Snippet: The Ly49C·H2-Q10 Interface

    Article Snippet: Alignment of the sequence of the H2-Q10 heavy chain with classical MHC-encoded heavy chains identified regions of sequence homology between H2-Q10 (National Center for Biotechnology Information (NCBI) accession code ), H-2Kb (NCBI accession code 1VAC_A) and H-2Dd (NCBI accession code 1BII_A), both of which are known ligands for Ly49C ( A ).

    Techniques:

    H2-Q10 is a bone-fide ligand for C57BL/6 Ly49C. A , sequence alignment of H2-Q10, H-2K b , H-2D d , and Qa-1 b . Residues conserved in at least three of the sequences are shown as dots . Binding sites for Ly49C on H-2K b are shown within boxes. B , tetramer staining

    Journal:

    Article Title: Recognition of the Major Histocompatibility Complex (MHC) Class Ib Molecule H2-Q10 by the Natural Killer Cell Receptor Ly49C

    doi: 10.1074/jbc.M116.737130

    Figure Lengend Snippet: H2-Q10 is a bone-fide ligand for C57BL/6 Ly49C. A , sequence alignment of H2-Q10, H-2K b , H-2D d , and Qa-1 b . Residues conserved in at least three of the sequences are shown as dots . Binding sites for Ly49C on H-2K b are shown within boxes. B , tetramer staining

    Article Snippet: Alignment of the sequence of the H2-Q10 heavy chain with classical MHC-encoded heavy chains identified regions of sequence homology between H2-Q10 (National Center for Biotechnology Information (NCBI) accession code ), H-2Kb (NCBI accession code 1VAC_A) and H-2Dd (NCBI accession code 1BII_A), both of which are known ligands for Ly49C ( A ).

    Techniques: Sequencing, Binding Assay, Staining

    Variations in Ly49 configuration. A , the configuration of the Ly49 homo-dimer from the following structures is shown in ribbon representation with the α-helices in red and in two different orientations: Ly49C·H2-Q10 ( left ), Ly49C·H-2K

    Journal:

    Article Title: Recognition of the Major Histocompatibility Complex (MHC) Class Ib Molecule H2-Q10 by the Natural Killer Cell Receptor Ly49C

    doi: 10.1074/jbc.M116.737130

    Figure Lengend Snippet: Variations in Ly49 configuration. A , the configuration of the Ly49 homo-dimer from the following structures is shown in ribbon representation with the α-helices in red and in two different orientations: Ly49C·H2-Q10 ( left ), Ly49C·H-2K

    Article Snippet: Alignment of the sequence of the H2-Q10 heavy chain with classical MHC-encoded heavy chains identified regions of sequence homology between H2-Q10 (National Center for Biotechnology Information (NCBI) accession code ), H-2Kb (NCBI accession code 1VAC_A) and H-2Dd (NCBI accession code 1BII_A), both of which are known ligands for Ly49C ( A ).

    Techniques:

    Structure of unliganded H2-Q10. A , overview of the unliganded H2-Q10 (VGITNVDL) complex. The heavy chain is shown in violet , β2M is in cyan , and the VGITNVDL peptide is in green. B , the conformation of the bound peptide (stick representation).

    Journal:

    Article Title: Recognition of the Major Histocompatibility Complex (MHC) Class Ib Molecule H2-Q10 by the Natural Killer Cell Receptor Ly49C

    doi: 10.1074/jbc.M116.737130

    Figure Lengend Snippet: Structure of unliganded H2-Q10. A , overview of the unliganded H2-Q10 (VGITNVDL) complex. The heavy chain is shown in violet , β2M is in cyan , and the VGITNVDL peptide is in green. B , the conformation of the bound peptide (stick representation).

    Article Snippet: Alignment of the sequence of the H2-Q10 heavy chain with classical MHC-encoded heavy chains identified regions of sequence homology between H2-Q10 (National Center for Biotechnology Information (NCBI) accession code ), H-2Kb (NCBI accession code 1VAC_A) and H-2Dd (NCBI accession code 1BII_A), both of which are known ligands for Ly49C ( A ).

    Techniques:

    The molecular surface at the Ly49C·H2-Q10 interface. Electrostatic complementarity at the Ly49C·H2-Q10 interface is shown. Positive surfaces of Ly49C ( left ) and H2-Q10 ( right ) are shown in red (−5 kiloteslas), neutral surfaces

    Journal:

    Article Title: Recognition of the Major Histocompatibility Complex (MHC) Class Ib Molecule H2-Q10 by the Natural Killer Cell Receptor Ly49C

    doi: 10.1074/jbc.M116.737130

    Figure Lengend Snippet: The molecular surface at the Ly49C·H2-Q10 interface. Electrostatic complementarity at the Ly49C·H2-Q10 interface is shown. Positive surfaces of Ly49C ( left ) and H2-Q10 ( right ) are shown in red (−5 kiloteslas), neutral surfaces

    Article Snippet: Alignment of the sequence of the H2-Q10 heavy chain with classical MHC-encoded heavy chains identified regions of sequence homology between H2-Q10 (National Center for Biotechnology Information (NCBI) accession code ), H-2Kb (NCBI accession code 1VAC_A) and H-2Dd (NCBI accession code 1BII_A), both of which are known ligands for Ly49C ( A ).

    Techniques:

    Overview of the Ly49C H2-Q10 complex. A , the 2 F o − F c electron density map is shown contoured at 1σ and colored according to chain (H2-Q10 ( violet ); β2M ( cyan ); Ly49C ( magenta )). B , the Ly49C.H2-Q10 complex visible within the crystallographic

    Journal:

    Article Title: Recognition of the Major Histocompatibility Complex (MHC) Class Ib Molecule H2-Q10 by the Natural Killer Cell Receptor Ly49C

    doi: 10.1074/jbc.M116.737130

    Figure Lengend Snippet: Overview of the Ly49C H2-Q10 complex. A , the 2 F o − F c electron density map is shown contoured at 1σ and colored according to chain (H2-Q10 ( violet ); β2M ( cyan ); Ly49C ( magenta )). B , the Ly49C.H2-Q10 complex visible within the crystallographic

    Article Snippet: Alignment of the sequence of the H2-Q10 heavy chain with classical MHC-encoded heavy chains identified regions of sequence homology between H2-Q10 (National Center for Biotechnology Information (NCBI) accession code ), H-2Kb (NCBI accession code 1VAC_A) and H-2Dd (NCBI accession code 1BII_A), both of which are known ligands for Ly49C ( A ).

    Techniques:

    Comparison of the structure of H2-Q10 and H-2K b on Ly49C. A , comparison of the binding footprint on H2-Q10/H2-K b ( left ) and Ly49C ( right ). Contacting surface residues unique to Ly49C·H2-Q10 and Ly49C·H-2K b are shown in red and yellow ,

    Journal:

    Article Title: Recognition of the Major Histocompatibility Complex (MHC) Class Ib Molecule H2-Q10 by the Natural Killer Cell Receptor Ly49C

    doi: 10.1074/jbc.M116.737130

    Figure Lengend Snippet: Comparison of the structure of H2-Q10 and H-2K b on Ly49C. A , comparison of the binding footprint on H2-Q10/H2-K b ( left ) and Ly49C ( right ). Contacting surface residues unique to Ly49C·H2-Q10 and Ly49C·H-2K b are shown in red and yellow ,

    Article Snippet: Alignment of the sequence of the H2-Q10 heavy chain with classical MHC-encoded heavy chains identified regions of sequence homology between H2-Q10 (National Center for Biotechnology Information (NCBI) accession code ), H-2Kb (NCBI accession code 1VAC_A) and H-2Dd (NCBI accession code 1BII_A), both of which are known ligands for Ly49C ( A ).

    Techniques: Binding Assay

    Structure of Unliganded H2-Q10

    Journal:

    Article Title: Recognition of the Major Histocompatibility Complex (MHC) Class Ib Molecule H2-Q10 by the Natural Killer Cell Receptor Ly49C

    doi: 10.1074/jbc.M116.737130

    Figure Lengend Snippet: Structure of Unliganded H2-Q10

    Article Snippet: Alignment of the sequence of the H2-Q10 heavy chain with classical MHC-encoded heavy chains identified regions of sequence homology between H2-Q10 (National Center for Biotechnology Information (NCBI) accession code ), H-2Kb (NCBI accession code 1VAC_A) and H-2Dd (NCBI accession code 1BII_A), both of which are known ligands for Ly49C ( A ).

    Techniques:

    Molecular interaction at the Ly49C·H2-Q10 interface. The interactions of Ly49C ( magenta ) with the H2-Q10 α1/α2 domain ( violet ) ( A ) and the β2M subunit ( cyan ) ( B ) are shown. For comparison, the equivalent side chain residues

    Journal:

    Article Title: Recognition of the Major Histocompatibility Complex (MHC) Class Ib Molecule H2-Q10 by the Natural Killer Cell Receptor Ly49C

    doi: 10.1074/jbc.M116.737130

    Figure Lengend Snippet: Molecular interaction at the Ly49C·H2-Q10 interface. The interactions of Ly49C ( magenta ) with the H2-Q10 α1/α2 domain ( violet ) ( A ) and the β2M subunit ( cyan ) ( B ) are shown. For comparison, the equivalent side chain residues

    Article Snippet: Alignment of the sequence of the H2-Q10 heavy chain with classical MHC-encoded heavy chains identified regions of sequence homology between H2-Q10 (National Center for Biotechnology Information (NCBI) accession code ), H-2Kb (NCBI accession code 1VAC_A) and H-2Dd (NCBI accession code 1BII_A), both of which are known ligands for Ly49C ( A ).

    Techniques:

    Inhibition of ANXA2P3 in siRNA groups 2 and 3 attenuates the expression of ANXA2, whereas overexpression of ANXA2P3 increases the expression of ANXA2 in vitro . Data are presented as the means ± standard deviation (n=3). P-values were obtained by one-way analysis of variance followed by a post hoc Tukey’s test for multiple comparisons. # P

    Journal: International Journal of Molecular Medicine

    Article Title: Role of the long non-coding RNA-Annexin A2 pseudogene 3/Annexin A2 signaling pathway in biliary atresia-associated hepatic injury

    doi: 10.3892/ijmm.2018.4023

    Figure Lengend Snippet: Inhibition of ANXA2P3 in siRNA groups 2 and 3 attenuates the expression of ANXA2, whereas overexpression of ANXA2P3 increases the expression of ANXA2 in vitro . Data are presented as the means ± standard deviation (n=3). P-values were obtained by one-way analysis of variance followed by a post hoc Tukey’s test for multiple comparisons. # P

    Article Snippet: The siRNA sequences were as follows: si-NC for ANXA2P3: NR_001446.2 [National Center for Biotechnology Information (NCBI) reference sequence]; si-NC for ANXA2: NM_001002858.2 (NCBI reference sequence); scrambled negative control vector (OE-NC) for ANXA2: NM_001002858.2 (NCBI reference sequence); OE-NC for ANXA2P3: NR_001446.2 (NCBI reference sequence); si-ANXA2, 5′-GGA GTG AAG AGG AAA GGA ACT-3′; si-ANXA2P3-1, 5′-GGA TGG CTC TGT CG TTG ATT A-3′; si-ANXA2P3-2, 5′-GGT CAT CAC TCT ACA CCC TCA-3′; si-ANXA2P3-3, 5′-GGA GAG AGG ATG TTG CCT TTG-3′.

    Techniques: Inhibition, Expressing, Over Expression, In Vitro, Standard Deviation

    Results of flow cytometric analysis of cell cycle progression. (A) si-ANXA2P3-transfected liver cells exhibited cell cycle arrest in the G 1 phase compared with si-NC cells. si-ANXA2P3 group: 67.14% in G 1 phase, 19.27% in S phase and 18.66% in G 2 phase; si-NC group: 54.45, 23.3 and 23.83%, respectively. In the OE-ANXA2P3 group, the percentage of cells in G 1 phase, S phase and G 2 phase was 43.48, 22.40 and 34.36%, respectively. Conversely, in the OE-NC group, the percentage of cells in G 1 phase, S phase and G 2 phase was 54.04, 22.37 and 23.43%, respectively. (B) Summary of cell cycle distribution in transfected liver cells. Data are presented as the means ± standard deviation. P-values were obtained by Student’s t-test. For all experiments, n=3. *** P

    Journal: International Journal of Molecular Medicine

    Article Title: Role of the long non-coding RNA-Annexin A2 pseudogene 3/Annexin A2 signaling pathway in biliary atresia-associated hepatic injury

    doi: 10.3892/ijmm.2018.4023

    Figure Lengend Snippet: Results of flow cytometric analysis of cell cycle progression. (A) si-ANXA2P3-transfected liver cells exhibited cell cycle arrest in the G 1 phase compared with si-NC cells. si-ANXA2P3 group: 67.14% in G 1 phase, 19.27% in S phase and 18.66% in G 2 phase; si-NC group: 54.45, 23.3 and 23.83%, respectively. In the OE-ANXA2P3 group, the percentage of cells in G 1 phase, S phase and G 2 phase was 43.48, 22.40 and 34.36%, respectively. Conversely, in the OE-NC group, the percentage of cells in G 1 phase, S phase and G 2 phase was 54.04, 22.37 and 23.43%, respectively. (B) Summary of cell cycle distribution in transfected liver cells. Data are presented as the means ± standard deviation. P-values were obtained by Student’s t-test. For all experiments, n=3. *** P

    Article Snippet: The siRNA sequences were as follows: si-NC for ANXA2P3: NR_001446.2 [National Center for Biotechnology Information (NCBI) reference sequence]; si-NC for ANXA2: NM_001002858.2 (NCBI reference sequence); scrambled negative control vector (OE-NC) for ANXA2: NM_001002858.2 (NCBI reference sequence); OE-NC for ANXA2P3: NR_001446.2 (NCBI reference sequence); si-ANXA2, 5′-GGA GTG AAG AGG AAA GGA ACT-3′; si-ANXA2P3-1, 5′-GGA TGG CTC TGT CG TTG ATT A-3′; si-ANXA2P3-2, 5′-GGT CAT CAC TCT ACA CCC TCA-3′; si-ANXA2P3-3, 5′-GGA GAG AGG ATG TTG CCT TTG-3′.

    Techniques: Flow Cytometry, Transfection, Standard Deviation

    Cell Counting kit-8 assay was used to determine cell viability. Cell viability of (A) si-ANXA2- and (B) si-ANXA2P3-transfected cells was detected. ANXA2, Annexin A2; ANXA2P3, ANXA2 pseudogene 3; NC, negative control; OD, optical density; SI/siRNA, small interfering RNA.

    Journal: International Journal of Molecular Medicine

    Article Title: Role of the long non-coding RNA-Annexin A2 pseudogene 3/Annexin A2 signaling pathway in biliary atresia-associated hepatic injury

    doi: 10.3892/ijmm.2018.4023

    Figure Lengend Snippet: Cell Counting kit-8 assay was used to determine cell viability. Cell viability of (A) si-ANXA2- and (B) si-ANXA2P3-transfected cells was detected. ANXA2, Annexin A2; ANXA2P3, ANXA2 pseudogene 3; NC, negative control; OD, optical density; SI/siRNA, small interfering RNA.

    Article Snippet: The siRNA sequences were as follows: si-NC for ANXA2P3: NR_001446.2 [National Center for Biotechnology Information (NCBI) reference sequence]; si-NC for ANXA2: NM_001002858.2 (NCBI reference sequence); scrambled negative control vector (OE-NC) for ANXA2: NM_001002858.2 (NCBI reference sequence); OE-NC for ANXA2P3: NR_001446.2 (NCBI reference sequence); si-ANXA2, 5′-GGA GTG AAG AGG AAA GGA ACT-3′; si-ANXA2P3-1, 5′-GGA TGG CTC TGT CG TTG ATT A-3′; si-ANXA2P3-2, 5′-GGT CAT CAC TCT ACA CCC TCA-3′; si-ANXA2P3-3, 5′-GGA GAG AGG ATG TTG CCT TTG-3′.

    Techniques: Cell Counting, Transfection, Negative Control, Small Interfering RNA

    ANXA2 and ANXA2P3 expression was detected in liver tissues from patients with BA. (A) Liver tissue from patients with BA (n=20) exhibited significantly higher ANXA2 and ANXA2P3 expression compared with in liver tissue from patients with hepatoblastoma (n=6). * P

    Journal: International Journal of Molecular Medicine

    Article Title: Role of the long non-coding RNA-Annexin A2 pseudogene 3/Annexin A2 signaling pathway in biliary atresia-associated hepatic injury

    doi: 10.3892/ijmm.2018.4023

    Figure Lengend Snippet: ANXA2 and ANXA2P3 expression was detected in liver tissues from patients with BA. (A) Liver tissue from patients with BA (n=20) exhibited significantly higher ANXA2 and ANXA2P3 expression compared with in liver tissue from patients with hepatoblastoma (n=6). * P

    Article Snippet: The siRNA sequences were as follows: si-NC for ANXA2P3: NR_001446.2 [National Center for Biotechnology Information (NCBI) reference sequence]; si-NC for ANXA2: NM_001002858.2 (NCBI reference sequence); scrambled negative control vector (OE-NC) for ANXA2: NM_001002858.2 (NCBI reference sequence); OE-NC for ANXA2P3: NR_001446.2 (NCBI reference sequence); si-ANXA2, 5′-GGA GTG AAG AGG AAA GGA ACT-3′; si-ANXA2P3-1, 5′-GGA TGG CTC TGT CG TTG ATT A-3′; si-ANXA2P3-2, 5′-GGT CAT CAC TCT ACA CCC TCA-3′; si-ANXA2P3-3, 5′-GGA GAG AGG ATG TTG CCT TTG-3′.

    Techniques: Expressing

    Detection of green fluorescent protein in transfected liver cells. (A) ANXA2 and (B) ANXA2P3 siRNA groups. (C) ANXA2 and (D) ANXA2P3 overexpressed groups. ANXA2, Annexin A2; ANXA2P3, ANXA2 pseudogene 3; NC, negative control; siRNA, small interfering RNA.

    Journal: International Journal of Molecular Medicine

    Article Title: Role of the long non-coding RNA-Annexin A2 pseudogene 3/Annexin A2 signaling pathway in biliary atresia-associated hepatic injury

    doi: 10.3892/ijmm.2018.4023

    Figure Lengend Snippet: Detection of green fluorescent protein in transfected liver cells. (A) ANXA2 and (B) ANXA2P3 siRNA groups. (C) ANXA2 and (D) ANXA2P3 overexpressed groups. ANXA2, Annexin A2; ANXA2P3, ANXA2 pseudogene 3; NC, negative control; siRNA, small interfering RNA.

    Article Snippet: The siRNA sequences were as follows: si-NC for ANXA2P3: NR_001446.2 [National Center for Biotechnology Information (NCBI) reference sequence]; si-NC for ANXA2: NM_001002858.2 (NCBI reference sequence); scrambled negative control vector (OE-NC) for ANXA2: NM_001002858.2 (NCBI reference sequence); OE-NC for ANXA2P3: NR_001446.2 (NCBI reference sequence); si-ANXA2, 5′-GGA GTG AAG AGG AAA GGA ACT-3′; si-ANXA2P3-1, 5′-GGA TGG CTC TGT CG TTG ATT A-3′; si-ANXA2P3-2, 5′-GGT CAT CAC TCT ACA CCC TCA-3′; si-ANXA2P3-3, 5′-GGA GAG AGG ATG TTG CCT TTG-3′.

    Techniques: Transfection, Negative Control, Small Interfering RNA

    ANXA2 and ANXA2P3 expression was detected using reverse transcription-quantitative polymerase chain reaction. (A) Expression levels of ANXA2 and (B) ANXA2P3 in siRNA- and pcDNA3.1 plasmid vector-transfected liver cells. Data are presented as the means ± standard deviation. P-values were obtained by Student’s t-test. For all experiments, n=3. ** P

    Journal: International Journal of Molecular Medicine

    Article Title: Role of the long non-coding RNA-Annexin A2 pseudogene 3/Annexin A2 signaling pathway in biliary atresia-associated hepatic injury

    doi: 10.3892/ijmm.2018.4023

    Figure Lengend Snippet: ANXA2 and ANXA2P3 expression was detected using reverse transcription-quantitative polymerase chain reaction. (A) Expression levels of ANXA2 and (B) ANXA2P3 in siRNA- and pcDNA3.1 plasmid vector-transfected liver cells. Data are presented as the means ± standard deviation. P-values were obtained by Student’s t-test. For all experiments, n=3. ** P

    Article Snippet: The siRNA sequences were as follows: si-NC for ANXA2P3: NR_001446.2 [National Center for Biotechnology Information (NCBI) reference sequence]; si-NC for ANXA2: NM_001002858.2 (NCBI reference sequence); scrambled negative control vector (OE-NC) for ANXA2: NM_001002858.2 (NCBI reference sequence); OE-NC for ANXA2P3: NR_001446.2 (NCBI reference sequence); si-ANXA2, 5′-GGA GTG AAG AGG AAA GGA ACT-3′; si-ANXA2P3-1, 5′-GGA TGG CTC TGT CG TTG ATT A-3′; si-ANXA2P3-2, 5′-GGT CAT CAC TCT ACA CCC TCA-3′; si-ANXA2P3-3, 5′-GGA GAG AGG ATG TTG CCT TTG-3′.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Transfection, Standard Deviation

    Results of flow cytometric analysis of apoptosis. (A) Apoptotic rate of si-ANXA2P3-transfected liver cells was increased compared with si-NC-transfected liver cells. In the si-ANXA2P3 group, the values were as follows: Normal cells, 74.1%; early apoptotic cells, 14.6%; late apoptotic cells, 7.8%; and necrotic cells, 3.5%. Conversely, in the si-NC group, the values were as follows: Normal cells, 93.6%; early apoptotic cells, 4.1%; late apoptotic cells, 1.0%; and necrotic cells, 1.3%. In the OE-ANXA2P3 group, the values were as follows: Normal cells, 97.2%; early apoptotic cells, 2.7%; late apoptotic cells, 0.1%; and necrotic cells, 0.1%. Conversely, in the OE-NC group, the values were as follows: Normal cells, 93.0%; early apoptotic cells, 5.5%; late apoptotic cells, 0.6%; and necrotic cells and 0.8%. (B) Summary of cell apoptotic rates. Cells in the upper right quadrant are late apoptotic cells and cells in the lower right quadrant are early apoptotic cells. The total apoptotic rate of cells is the sum of the apoptotic rates of the upper right and lower right quadrants. Data are presented as the means ± standard deviation. P-values were obtained by Student’s t-test. For all experiments, n=3. ** P

    Journal: International Journal of Molecular Medicine

    Article Title: Role of the long non-coding RNA-Annexin A2 pseudogene 3/Annexin A2 signaling pathway in biliary atresia-associated hepatic injury

    doi: 10.3892/ijmm.2018.4023

    Figure Lengend Snippet: Results of flow cytometric analysis of apoptosis. (A) Apoptotic rate of si-ANXA2P3-transfected liver cells was increased compared with si-NC-transfected liver cells. In the si-ANXA2P3 group, the values were as follows: Normal cells, 74.1%; early apoptotic cells, 14.6%; late apoptotic cells, 7.8%; and necrotic cells, 3.5%. Conversely, in the si-NC group, the values were as follows: Normal cells, 93.6%; early apoptotic cells, 4.1%; late apoptotic cells, 1.0%; and necrotic cells, 1.3%. In the OE-ANXA2P3 group, the values were as follows: Normal cells, 97.2%; early apoptotic cells, 2.7%; late apoptotic cells, 0.1%; and necrotic cells, 0.1%. Conversely, in the OE-NC group, the values were as follows: Normal cells, 93.0%; early apoptotic cells, 5.5%; late apoptotic cells, 0.6%; and necrotic cells and 0.8%. (B) Summary of cell apoptotic rates. Cells in the upper right quadrant are late apoptotic cells and cells in the lower right quadrant are early apoptotic cells. The total apoptotic rate of cells is the sum of the apoptotic rates of the upper right and lower right quadrants. Data are presented as the means ± standard deviation. P-values were obtained by Student’s t-test. For all experiments, n=3. ** P

    Article Snippet: The siRNA sequences were as follows: si-NC for ANXA2P3: NR_001446.2 [National Center for Biotechnology Information (NCBI) reference sequence]; si-NC for ANXA2: NM_001002858.2 (NCBI reference sequence); scrambled negative control vector (OE-NC) for ANXA2: NM_001002858.2 (NCBI reference sequence); OE-NC for ANXA2P3: NR_001446.2 (NCBI reference sequence); si-ANXA2, 5′-GGA GTG AAG AGG AAA GGA ACT-3′; si-ANXA2P3-1, 5′-GGA TGG CTC TGT CG TTG ATT A-3′; si-ANXA2P3-2, 5′-GGT CAT CAC TCT ACA CCC TCA-3′; si-ANXA2P3-3, 5′-GGA GAG AGG ATG TTG CCT TTG-3′.

    Techniques: Flow Cytometry, Transfection, Standard Deviation

    Characterization of global transcriptome in Corynebacterium glutamicum ATCC21300 and ATCC13032. ( A ) The total number of mRNA-Seq (sequencing) reads mapped in each C. glutamicum strain library; and ( B ) differentially expressed genes (DEGs) in C. glutamicum ATCC21300. Detailed information is shown in Table S2 . CDS: coding sequencing.

    Journal: International Journal of Molecular Sciences

    Article Title: Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect l-Lysine Production in Corynebacterium glutamicum

    doi: 10.3390/ijms17030353

    Figure Lengend Snippet: Characterization of global transcriptome in Corynebacterium glutamicum ATCC21300 and ATCC13032. ( A ) The total number of mRNA-Seq (sequencing) reads mapped in each C. glutamicum strain library; and ( B ) differentially expressed genes (DEGs) in C. glutamicum ATCC21300. Detailed information is shown in Table S2 . CDS: coding sequencing.

    Article Snippet: A total of 23,799,828 and 29,850,918 reads, generated from C. glutamicum ATCC21300 and ATCC13032 libraries, respectively, were mapped to the C. glutamicum ATCC13032 genome sequence (National Center for Biotechnology Information (NCBI) reference sequence: NC_003450); 66.2% and 63.7% of reads, respectively, were matched to annotated CDS (coding sequencing) regions ( A).

    Techniques: Sequencing

    The expression patterns of genes associated with biotin synthesis and transport in Corynebacterium glutamicum ATCC13032 and ATCC21300. ( A ) The expression pattern of bioB ; ( B ) the expression pattern of bioA ; ( C ) the expression pattern of bioD ; ( D ) the expression pattern of the bioY homolog; ( E ) the expression pattern of the bioM homolog; and ( F ) the expression pattern of the ABC transporter permease. The gene expression level (arbitrary units) was normalized using the 16s RNA level as an internal reference. Gene expression levels were quantified by real-time RT-PCR.

    Journal: International Journal of Molecular Sciences

    Article Title: Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect l-Lysine Production in Corynebacterium glutamicum

    doi: 10.3390/ijms17030353

    Figure Lengend Snippet: The expression patterns of genes associated with biotin synthesis and transport in Corynebacterium glutamicum ATCC13032 and ATCC21300. ( A ) The expression pattern of bioB ; ( B ) the expression pattern of bioA ; ( C ) the expression pattern of bioD ; ( D ) the expression pattern of the bioY homolog; ( E ) the expression pattern of the bioM homolog; and ( F ) the expression pattern of the ABC transporter permease. The gene expression level (arbitrary units) was normalized using the 16s RNA level as an internal reference. Gene expression levels were quantified by real-time RT-PCR.

    Article Snippet: A total of 23,799,828 and 29,850,918 reads, generated from C. glutamicum ATCC21300 and ATCC13032 libraries, respectively, were mapped to the C. glutamicum ATCC13032 genome sequence (National Center for Biotechnology Information (NCBI) reference sequence: NC_003450); 66.2% and 63.7% of reads, respectively, were matched to annotated CDS (coding sequencing) regions ( A).

    Techniques: Expressing, Quantitative RT-PCR

    l -Lysine production of Corynebacterium glutamicum ATCC13032, ATCC21300, and the bioB mutant (of ATCC21300). Values shown are averages based on results obtained from at least three independent experiments, in which the standard deviation was

    Journal: International Journal of Molecular Sciences

    Article Title: Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect l-Lysine Production in Corynebacterium glutamicum

    doi: 10.3390/ijms17030353

    Figure Lengend Snippet: l -Lysine production of Corynebacterium glutamicum ATCC13032, ATCC21300, and the bioB mutant (of ATCC21300). Values shown are averages based on results obtained from at least three independent experiments, in which the standard deviation was

    Article Snippet: A total of 23,799,828 and 29,850,918 reads, generated from C. glutamicum ATCC21300 and ATCC13032 libraries, respectively, were mapped to the C. glutamicum ATCC13032 genome sequence (National Center for Biotechnology Information (NCBI) reference sequence: NC_003450); 66.2% and 63.7% of reads, respectively, were matched to annotated CDS (coding sequencing) regions ( A).

    Techniques: Mutagenesis, Standard Deviation

    Growth rate of Corynebacterium glutamicum ATCC13032, ATCC21300, and the bioB mutant of ATCC21300.

    Journal: International Journal of Molecular Sciences

    Article Title: Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect l-Lysine Production in Corynebacterium glutamicum

    doi: 10.3390/ijms17030353

    Figure Lengend Snippet: Growth rate of Corynebacterium glutamicum ATCC13032, ATCC21300, and the bioB mutant of ATCC21300.

    Article Snippet: A total of 23,799,828 and 29,850,918 reads, generated from C. glutamicum ATCC21300 and ATCC13032 libraries, respectively, were mapped to the C. glutamicum ATCC13032 genome sequence (National Center for Biotechnology Information (NCBI) reference sequence: NC_003450); 66.2% and 63.7% of reads, respectively, were matched to annotated CDS (coding sequencing) regions ( A).

    Techniques: Mutagenesis

    The expression patterns of genes associated with biotin transport in Corynebacterium glutamicum ATCC13032, ATCC21300, and the bioB mutant (of ATCC21300). ( A ) The expression pattern of the bioY homolog; ( B ) the expression pattern of the bioM homolog; ( C ) the expression pattern of the ABC transporter permease. The gene expression level (arbitrary units) was normalized using the 16s RNA level as an internal reference. Gene expression levels were quantified by real-time RT-PCR.

    Journal: International Journal of Molecular Sciences

    Article Title: Transcriptome and Gene Ontology (GO) Enrichment Analysis Reveals Genes Involved in Biotin Metabolism That Affect l-Lysine Production in Corynebacterium glutamicum

    doi: 10.3390/ijms17030353

    Figure Lengend Snippet: The expression patterns of genes associated with biotin transport in Corynebacterium glutamicum ATCC13032, ATCC21300, and the bioB mutant (of ATCC21300). ( A ) The expression pattern of the bioY homolog; ( B ) the expression pattern of the bioM homolog; ( C ) the expression pattern of the ABC transporter permease. The gene expression level (arbitrary units) was normalized using the 16s RNA level as an internal reference. Gene expression levels were quantified by real-time RT-PCR.

    Article Snippet: A total of 23,799,828 and 29,850,918 reads, generated from C. glutamicum ATCC21300 and ATCC13032 libraries, respectively, were mapped to the C. glutamicum ATCC13032 genome sequence (National Center for Biotechnology Information (NCBI) reference sequence: NC_003450); 66.2% and 63.7% of reads, respectively, were matched to annotated CDS (coding sequencing) regions ( A).

    Techniques: Expressing, Mutagenesis, Quantitative RT-PCR

    Quantitative analysis of arabinose, xylose, and XOS (A)XOS breakdown and metabolite production as a function of time with Bifidobacterium longum NCC2705 (A and B) and Eubacterium rectale ATCC 33656 (C and D) in the presence of (A)XOS during monoculture

    Journal: Applied and Environmental Microbiology

    Article Title: Mutual Cross-Feeding Interactions between Bifidobacterium longum subsp. longum NCC2705 and Eubacterium rectale ATCC 33656 Explain the Bifidogenic and Butyrogenic Effects of Arabinoxylan Oligosaccharides

    doi: 10.1128/AEM.02089-15

    Figure Lengend Snippet: Quantitative analysis of arabinose, xylose, and XOS (A)XOS breakdown and metabolite production as a function of time with Bifidobacterium longum NCC2705 (A and B) and Eubacterium rectale ATCC 33656 (C and D) in the presence of (A)XOS during monoculture

    Article Snippet: These assays were based on the whole-genome sequences of B. longum NCC2705 (National Center for Biotechnology Information [NCBI] accession number , accessed in January 2014) and E. rectale ATCC 33656 (NCBI accession number , accessed in January 2014).

    Techniques:

    Qualitative analysis of AXOS breakdown as a function of time by Bifidobacterium longum NCC2705 (A) and Eubacterium rectale ATCC 33656 (B) in the presence of (A)XOS during monoculture fermentations in MCB and mMCB, respectively, and by both strains (C)

    Journal: Applied and Environmental Microbiology

    Article Title: Mutual Cross-Feeding Interactions between Bifidobacterium longum subsp. longum NCC2705 and Eubacterium rectale ATCC 33656 Explain the Bifidogenic and Butyrogenic Effects of Arabinoxylan Oligosaccharides

    doi: 10.1128/AEM.02089-15

    Figure Lengend Snippet: Qualitative analysis of AXOS breakdown as a function of time by Bifidobacterium longum NCC2705 (A) and Eubacterium rectale ATCC 33656 (B) in the presence of (A)XOS during monoculture fermentations in MCB and mMCB, respectively, and by both strains (C)

    Article Snippet: These assays were based on the whole-genome sequences of B. longum NCC2705 (National Center for Biotechnology Information [NCBI] accession number , accessed in January 2014) and E. rectale ATCC 33656 (NCBI accession number , accessed in January 2014).

    Techniques:

    Effect of cocultivation on gene expression. (i) Effect of cocultivation on gene expression in Bifidobacterium longum NCC2705.

    Journal: Applied and Environmental Microbiology

    Article Title: Mutual Cross-Feeding Interactions between Bifidobacterium longum subsp. longum NCC2705 and Eubacterium rectale ATCC 33656 Explain the Bifidogenic and Butyrogenic Effects of Arabinoxylan Oligosaccharides

    doi: 10.1128/AEM.02089-15

    Figure Lengend Snippet: Effect of cocultivation on gene expression. (i) Effect of cocultivation on gene expression in Bifidobacterium longum NCC2705.

    Article Snippet: These assays were based on the whole-genome sequences of B. longum NCC2705 (National Center for Biotechnology Information [NCBI] accession number , accessed in January 2014) and E. rectale ATCC 33656 (NCBI accession number , accessed in January 2014).

    Techniques: Expressing

    Metabolic pathways and genes (reconstructed from the genome annotations based on information obtained from the NCBI, KEGG, and BioCyc databases) studied in Bifidobacterium longum NCC2705 and Eubacterium rectale ATCC 33656 by RT-qPCR. The mutual cross-feeding

    Journal: Applied and Environmental Microbiology

    Article Title: Mutual Cross-Feeding Interactions between Bifidobacterium longum subsp. longum NCC2705 and Eubacterium rectale ATCC 33656 Explain the Bifidogenic and Butyrogenic Effects of Arabinoxylan Oligosaccharides

    doi: 10.1128/AEM.02089-15

    Figure Lengend Snippet: Metabolic pathways and genes (reconstructed from the genome annotations based on information obtained from the NCBI, KEGG, and BioCyc databases) studied in Bifidobacterium longum NCC2705 and Eubacterium rectale ATCC 33656 by RT-qPCR. The mutual cross-feeding

    Article Snippet: These assays were based on the whole-genome sequences of B. longum NCC2705 (National Center for Biotechnology Information [NCBI] accession number , accessed in January 2014) and E. rectale ATCC 33656 (NCBI accession number , accessed in January 2014).

    Techniques: Quantitative RT-PCR

    Monitoring of the growth of Bifidobacterium longum NCC2705 (triangles) and Eubacterium rectale ATCC 33656 (squares) as a function of time in the presence of (A)XOS during monoculture fermentations in MCB and mMCB, respectively, and of both strains (circles)

    Journal: Applied and Environmental Microbiology

    Article Title: Mutual Cross-Feeding Interactions between Bifidobacterium longum subsp. longum NCC2705 and Eubacterium rectale ATCC 33656 Explain the Bifidogenic and Butyrogenic Effects of Arabinoxylan Oligosaccharides

    doi: 10.1128/AEM.02089-15

    Figure Lengend Snippet: Monitoring of the growth of Bifidobacterium longum NCC2705 (triangles) and Eubacterium rectale ATCC 33656 (squares) as a function of time in the presence of (A)XOS during monoculture fermentations in MCB and mMCB, respectively, and of both strains (circles)

    Article Snippet: These assays were based on the whole-genome sequences of B. longum NCC2705 (National Center for Biotechnology Information [NCBI] accession number , accessed in January 2014) and E. rectale ATCC 33656 (NCBI accession number , accessed in January 2014).

    Techniques:

    Expression of Slc26a6 in the duodenal and renal tissues of rats among different groups. (A) Western analysis of duodenal tissue showed no difference in the expression of SLC26A6 among lv-Slc26a6, siRNA-Slc26a6, control, and vector groups. The data are expressed as means ± SD ( n = 4 rats/group). (B) Evaluation of the expression of renal Slc26a6. The lv-Slc26a6 group showed significantly higher expression compared with the control group, while the level reduced in the siRNA-Slc26a6 group. No significant difference was observed in the expression in the vector and control groups. The data are expressed as means ± SD ( n = 4 rats/group); * P

    Journal: PeerJ

    Article Title: High expression of SLC26A6 in the kidney may contribute to renal calcification via an SLC26A6-dependent mechanism

    doi: 10.7717/peerj.5192

    Figure Lengend Snippet: Expression of Slc26a6 in the duodenal and renal tissues of rats among different groups. (A) Western analysis of duodenal tissue showed no difference in the expression of SLC26A6 among lv-Slc26a6, siRNA-Slc26a6, control, and vector groups. The data are expressed as means ± SD ( n = 4 rats/group). (B) Evaluation of the expression of renal Slc26a6. The lv-Slc26a6 group showed significantly higher expression compared with the control group, while the level reduced in the siRNA-Slc26a6 group. No significant difference was observed in the expression in the vector and control groups. The data are expressed as means ± SD ( n = 4 rats/group); * P

    Article Snippet: The Slc26a6 sequence (NCBI Gene ID: 301010; National Center for Biotechnology Information, Rockville, MD, USA) was selected according to the NCBI GeneBank.

    Techniques: Expressing, Western Blot, Plasmid Preparation

    Transfection with Slc26a6 or siRNA-Slc26a6 led to different urinary oxalate and crystal formation in rats. (A) Supplementation of the drinking water with 1.0% EG induced hyperoxaluria; 24-h urine was collected by putting the rats in a metabolic cage. The oxalate level in urine was measured by ion chromatography, and the result showed that Slc26a6-lentivirus-transfected rats had significantly higher urinary oxalate excretion compared with the vector and control groups. siRNA-Slc26a6-transfected rats had less urinary oxalate compared with the control group. The data are expressed as means ± SD ( n = 6 rats/group); * P

    Journal: PeerJ

    Article Title: High expression of SLC26A6 in the kidney may contribute to renal calcification via an SLC26A6-dependent mechanism

    doi: 10.7717/peerj.5192

    Figure Lengend Snippet: Transfection with Slc26a6 or siRNA-Slc26a6 led to different urinary oxalate and crystal formation in rats. (A) Supplementation of the drinking water with 1.0% EG induced hyperoxaluria; 24-h urine was collected by putting the rats in a metabolic cage. The oxalate level in urine was measured by ion chromatography, and the result showed that Slc26a6-lentivirus-transfected rats had significantly higher urinary oxalate excretion compared with the vector and control groups. siRNA-Slc26a6-transfected rats had less urinary oxalate compared with the control group. The data are expressed as means ± SD ( n = 6 rats/group); * P

    Article Snippet: The Slc26a6 sequence (NCBI Gene ID: 301010; National Center for Biotechnology Information, Rockville, MD, USA) was selected according to the NCBI GeneBank.

    Techniques: Transfection, Ion Chromatography, Plasmid Preparation

    24-h urine analysis and the expression of SLC26A6 in the kidneys of stone formers and nonstone formers. (A) 24-h urinary analysis of stone formers and nonstone formers. n = 10. Compared with the nonstone formers, stone formers had a higher level of urinary oxalate, calcium and a lower urinary citrate. No significant difference was observed in urinary phosphorus, magnesium and pH between the two groups. (B, C) Western blot analysis of expression of SLC26A6 from kidney of stone formers and nonstone formers. Densitometry values were normalized to respective β-actin values before statistical analysis. n = 10. (D, E) One representative image for each group. A similar trend was seen in the IHC analysis (upper magnification: ×200, lower magnification: ×800). The asterisk (*) denotes glomerulus, and the arrow points to SLC26A6 protein. The mean percentage of SLC26A6 is shown in the column diagram. n = 10. The data are expressed as means ± standard deviation (SD); * P

    Journal: PeerJ

    Article Title: High expression of SLC26A6 in the kidney may contribute to renal calcification via an SLC26A6-dependent mechanism

    doi: 10.7717/peerj.5192

    Figure Lengend Snippet: 24-h urine analysis and the expression of SLC26A6 in the kidneys of stone formers and nonstone formers. (A) 24-h urinary analysis of stone formers and nonstone formers. n = 10. Compared with the nonstone formers, stone formers had a higher level of urinary oxalate, calcium and a lower urinary citrate. No significant difference was observed in urinary phosphorus, magnesium and pH between the two groups. (B, C) Western blot analysis of expression of SLC26A6 from kidney of stone formers and nonstone formers. Densitometry values were normalized to respective β-actin values before statistical analysis. n = 10. (D, E) One representative image for each group. A similar trend was seen in the IHC analysis (upper magnification: ×200, lower magnification: ×800). The asterisk (*) denotes glomerulus, and the arrow points to SLC26A6 protein. The mean percentage of SLC26A6 is shown in the column diagram. n = 10. The data are expressed as means ± standard deviation (SD); * P

    Article Snippet: The Slc26a6 sequence (NCBI Gene ID: 301010; National Center for Biotechnology Information, Rockville, MD, USA) was selected according to the NCBI GeneBank.

    Techniques: Expressing, Western Blot, Immunohistochemistry, Standard Deviation

    Proposed mechanisms of oxalate transport across the renal epithelium in the proximal tubule. In the renal proximal tubule cells, oxalate transport is associated with Slc26a1 expressed on basolateral membrane and Slc26a6 expressed on apical membrane. Slc26a1 mediates the uptake of oxalate in exchange for reabsorbed sulfate (or Cl − or HCO 3 − ). The high expression of Slc26a6 mediates more secretion by oxalate–Cl − exchange and reabsorption by sulfate–oxalate exchange. According to the results, oxalate–Cl − exchange possessed a dominant position resulting in the enhanced net secretion of oxalate.

    Journal: PeerJ

    Article Title: High expression of SLC26A6 in the kidney may contribute to renal calcification via an SLC26A6-dependent mechanism

    doi: 10.7717/peerj.5192

    Figure Lengend Snippet: Proposed mechanisms of oxalate transport across the renal epithelium in the proximal tubule. In the renal proximal tubule cells, oxalate transport is associated with Slc26a1 expressed on basolateral membrane and Slc26a6 expressed on apical membrane. Slc26a1 mediates the uptake of oxalate in exchange for reabsorbed sulfate (or Cl − or HCO 3 − ). The high expression of Slc26a6 mediates more secretion by oxalate–Cl − exchange and reabsorption by sulfate–oxalate exchange. According to the results, oxalate–Cl − exchange possessed a dominant position resulting in the enhanced net secretion of oxalate.

    Article Snippet: The Slc26a6 sequence (NCBI Gene ID: 301010; National Center for Biotechnology Information, Rockville, MD, USA) was selected according to the NCBI GeneBank.

    Techniques: Expressing

    Lentivirus preparation and transfection detection. (A) The Slc26a6 sequence was inserted into a lentiviral vector as pWSLV-05-Slc26a6. And ds-siRNA anti-Slc26a6 sequence was inserted into a lentiviral vector as pLenti-siRNA (Slc26a6)-RFP. (B) After successful lentivirus insertion into the kidneys of rats, frozen sections of kidneys and duodenums were observed under a fluorescence microscope. Cell nucleus were stained with DAPI. RFP was used as a marker to show successful insertion. Compared with the control group, transfection in the kidneys of the experimental groups (lv-Slc26a6 and siRNA-Slc26a6 groups) was successful as seen under a fluorescence microscope (magnification ×200). No difference of RFP expression was observed in the duodenum tissue between the control group and the experimental groups. The data are expressed as means ± SD ( n = 4 rats/group); * P

    Journal: PeerJ

    Article Title: High expression of SLC26A6 in the kidney may contribute to renal calcification via an SLC26A6-dependent mechanism

    doi: 10.7717/peerj.5192

    Figure Lengend Snippet: Lentivirus preparation and transfection detection. (A) The Slc26a6 sequence was inserted into a lentiviral vector as pWSLV-05-Slc26a6. And ds-siRNA anti-Slc26a6 sequence was inserted into a lentiviral vector as pLenti-siRNA (Slc26a6)-RFP. (B) After successful lentivirus insertion into the kidneys of rats, frozen sections of kidneys and duodenums were observed under a fluorescence microscope. Cell nucleus were stained with DAPI. RFP was used as a marker to show successful insertion. Compared with the control group, transfection in the kidneys of the experimental groups (lv-Slc26a6 and siRNA-Slc26a6 groups) was successful as seen under a fluorescence microscope (magnification ×200). No difference of RFP expression was observed in the duodenum tissue between the control group and the experimental groups. The data are expressed as means ± SD ( n = 4 rats/group); * P

    Article Snippet: The Slc26a6 sequence (NCBI Gene ID: 301010; National Center for Biotechnology Information, Rockville, MD, USA) was selected according to the NCBI GeneBank.

    Techniques: Transfection, Sequencing, Plasmid Preparation, Fluorescence, Microscopy, Staining, Marker, Expressing

    Activation Loop and Active State Stabilization Comparison of the activation segment arrangements of (A) DYRK1A, activation segment in orange, and (B) dual-phosphorylated ERK2, activation segment in green. The ERK2 structure is from PDB ID code 2ERK . Both DYRK1A and ERK2 have a completely ordered activation loop and glycine-rich loop, and active αC conformations. The activation loop in dual-phosphorylated active ERK2 forms an extensive hydrogen-bonding network around pT183. Phosphorylated Y185 is also stabilized through an extensive interaction network that is similar to the pY321 network formed by DYRK1A.

    Journal: Structure(London, England:1993)

    Article Title: Structures of Down Syndrome Kinases, DYRKs, Reveal Mechanisms of Kinase Activation and Substrate Recognition

    doi: 10.1016/j.str.2013.03.012

    Figure Lengend Snippet: Activation Loop and Active State Stabilization Comparison of the activation segment arrangements of (A) DYRK1A, activation segment in orange, and (B) dual-phosphorylated ERK2, activation segment in green. The ERK2 structure is from PDB ID code 2ERK . Both DYRK1A and ERK2 have a completely ordered activation loop and glycine-rich loop, and active αC conformations. The activation loop in dual-phosphorylated active ERK2 forms an extensive hydrogen-bonding network around pT183. Phosphorylated Y185 is also stabilized through an extensive interaction network that is similar to the pY321 network formed by DYRK1A.

    Article Snippet: The DYRK1A structure was determined from a construct expressing residues 127–485 of human DYRK1A (National Center for Biotechnology Information [NCBI] genInfo identifier [gi] number 18765758 ).

    Techniques: Activation Assay

    Phosphomapping and Autophosphorylation (A) Autophosphorylation kinetics of DYRK1A Y321F showing electrospray ionization-MS spectra recorded after 0 hr, 4 hr, and overnight. (B) Phosphorylation capability of DYRK1A Y321F mutant. The top panel shows a western blot of DYRK1A Y321F autophosphorylation probed by anti-phosphotyrosine antibody after the reaction times indicated. The bottom panel shows a quantitative control with equal amounts of sample run on the gel. Related to Figure S6 . (C) The autophosphorylation sites mapped for wild-type DYRK1A are shown on the structure as green sticks. (D) Thermal unfolding of DYRK1A wild-type and Y321F mutant. See also Figure S6 .

    Journal: Structure(London, England:1993)

    Article Title: Structures of Down Syndrome Kinases, DYRKs, Reveal Mechanisms of Kinase Activation and Substrate Recognition

    doi: 10.1016/j.str.2013.03.012

    Figure Lengend Snippet: Phosphomapping and Autophosphorylation (A) Autophosphorylation kinetics of DYRK1A Y321F showing electrospray ionization-MS spectra recorded after 0 hr, 4 hr, and overnight. (B) Phosphorylation capability of DYRK1A Y321F mutant. The top panel shows a western blot of DYRK1A Y321F autophosphorylation probed by anti-phosphotyrosine antibody after the reaction times indicated. The bottom panel shows a quantitative control with equal amounts of sample run on the gel. Related to Figure S6 . (C) The autophosphorylation sites mapped for wild-type DYRK1A are shown on the structure as green sticks. (D) Thermal unfolding of DYRK1A wild-type and Y321F mutant. See also Figure S6 .

    Article Snippet: The DYRK1A structure was determined from a construct expressing residues 127–485 of human DYRK1A (National Center for Biotechnology Information [NCBI] genInfo identifier [gi] number 18765758 ).

    Techniques: Mass Spectrometry, Mutagenesis, Western Blot

    Structures of DYRK1A and DYRK2 (A) Structure of DYRK1A kinase domain and DYRK homology box with the inhibitor DJM2005 bound in the ATP binding site. The DH box and CMGC-specific inset are shown in magenta and the activation segment in orange. (B) Similar view of DYRK2 showing the NAPA region and DH box in magenta. (C) Active site of DYRK1A with inhibitor DJM2005 bound. Part of strand β1 has been removed for clarity. The inhibitor is colored yellow, the protein is colored as in (A), and hydrogen bonds are shown as dashed red lines. See also Figure S3 . (D) Correlation of the binding of various kinase inhibitors (measured by ΔT m ) to DYRK1A and DYRK2 showing that whereas some inhibitors bind both proteins, the active site differences allow for DYRK1A- or DYRK2-specific inhibitors.

    Journal: Structure(London, England:1993)

    Article Title: Structures of Down Syndrome Kinases, DYRKs, Reveal Mechanisms of Kinase Activation and Substrate Recognition

    doi: 10.1016/j.str.2013.03.012

    Figure Lengend Snippet: Structures of DYRK1A and DYRK2 (A) Structure of DYRK1A kinase domain and DYRK homology box with the inhibitor DJM2005 bound in the ATP binding site. The DH box and CMGC-specific inset are shown in magenta and the activation segment in orange. (B) Similar view of DYRK2 showing the NAPA region and DH box in magenta. (C) Active site of DYRK1A with inhibitor DJM2005 bound. Part of strand β1 has been removed for clarity. The inhibitor is colored yellow, the protein is colored as in (A), and hydrogen bonds are shown as dashed red lines. See also Figure S3 . (D) Correlation of the binding of various kinase inhibitors (measured by ΔT m ) to DYRK1A and DYRK2 showing that whereas some inhibitors bind both proteins, the active site differences allow for DYRK1A- or DYRK2-specific inhibitors.

    Article Snippet: The DYRK1A structure was determined from a construct expressing residues 127–485 of human DYRK1A (National Center for Biotechnology Information [NCBI] genInfo identifier [gi] number 18765758 ).

    Techniques: Binding Assay, Activation Assay

    The N-Terminal NAPA and DH Box Regions The DH box region is in dark blue, the NAPA1 region for DYRK2 is in green, and the NAPA2 regions for DYRK1A and DYRK2 are in cyan. Residues in these motifs that are highly conserved across DYRK kinases from different species ( Kinstrie et al., 2010 ) are labeled. (A) DH box region of DYRK1A. (B) DH box region of DYRK2. (C) Overview of the N terminus of DYRK2 showing NAPA and DH box motifs. (D) NAPA1 and NAPA2 regions of DYRK2 showing their folded, assembled state. See also Figure S4 .

    Journal: Structure(London, England:1993)

    Article Title: Structures of Down Syndrome Kinases, DYRKs, Reveal Mechanisms of Kinase Activation and Substrate Recognition

    doi: 10.1016/j.str.2013.03.012

    Figure Lengend Snippet: The N-Terminal NAPA and DH Box Regions The DH box region is in dark blue, the NAPA1 region for DYRK2 is in green, and the NAPA2 regions for DYRK1A and DYRK2 are in cyan. Residues in these motifs that are highly conserved across DYRK kinases from different species ( Kinstrie et al., 2010 ) are labeled. (A) DH box region of DYRK1A. (B) DH box region of DYRK2. (C) Overview of the N terminus of DYRK2 showing NAPA and DH box motifs. (D) NAPA1 and NAPA2 regions of DYRK2 showing their folded, assembled state. See also Figure S4 .

    Article Snippet: The DYRK1A structure was determined from a construct expressing residues 127–485 of human DYRK1A (National Center for Biotechnology Information [NCBI] genInfo identifier [gi] number 18765758 ).

    Techniques: Labeling

    Domain Arrangement of Human DYRK Family Kinases The construct boundaries for the crystallized DYRK1A and DYRK2 proteins are indicated. NLS, nuclear localization signal; PEST, PEST domain. See also Figure S1 .

    Journal: Structure(London, England:1993)

    Article Title: Structures of Down Syndrome Kinases, DYRKs, Reveal Mechanisms of Kinase Activation and Substrate Recognition

    doi: 10.1016/j.str.2013.03.012

    Figure Lengend Snippet: Domain Arrangement of Human DYRK Family Kinases The construct boundaries for the crystallized DYRK1A and DYRK2 proteins are indicated. NLS, nuclear localization signal; PEST, PEST domain. See also Figure S1 .

    Article Snippet: The DYRK1A structure was determined from a construct expressing residues 127–485 of human DYRK1A (National Center for Biotechnology Information [NCBI] genInfo identifier [gi] number 18765758 ).

    Techniques: Construct

    Substrate Binding of DYRK1A (A) Representative panels of DYRK1A substrate specificity defining peptide residues identified using in vivo isolation. Each panel represents a separate clustering of peptide sequences, with the most commonly observed residues at each position at the top of each letter stack. Within each clustering, the fraction of the height occupied by each residue represents its predominance at that position. (B) Ternary complex of DYRK1A substrate peptide and inhibitor DJM2005 bound to DYRK1A. The substrate peptide is shown bound between the two lobes of the kinase in the binding cleft extending from the ATP site toward helix αC. The DH box and kinase domain of DYRK1A are shown as an electrostatic surface representation, with the substrate peptide in white balls and sticks and residues of substrates labeled with reference to the phosphoacceptor residue (threonine T0). (C) Electron density map of the DYRK1A substrate peptide for the 8 out of 11 residues for which the density was visible in the structure, numbered with respect to phosphoacceptor threonine T0. (D) Close-up view of the atomic arrangement around the phosphoacceptor residue threonine T0. (E) Close-up view of the arginine binding pocket of DYRK1A, where the arginine at position −3 of the substrate binds to negatively charged residues of the C lobe (red-colored surface) and forms an extensive bonding network with residues from the activation loop, αD and αF. (F) Stick representation similar to (E). See also Figure S7 .

    Journal: Structure(London, England:1993)

    Article Title: Structures of Down Syndrome Kinases, DYRKs, Reveal Mechanisms of Kinase Activation and Substrate Recognition

    doi: 10.1016/j.str.2013.03.012

    Figure Lengend Snippet: Substrate Binding of DYRK1A (A) Representative panels of DYRK1A substrate specificity defining peptide residues identified using in vivo isolation. Each panel represents a separate clustering of peptide sequences, with the most commonly observed residues at each position at the top of each letter stack. Within each clustering, the fraction of the height occupied by each residue represents its predominance at that position. (B) Ternary complex of DYRK1A substrate peptide and inhibitor DJM2005 bound to DYRK1A. The substrate peptide is shown bound between the two lobes of the kinase in the binding cleft extending from the ATP site toward helix αC. The DH box and kinase domain of DYRK1A are shown as an electrostatic surface representation, with the substrate peptide in white balls and sticks and residues of substrates labeled with reference to the phosphoacceptor residue (threonine T0). (C) Electron density map of the DYRK1A substrate peptide for the 8 out of 11 residues for which the density was visible in the structure, numbered with respect to phosphoacceptor threonine T0. (D) Close-up view of the atomic arrangement around the phosphoacceptor residue threonine T0. (E) Close-up view of the arginine binding pocket of DYRK1A, where the arginine at position −3 of the substrate binds to negatively charged residues of the C lobe (red-colored surface) and forms an extensive bonding network with residues from the activation loop, αD and αF. (F) Stick representation similar to (E). See also Figure S7 .

    Article Snippet: The DYRK1A structure was determined from a construct expressing residues 127–485 of human DYRK1A (National Center for Biotechnology Information [NCBI] genInfo identifier [gi] number 18765758 ).

    Techniques: Binding Assay, In Vivo, Isolation, Labeling, Activation Assay

    Mini-dysferlin C72 formation requires ∼200 μM extracellular calcium, broadly correlating with the extracellular calcium concentration required for calcium-dependent membrane repair of injured muscle cells. (A) Development of a flow cytometry membrane repair assay reveals 100–200 μM as the activating concentration of extracellular Ca 2+ required for calcium-dependent membrane repair pathways in cultured human muscle cells. (B) Treatment of primary human muscle cells with the calpain inhibitor calpeptin shows dose-dependent inhibition of cell survival, with an IC 50 of 11.8 ± 5.8 μM (a representative dose–response curve is shown; the calculated IC 50 is derived from four independent dose–response curves performed on different days, one with singlet samples at each dose, three in duplicate). C) Representative Western blot of a dose–response curve showing increasing formation of cleaved mini-dysferlin C72 with increasing concentrations of extracellular calcium. (D) Pooled densitometric quantification of levels of cleaved mini-dysferlin C72 from five calcium dose–response curves ( EC 50 of ∼ 250 μM Ca 2+ , 95% confidence interval). (E, F) In vitro digestion of dysferlin-exon 40a with 0.2 A.U. of purified calpain-1 (E) and calpain-2 (F). Mini-dysferlin C72 is indicated with black arrows.

    Journal: Molecular Biology of the Cell

    Article Title: Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair

    doi: 10.1091/mbc.E14-04-0947

    Figure Lengend Snippet: Mini-dysferlin C72 formation requires ∼200 μM extracellular calcium, broadly correlating with the extracellular calcium concentration required for calcium-dependent membrane repair of injured muscle cells. (A) Development of a flow cytometry membrane repair assay reveals 100–200 μM as the activating concentration of extracellular Ca 2+ required for calcium-dependent membrane repair pathways in cultured human muscle cells. (B) Treatment of primary human muscle cells with the calpain inhibitor calpeptin shows dose-dependent inhibition of cell survival, with an IC 50 of 11.8 ± 5.8 μM (a representative dose–response curve is shown; the calculated IC 50 is derived from four independent dose–response curves performed on different days, one with singlet samples at each dose, three in duplicate). C) Representative Western blot of a dose–response curve showing increasing formation of cleaved mini-dysferlin C72 with increasing concentrations of extracellular calcium. (D) Pooled densitometric quantification of levels of cleaved mini-dysferlin C72 from five calcium dose–response curves ( EC 50 of ∼ 250 μM Ca 2+ , 95% confidence interval). (E, F) In vitro digestion of dysferlin-exon 40a with 0.2 A.U. of purified calpain-1 (E) and calpain-2 (F). Mini-dysferlin C72 is indicated with black arrows.

    Article Snippet: Our dysferlin construct (EGFP-FL-DYSF pcDNA3.1, National Center for Biotechnology Information [NCBI] reference sequence NP_003485.1) was a generous gift from Kate Bushby (Institute of Human Genetics, International Centre for Life, Newcastle upon Tyne, UK) and subcloned into pIRES2 EGFP (OriGene).

    Techniques: Concentration Assay, Flow Cytometry, Cytometry, Cell Culture, Inhibition, Derivative Assay, Western Blot, In Vitro, Purification

    Dysferlin is cleaved in multiple cells types independent of MG53. (A, B) Injury-activated formation of mini-dysferlin C72 is calcium dependent and blocked by calpeptin and occurs in multiple cell lineages. (A) Cells were cultured to confluence and damaged by scraping in the presence or absence of Ca 2+ or the presence of Ca 2+ plus the calpain inhibitor calpeptin (Calp). Cell pellets were lysed in RIPA, and 10 μg of protein was separated by SDS–PAGE and transferred onto PVDF membrane. One PVDF membrane was probed with Hamlet-1, which detects the dysferlin C-terminus and mini-dysferlin C72 (black arrowhead). The duplicate PVDF membrane was probed with Romeo, detecting the dysferlin N-terminus and corresponding cleaved N-terminal fragment (gray arrowhead). Membranes were reprobed with anti-MG53 or anti-GAPDH to show equal loading. (B) Mouse astrocytes and human umbilical vein endothelial cells do not express MG53, and thus formation of mini-dysferlin C72 occurs independently of MG53.

    Journal: Molecular Biology of the Cell

    Article Title: Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair

    doi: 10.1091/mbc.E14-04-0947

    Figure Lengend Snippet: Dysferlin is cleaved in multiple cells types independent of MG53. (A, B) Injury-activated formation of mini-dysferlin C72 is calcium dependent and blocked by calpeptin and occurs in multiple cell lineages. (A) Cells were cultured to confluence and damaged by scraping in the presence or absence of Ca 2+ or the presence of Ca 2+ plus the calpain inhibitor calpeptin (Calp). Cell pellets were lysed in RIPA, and 10 μg of protein was separated by SDS–PAGE and transferred onto PVDF membrane. One PVDF membrane was probed with Hamlet-1, which detects the dysferlin C-terminus and mini-dysferlin C72 (black arrowhead). The duplicate PVDF membrane was probed with Romeo, detecting the dysferlin N-terminus and corresponding cleaved N-terminal fragment (gray arrowhead). Membranes were reprobed with anti-MG53 or anti-GAPDH to show equal loading. (B) Mouse astrocytes and human umbilical vein endothelial cells do not express MG53, and thus formation of mini-dysferlin C72 occurs independently of MG53.

    Article Snippet: Our dysferlin construct (EGFP-FL-DYSF pcDNA3.1, National Center for Biotechnology Information [NCBI] reference sequence NP_003485.1) was a generous gift from Kate Bushby (Institute of Human Genetics, International Centre for Life, Newcastle upon Tyne, UK) and subcloned into pIRES2 EGFP (OriGene).

    Techniques: Cell Culture, SDS Page

    The calpain cleavage site in dysferlin is predicted to reside in exon 40a. (A) The apparent molecular weight of mini-dysferlin C72 (72 kDa) predicts that cleavage of dysferlin occurs between exons 40 and 41, between C2DE and C2E. (B) Exon 40a bears a consensus site for calpain cleavage (GPS-CCD, ccd.biocuckoo.org; Liu et al. , 2011 ). (C) Alignment of exon 40a between dysferlin paralogues reveals only moderate preservation of amino acid sequence. However, exon 40a sequences in all species possess a putative calpain cleavage site, in each case with maximum likelihood of cleavage LAPTNTA–SPPSSPH.

    Journal: Molecular Biology of the Cell

    Article Title: Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair

    doi: 10.1091/mbc.E14-04-0947

    Figure Lengend Snippet: The calpain cleavage site in dysferlin is predicted to reside in exon 40a. (A) The apparent molecular weight of mini-dysferlin C72 (72 kDa) predicts that cleavage of dysferlin occurs between exons 40 and 41, between C2DE and C2E. (B) Exon 40a bears a consensus site for calpain cleavage (GPS-CCD, ccd.biocuckoo.org; Liu et al. , 2011 ). (C) Alignment of exon 40a between dysferlin paralogues reveals only moderate preservation of amino acid sequence. However, exon 40a sequences in all species possess a putative calpain cleavage site, in each case with maximum likelihood of cleavage LAPTNTA–SPPSSPH.

    Article Snippet: Our dysferlin construct (EGFP-FL-DYSF pcDNA3.1, National Center for Biotechnology Information [NCBI] reference sequence NP_003485.1) was a generous gift from Kate Bushby (Institute of Human Genetics, International Centre for Life, Newcastle upon Tyne, UK) and subcloned into pIRES2 EGFP (OriGene).

    Techniques: Molecular Weight, Preserving, Sequencing

    Exon 40a–containing dysferlin is ubiquitously expressed, and mini-dysferlin C72 can be generated in multiple tissues. (A) Exon 40a is widely expressed in human tissues (∼40–60% transcripts), with lower relative levels in skeletal muscle, heart, and brain (∼10–15% transcripts). Dysferlin alternately spliced exons 5a, 17, and 40a were PCR amplified from a human tissue cDNA panel (Clontech) using primers flanking each of the exons. PCR amplification was performed for 30, 35, and 40 cycles to derive a simple standard curve and control for saturation. Ctrl; plasmid control. (B) Endogenous dysferlin from multiple tissues is cleaved by calpains in vitro, releasing mini-dysferlin C72 . Mouse tissues were sectioned and lysed in RIPA, and endogenous dysferlin was immunoprecipitated with Romeo and protein G–Sepharose. Dysferlin-bound Sepharose beads were incubated with 0.2 active unit (A.U.) of purified recombinant calpain-1 at 30°C for 10 s in the presence of 2 mM CaCl 2 . Dysferlin was detected by Western analysis with the C-terminal antibody Hamlet-1. Mini-dysferlin C72­ is indicated with a black arrow. (C) An anti–exon 40a antibody (α-40a) is specific to exon 40a-containing dysferlin in transfected HEK293 cells. Membranes were probed with anti–exon 40a and then reprobed with Hamlet-1 to reveal total dysferlin expression. GAPDH indicates even loading. (D) Anti–exon 40a antibody recognizes full-length dysferlin-exon 40a and cleaved mini-dysferlin C72 but not the N-terminal counterfragment. Dysferlin was immunopurified from transfected HEK293 cells and subject to in vitro calpain cleavage. R1 (Romeo) reveals the N-terminal counterfragment, H1 reveals mini-dysferlin C72 , and α-40a shows reactivity to full-length dysferlin and mini-dysferlin C72 . (E) Dysferlin exon 40a is expressed at similar levels in human muscle and heart. Total dysferlin was immunoprecipitated with Hamlet-1 from three control human muscles (1–3, ages 5, 18, and 37 yr, respectively, from young adults subject to testing for malignant hypothermia and shown to be normal) and two human hearts (1 and 2, donor hearts from young adults). Dysferlin-exon 40a was identified by Western blot with pAb α-40a. Membranes were reprobed with Romeo to reveal total immunoprecipitated dysferlin.

    Journal: Molecular Biology of the Cell

    Article Title: Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair

    doi: 10.1091/mbc.E14-04-0947

    Figure Lengend Snippet: Exon 40a–containing dysferlin is ubiquitously expressed, and mini-dysferlin C72 can be generated in multiple tissues. (A) Exon 40a is widely expressed in human tissues (∼40–60% transcripts), with lower relative levels in skeletal muscle, heart, and brain (∼10–15% transcripts). Dysferlin alternately spliced exons 5a, 17, and 40a were PCR amplified from a human tissue cDNA panel (Clontech) using primers flanking each of the exons. PCR amplification was performed for 30, 35, and 40 cycles to derive a simple standard curve and control for saturation. Ctrl; plasmid control. (B) Endogenous dysferlin from multiple tissues is cleaved by calpains in vitro, releasing mini-dysferlin C72 . Mouse tissues were sectioned and lysed in RIPA, and endogenous dysferlin was immunoprecipitated with Romeo and protein G–Sepharose. Dysferlin-bound Sepharose beads were incubated with 0.2 active unit (A.U.) of purified recombinant calpain-1 at 30°C for 10 s in the presence of 2 mM CaCl 2 . Dysferlin was detected by Western analysis with the C-terminal antibody Hamlet-1. Mini-dysferlin C72­ is indicated with a black arrow. (C) An anti–exon 40a antibody (α-40a) is specific to exon 40a-containing dysferlin in transfected HEK293 cells. Membranes were probed with anti–exon 40a and then reprobed with Hamlet-1 to reveal total dysferlin expression. GAPDH indicates even loading. (D) Anti–exon 40a antibody recognizes full-length dysferlin-exon 40a and cleaved mini-dysferlin C72 but not the N-terminal counterfragment. Dysferlin was immunopurified from transfected HEK293 cells and subject to in vitro calpain cleavage. R1 (Romeo) reveals the N-terminal counterfragment, H1 reveals mini-dysferlin C72 , and α-40a shows reactivity to full-length dysferlin and mini-dysferlin C72 . (E) Dysferlin exon 40a is expressed at similar levels in human muscle and heart. Total dysferlin was immunoprecipitated with Hamlet-1 from three control human muscles (1–3, ages 5, 18, and 37 yr, respectively, from young adults subject to testing for malignant hypothermia and shown to be normal) and two human hearts (1 and 2, donor hearts from young adults). Dysferlin-exon 40a was identified by Western blot with pAb α-40a. Membranes were reprobed with Romeo to reveal total immunoprecipitated dysferlin.

    Article Snippet: Our dysferlin construct (EGFP-FL-DYSF pcDNA3.1, National Center for Biotechnology Information [NCBI] reference sequence NP_003485.1) was a generous gift from Kate Bushby (Institute of Human Genetics, International Centre for Life, Newcastle upon Tyne, UK) and subcloned into pIRES2 EGFP (OriGene).

    Techniques: Generated, Polymerase Chain Reaction, Amplification, Plasmid Preparation, In Vitro, Immunoprecipitation, Incubation, Purification, Recombinant, Western Blot, Transfection, Expressing

    Dysferlin exon 40a and calpain recruit to sites of membrane injury. Cultured MO3.13 secondary oligodendrocytes (row 1) and primary human myotubes (row 2) were shot with 4-μm silica beads using a Bio-Rad Helios Gene Gun, fixed at 10 s postinjury in cold 3% paraformaldehyde, and then permeabilized and immunolabeled (see Materials and Methods ). Romeo was applied for 2 h before Hamlet-1 to bias the detection of the N-terminal dysferlin epitope. Dysferlin was detectable only at sites of membrane injury with Hamlet-1 (rows 1 and 2). Staining with an antibody raised to dysferlin exon 40a revealed exon 40a–containing dysferlin recruits to sites of injury within 10 s (row 3). Calpain-2 was detectable at sites of membrane injury at 2 s (T2, row 4) and 10 s postdamage (T10, row 5). Large-injury sites often showed a void of negative labeling for calpain-2 (T10, row 6), suggesting that calpain might be extracted or escape from large injuries. Scale bar, 5 μm.

    Journal: Molecular Biology of the Cell

    Article Title: Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair

    doi: 10.1091/mbc.E14-04-0947

    Figure Lengend Snippet: Dysferlin exon 40a and calpain recruit to sites of membrane injury. Cultured MO3.13 secondary oligodendrocytes (row 1) and primary human myotubes (row 2) were shot with 4-μm silica beads using a Bio-Rad Helios Gene Gun, fixed at 10 s postinjury in cold 3% paraformaldehyde, and then permeabilized and immunolabeled (see Materials and Methods ). Romeo was applied for 2 h before Hamlet-1 to bias the detection of the N-terminal dysferlin epitope. Dysferlin was detectable only at sites of membrane injury with Hamlet-1 (rows 1 and 2). Staining with an antibody raised to dysferlin exon 40a revealed exon 40a–containing dysferlin recruits to sites of injury within 10 s (row 3). Calpain-2 was detectable at sites of membrane injury at 2 s (T2, row 4) and 10 s postdamage (T10, row 5). Large-injury sites often showed a void of negative labeling for calpain-2 (T10, row 6), suggesting that calpain might be extracted or escape from large injuries. Scale bar, 5 μm.

    Article Snippet: Our dysferlin construct (EGFP-FL-DYSF pcDNA3.1, National Center for Biotechnology Information [NCBI] reference sequence NP_003485.1) was a generous gift from Kate Bushby (Institute of Human Genetics, International Centre for Life, Newcastle upon Tyne, UK) and subcloned into pIRES2 EGFP (OriGene).

    Techniques: Cell Culture, Immunolabeling, Staining, Labeling

    Cleavage of dysferlin to form mini-dysferlin C72 is conferred by exon 40a. (A) Untransfected HEK293 cells, as well as HEK293 transfected with dysferlin expression constructs with (+40a) or without exon 40a, were subjected to scrape injury 24 h posttransfection in the presence or absence of calcium. Only dysferlin expression constructs bearing exon 40a demonstrate injury-activated, calcium-dependent formation of the C-terminal mini-dysferlin C72 fragment (lane 6, Hamlet-1 and anti-Myc, black arrows). The N-terminal counterfragment can be detected with Romeo-1 (lane 6, gray arrow). Membranes were reprobed for loading controls GAPDH and β-tubulin. (B) Ubiquitous calpains specifically cleave exon 40a–containing dysferlin. MEFs were transfected by electroporation with dysferlin expression constructs with or without exon 40a and harvested 24 h posttransfection via scrape injury in the presence of calcium. Injury-activated formation of mini-dysferlin C72 requires exon 40a and is observed in wild-type MEFs (WT) but not in MEFs from CAPNS1 -knockout mice (−/−) deficient for calpain-1 and -2. Retroviral rescue of CAPNS1 in knockout (−/−R) MEFs restores calpain expression (see CAPN2 immunoblot) to levels exceeding that in WT cells and increases injury-induced dysferlin cleavage. Mini-dysferlin C72 is indicated with asterisks. (C) Dysferlin bearing exon 40a is specifically cleaved by either calpain-1 or -2 in vitro, forming mini-dysferlin C72 . Enhanced GFP–dysferlin FLAG was immunoprecipitated with anti-dysferlin (Romeo) and protein G–Sepharose (see Materials and Methods ). Sepharose beads were incubated with the indicated dilutions of purified recombinant calpain-1 or -2 at 30°C for 3 min in the presence of 2 mM CaCl 2 . Digested samples were analyzed by SDS–PAGE and Western blot. Dysferlin bearing exon 40a is specifically cleaved by both calpain-1 and -2 to form mini-dysferlin C72 (black arrowhead), whereas dysferlin without exon 40a remains uncleaved.

    Journal: Molecular Biology of the Cell

    Article Title: Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair

    doi: 10.1091/mbc.E14-04-0947

    Figure Lengend Snippet: Cleavage of dysferlin to form mini-dysferlin C72 is conferred by exon 40a. (A) Untransfected HEK293 cells, as well as HEK293 transfected with dysferlin expression constructs with (+40a) or without exon 40a, were subjected to scrape injury 24 h posttransfection in the presence or absence of calcium. Only dysferlin expression constructs bearing exon 40a demonstrate injury-activated, calcium-dependent formation of the C-terminal mini-dysferlin C72 fragment (lane 6, Hamlet-1 and anti-Myc, black arrows). The N-terminal counterfragment can be detected with Romeo-1 (lane 6, gray arrow). Membranes were reprobed for loading controls GAPDH and β-tubulin. (B) Ubiquitous calpains specifically cleave exon 40a–containing dysferlin. MEFs were transfected by electroporation with dysferlin expression constructs with or without exon 40a and harvested 24 h posttransfection via scrape injury in the presence of calcium. Injury-activated formation of mini-dysferlin C72 requires exon 40a and is observed in wild-type MEFs (WT) but not in MEFs from CAPNS1 -knockout mice (−/−) deficient for calpain-1 and -2. Retroviral rescue of CAPNS1 in knockout (−/−R) MEFs restores calpain expression (see CAPN2 immunoblot) to levels exceeding that in WT cells and increases injury-induced dysferlin cleavage. Mini-dysferlin C72 is indicated with asterisks. (C) Dysferlin bearing exon 40a is specifically cleaved by either calpain-1 or -2 in vitro, forming mini-dysferlin C72 . Enhanced GFP–dysferlin FLAG was immunoprecipitated with anti-dysferlin (Romeo) and protein G–Sepharose (see Materials and Methods ). Sepharose beads were incubated with the indicated dilutions of purified recombinant calpain-1 or -2 at 30°C for 3 min in the presence of 2 mM CaCl 2 . Digested samples were analyzed by SDS–PAGE and Western blot. Dysferlin bearing exon 40a is specifically cleaved by both calpain-1 and -2 to form mini-dysferlin C72 (black arrowhead), whereas dysferlin without exon 40a remains uncleaved.

    Article Snippet: Our dysferlin construct (EGFP-FL-DYSF pcDNA3.1, National Center for Biotechnology Information [NCBI] reference sequence NP_003485.1) was a generous gift from Kate Bushby (Institute of Human Genetics, International Centre for Life, Newcastle upon Tyne, UK) and subcloned into pIRES2 EGFP (OriGene).

    Techniques: Transfection, Expressing, Construct, Electroporation, Knock-Out, Mouse Assay, In Vitro, Immunoprecipitation, Incubation, Purification, Recombinant, SDS Page, Western Blot

    Calpain cleaves otoferlin and myoferlin in addition to dysferlin. (A) Calpain rapidly cleaves immunoprecipitated ferlin proteins in vitro. Dysferlin MycHis , otoferlin MycFlag , and myoferlin MycFlag were immunoprecipitated with anti-myc and protein G–Sepharose (see Materials and Methods ). Dysferlin-bound Sepharose beads were incubated with purified 0.2 A.U. of recombinant calpain-1 at 30°C for 2 or 10 s in the presence of 2 mM CaCl 2. Proteolysis was rapidly inhibited by reconstitution of the reaction in SDS lysis buffer and heating to 94°C. Digested samples were analyzed by SDS–PAGE and Western blot. Top, C-terminal fragments detected with anti-myc (dysferlin) or anti-Flag (myoferlin and otoferlin). Bottom, N-terminal fragments detected by N-terminal (Romeo-dysferlin) or internal antibodies (7D2, myoferlin; C12, otoferlin). (B) Dysferlin and otoferlin display damage-dependent cleavage, whereas myoferlin cleavage appears to be constitutive. HEK293 cells were transfected with dysferlin MycHis , otoferlin MycFlag , and myoferlin MycFlag and lysed in calcium-free RIPA (lane 1), RIPA containing 900 μM calcium (permissive for calpain cleavage), or damaged by scraping in the presence of calcium. Scraped cell pellets were lysed in calcium-free RIPA, and 10 μg of protein was separated by SDS–PAGE and transferred onto PVDF membrane. Dysferlin was detected with anti-Myc; otoferlin and myoferlin were detected with anti-Flag. (C) Diagram of the predicted calpain cleavage sites within dysferlin, otoferlin, and myoferlin (schematic produced using DOG 2.0; Ren et al. , 2009 ). Molecular weight calculation of the cleaved C-terminal modules was used to elucidate the most likely calpain cleavage site ( ccd.biocuckoo.org ). In each case, the C-terminal fragments released by calpain cleavage represent transmembrane-anchored, dual-C2-domain modules.

    Journal: Molecular Biology of the Cell

    Article Title: Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair

    doi: 10.1091/mbc.E14-04-0947

    Figure Lengend Snippet: Calpain cleaves otoferlin and myoferlin in addition to dysferlin. (A) Calpain rapidly cleaves immunoprecipitated ferlin proteins in vitro. Dysferlin MycHis , otoferlin MycFlag , and myoferlin MycFlag were immunoprecipitated with anti-myc and protein G–Sepharose (see Materials and Methods ). Dysferlin-bound Sepharose beads were incubated with purified 0.2 A.U. of recombinant calpain-1 at 30°C for 2 or 10 s in the presence of 2 mM CaCl 2. Proteolysis was rapidly inhibited by reconstitution of the reaction in SDS lysis buffer and heating to 94°C. Digested samples were analyzed by SDS–PAGE and Western blot. Top, C-terminal fragments detected with anti-myc (dysferlin) or anti-Flag (myoferlin and otoferlin). Bottom, N-terminal fragments detected by N-terminal (Romeo-dysferlin) or internal antibodies (7D2, myoferlin; C12, otoferlin). (B) Dysferlin and otoferlin display damage-dependent cleavage, whereas myoferlin cleavage appears to be constitutive. HEK293 cells were transfected with dysferlin MycHis , otoferlin MycFlag , and myoferlin MycFlag and lysed in calcium-free RIPA (lane 1), RIPA containing 900 μM calcium (permissive for calpain cleavage), or damaged by scraping in the presence of calcium. Scraped cell pellets were lysed in calcium-free RIPA, and 10 μg of protein was separated by SDS–PAGE and transferred onto PVDF membrane. Dysferlin was detected with anti-Myc; otoferlin and myoferlin were detected with anti-Flag. (C) Diagram of the predicted calpain cleavage sites within dysferlin, otoferlin, and myoferlin (schematic produced using DOG 2.0; Ren et al. , 2009 ). Molecular weight calculation of the cleaved C-terminal modules was used to elucidate the most likely calpain cleavage site ( ccd.biocuckoo.org ). In each case, the C-terminal fragments released by calpain cleavage represent transmembrane-anchored, dual-C2-domain modules.

    Article Snippet: Our dysferlin construct (EGFP-FL-DYSF pcDNA3.1, National Center for Biotechnology Information [NCBI] reference sequence NP_003485.1) was a generous gift from Kate Bushby (Institute of Human Genetics, International Centre for Life, Newcastle upon Tyne, UK) and subcloned into pIRES2 EGFP (OriGene).

    Techniques: Immunoprecipitation, In Vitro, Incubation, Purification, Recombinant, Lysis, SDS Page, Western Blot, Transfection, Produced, Molecular Weight

    Kcne3 deletion age-dependently delays ventricular repolarization. A ) Left panel: representative surface ECGs of male 9-mo-old Kcne3 +/+ and Kcne3 −/− mice. Right panel: mean ECG parameters of male Kcne3 +/+ (5 mo old, n =8; 9 mo old, n =10)

    Journal:

    Article Title: Kcne3 deletion initiates extracardiac arrhythmogenesis in mice

    doi: 10.1096/fj.13-241828

    Figure Lengend Snippet: Kcne3 deletion age-dependently delays ventricular repolarization. A ) Left panel: representative surface ECGs of male 9-mo-old Kcne3 +/+ and Kcne3 −/− mice. Right panel: mean ECG parameters of male Kcne3 +/+ (5 mo old, n =8; 9 mo old, n =10)

    Article Snippet: Primer pairs for target genes Kcne3 [U.S. National Center for Biotechnology Information (NCBI; Bethesda, MD, USA) GeneID 57442] and Gapdh (NCBI GeneID 14433) produced amplicons of 143 and 123 bp, respectively.

    Techniques: Mouse Assay

    Kcne3 deletion causes adrenal lymphocyte infiltration. A ) Heat map indicating microarray analysis differentiation between adrenal transcript expression profiles of male Kcne3 +/+ vs. Kcne3 −/− mice ( n =4/genotype). B ) Kcne3 −/−

    Journal:

    Article Title: Kcne3 deletion initiates extracardiac arrhythmogenesis in mice

    doi: 10.1096/fj.13-241828

    Figure Lengend Snippet: Kcne3 deletion causes adrenal lymphocyte infiltration. A ) Heat map indicating microarray analysis differentiation between adrenal transcript expression profiles of male Kcne3 +/+ vs. Kcne3 −/− mice ( n =4/genotype). B ) Kcne3 −/−

    Article Snippet: Primer pairs for target genes Kcne3 [U.S. National Center for Biotechnology Information (NCBI; Bethesda, MD, USA) GeneID 57442] and Gapdh (NCBI GeneID 14433) produced amplicons of 143 and 123 bp, respectively.

    Techniques: Microarray, Expressing, Mouse Assay

    Aldosterone receptor antagonism eliminates QT prolongation arising from Kcne3 deletion. A ) Representative (of n =9–10 mice) baseline ECGs recorded from 9-mo-old Kcne3 +/+ and Kcne3 −/− mice pretreated with spironolactone. B ) Mean

    Journal:

    Article Title: Kcne3 deletion initiates extracardiac arrhythmogenesis in mice

    doi: 10.1096/fj.13-241828

    Figure Lengend Snippet: Aldosterone receptor antagonism eliminates QT prolongation arising from Kcne3 deletion. A ) Representative (of n =9–10 mice) baseline ECGs recorded from 9-mo-old Kcne3 +/+ and Kcne3 −/− mice pretreated with spironolactone. B ) Mean

    Article Snippet: Primer pairs for target genes Kcne3 [U.S. National Center for Biotechnology Information (NCBI; Bethesda, MD, USA) GeneID 57442] and Gapdh (NCBI GeneID 14433) produced amplicons of 143 and 123 bp, respectively.

    Techniques: Mouse Assay

    Kcne3 deletion causes hyperaldosteronism and hypercholesterolemia. A ) Mean Kcne3 transcript expression (by real-time qPCR) in colon, 4 chambers of the heart, and adrenal glands of female and male Kcne3 +/+ mice, with similar analysis of Kcne3 −/−

    Journal:

    Article Title: Kcne3 deletion initiates extracardiac arrhythmogenesis in mice

    doi: 10.1096/fj.13-241828

    Figure Lengend Snippet: Kcne3 deletion causes hyperaldosteronism and hypercholesterolemia. A ) Mean Kcne3 transcript expression (by real-time qPCR) in colon, 4 chambers of the heart, and adrenal glands of female and male Kcne3 +/+ mice, with similar analysis of Kcne3 −/−

    Article Snippet: Primer pairs for target genes Kcne3 [U.S. National Center for Biotechnology Information (NCBI; Bethesda, MD, USA) GeneID 57442] and Gapdh (NCBI GeneID 14433) produced amplicons of 143 and 123 bp, respectively.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Mouse Assay

    Kcne3 deletion has minimal effects on isolated ventricular myocyte K + currents. A ) Left panel: representative current traces recorded from myocytes isolated from the ventricular apex of female Kcne3 +/+ and Kcne3 −/− mice as indicated. Whole-cell

    Journal:

    Article Title: Kcne3 deletion initiates extracardiac arrhythmogenesis in mice

    doi: 10.1096/fj.13-241828

    Figure Lengend Snippet: Kcne3 deletion has minimal effects on isolated ventricular myocyte K + currents. A ) Left panel: representative current traces recorded from myocytes isolated from the ventricular apex of female Kcne3 +/+ and Kcne3 −/− mice as indicated. Whole-cell

    Article Snippet: Primer pairs for target genes Kcne3 [U.S. National Center for Biotechnology Information (NCBI; Bethesda, MD, USA) GeneID 57442] and Gapdh (NCBI GeneID 14433) produced amplicons of 143 and 123 bp, respectively.

    Techniques: Isolation, Mouse Assay

    Kcne3 deletion causes an aldosterone-dependent predisposition to postischemia reperfusion VT. A ) Representative (of n =9–10 mice) ECG traces recorded during reperfusion in untreated or spironolactone (spiro)-pretreated 9-mo-old Kcne3 +/+ and Kcne3

    Journal:

    Article Title: Kcne3 deletion initiates extracardiac arrhythmogenesis in mice

    doi: 10.1096/fj.13-241828

    Figure Lengend Snippet: Kcne3 deletion causes an aldosterone-dependent predisposition to postischemia reperfusion VT. A ) Representative (of n =9–10 mice) ECG traces recorded during reperfusion in untreated or spironolactone (spiro)-pretreated 9-mo-old Kcne3 +/+ and Kcne3

    Article Snippet: Primer pairs for target genes Kcne3 [U.S. National Center for Biotechnology Information (NCBI; Bethesda, MD, USA) GeneID 57442] and Gapdh (NCBI GeneID 14433) produced amplicons of 143 and 123 bp, respectively.

    Techniques: Mouse Assay

    Sequence analysis of Rom1 . The position of the point mutation is indicated by red arrowheads on sequence chromatograms from the Control DBF1 mouse (top) and the M-1156 mouse (bottom). A T→C single base substitution at position 1,195 of Rom1 ( M96760 , National Center for Biotechnology Information [NCBI]) was identified in the M-1156 mouse line.

    Journal: Molecular Vision

    Article Title: A monogenic dominant mutation in Rom1 generated by N-ethyl-N-nitrosourea mutagenesis causes retinal degeneration in mice

    doi:

    Figure Lengend Snippet: Sequence analysis of Rom1 . The position of the point mutation is indicated by red arrowheads on sequence chromatograms from the Control DBF1 mouse (top) and the M-1156 mouse (bottom). A T→C single base substitution at position 1,195 of Rom1 ( M96760 , National Center for Biotechnology Information [NCBI]) was identified in the M-1156 mouse line.

    Article Snippet: Sequence analysis demonstrated that the mutation consisted of a single base T→C substitution at position 1,195 in Rom1 (M96760, National Center for Biotechnology Information [NCBI]) and that the mutant allele was expressed.

    Techniques: Sequencing, Mutagenesis

    Photopic electroretinograms of 7-week-old Rom1 +/+ , Rom1 Rgsc1156/+ , and Rom1 Rgsc1156/Rgsc1156 mice (n=4 each). There were no significant differences in amplitude of the b-wave among the genotype s . Data shown are means±standard error.

    Journal: Molecular Vision

    Article Title: A monogenic dominant mutation in Rom1 generated by N-ethyl-N-nitrosourea mutagenesis causes retinal degeneration in mice

    doi:

    Figure Lengend Snippet: Photopic electroretinograms of 7-week-old Rom1 +/+ , Rom1 Rgsc1156/+ , and Rom1 Rgsc1156/Rgsc1156 mice (n=4 each). There were no significant differences in amplitude of the b-wave among the genotype s . Data shown are means±standard error.

    Article Snippet: Sequence analysis demonstrated that the mutation consisted of a single base T→C substitution at position 1,195 in Rom1 (M96760, National Center for Biotechnology Information [NCBI]) and that the mutant allele was expressed.

    Techniques: Mouse Assay

    Western blot analysis of retinal outer segment membrane protein 1 (Rom1), Prph2, and Rho in retinas from 3-week-old Rom1 +/+ , Rom1 Rgsc1156/+ , and Rom1 Rgsc1156/Rgsc1156 mice (n=6 each). A : Western blots of 10 µg of retinal protein with antibodies to Rom1, Prph2, and β-actin. The stripped membrane was reprobed with antibodies to Prph2 and β-actin. B : Western blots of 0.1 µg of retinal protein with antibodies to Rho and β-actin. The stripped membrane was reprobed with β-actin antibodies. C : Semiquantitative analysis of Rom1, Prph2, and Rho levels. The western blot band intensities were measured with ImageJ software. Rom1, Prph2, and Rho protein levels were normalized to β-actin levels. The intensity (mean±standard error) was normalized to wild-type values, which were set equal to 100%. Protein levels differed significantly among the genotypes (* indicates p

    Journal: Molecular Vision

    Article Title: A monogenic dominant mutation in Rom1 generated by N-ethyl-N-nitrosourea mutagenesis causes retinal degeneration in mice

    doi:

    Figure Lengend Snippet: Western blot analysis of retinal outer segment membrane protein 1 (Rom1), Prph2, and Rho in retinas from 3-week-old Rom1 +/+ , Rom1 Rgsc1156/+ , and Rom1 Rgsc1156/Rgsc1156 mice (n=6 each). A : Western blots of 10 µg of retinal protein with antibodies to Rom1, Prph2, and β-actin. The stripped membrane was reprobed with antibodies to Prph2 and β-actin. B : Western blots of 0.1 µg of retinal protein with antibodies to Rho and β-actin. The stripped membrane was reprobed with β-actin antibodies. C : Semiquantitative analysis of Rom1, Prph2, and Rho levels. The western blot band intensities were measured with ImageJ software. Rom1, Prph2, and Rho protein levels were normalized to β-actin levels. The intensity (mean±standard error) was normalized to wild-type values, which were set equal to 100%. Protein levels differed significantly among the genotypes (* indicates p

    Article Snippet: Sequence analysis demonstrated that the mutation consisted of a single base T→C substitution at position 1,195 in Rom1 (M96760, National Center for Biotechnology Information [NCBI]) and that the mutant allele was expressed.

    Techniques: Western Blot, Mouse Assay, Software

    Expression levels of Rom1 and Prph2 in mouse retinas from 8-week-old Rom1 +/+ , Rom1 Rgsc1156/+ , and Rom1 Rgsc1156/Rgsc1156 mice (n=3 each). A : Rom1 and Prph2 mRNA levels are expressed relative to glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) mRNA level. The relative Rom1 and Prph2 mRNA levels differed significantly among the genotypes (* indicates p

    Journal: Molecular Vision

    Article Title: A monogenic dominant mutation in Rom1 generated by N-ethyl-N-nitrosourea mutagenesis causes retinal degeneration in mice

    doi:

    Figure Lengend Snippet: Expression levels of Rom1 and Prph2 in mouse retinas from 8-week-old Rom1 +/+ , Rom1 Rgsc1156/+ , and Rom1 Rgsc1156/Rgsc1156 mice (n=3 each). A : Rom1 and Prph2 mRNA levels are expressed relative to glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) mRNA level. The relative Rom1 and Prph2 mRNA levels differed significantly among the genotypes (* indicates p

    Article Snippet: Sequence analysis demonstrated that the mutation consisted of a single base T→C substitution at position 1,195 in Rom1 (M96760, National Center for Biotechnology Information [NCBI]) and that the mutant allele was expressed.

    Techniques: Expressing, Mouse Assay

    Photomicrographs of retinas from 7- ( A, B, C ) and 35- ( D, E, F ) week-old Rom1 +/+ , Rom1 Rgsc1156/+ , and Rom1 Rgsc1156/Rgsc1156 mice (n=3 or 4 each). The outer nuclear layer of the heterozygotes ( B, E ) was thinner and the photoreceptor outer segments were shorter than in wild-type mice ( A, D ). The alterations were even greater in the homozygotes ( C, F ). Differences were more marked at 35 weeks of age than at 7 weeks of age. GCL, IPL, INL, OPL, ONL, IS, OS, and RPE correspond to ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, inner segment, outer segment, and retinal pigment epithelium. The scale bar represents 50 µm.

    Journal: Molecular Vision

    Article Title: A monogenic dominant mutation in Rom1 generated by N-ethyl-N-nitrosourea mutagenesis causes retinal degeneration in mice

    doi:

    Figure Lengend Snippet: Photomicrographs of retinas from 7- ( A, B, C ) and 35- ( D, E, F ) week-old Rom1 +/+ , Rom1 Rgsc1156/+ , and Rom1 Rgsc1156/Rgsc1156 mice (n=3 or 4 each). The outer nuclear layer of the heterozygotes ( B, E ) was thinner and the photoreceptor outer segments were shorter than in wild-type mice ( A, D ). The alterations were even greater in the homozygotes ( C, F ). Differences were more marked at 35 weeks of age than at 7 weeks of age. GCL, IPL, INL, OPL, ONL, IS, OS, and RPE correspond to ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, inner segment, outer segment, and retinal pigment epithelium. The scale bar represents 50 µm.

    Article Snippet: Sequence analysis demonstrated that the mutation consisted of a single base T→C substitution at position 1,195 in Rom1 (M96760, National Center for Biotechnology Information [NCBI]) and that the mutant allele was expressed.

    Techniques: Mouse Assay

    Electron micrographs of retinas from 35-week-old Rom1 +/+ , Rom1 Rgsc1156/+ , and Rom1 Rgsc1156/Rgsc1156 mice (n=3 each). The bar represents 10 µm in A, B, and C and 1 µm in D, E, and F . The photoreceptor outer segments were shorter in the heterozygotes ( B ) than in the wild-type mice ( A ). Remnants of the photoreceptor outer segments were observed in 35-week-old homozygotes ( C , arrow). At high magnification the diameters of the discs clearly varied in the heterozygotes ( E ), and the discs of the heterozygotes were more compactly stacked than in the wild-type ( D ). ( D-F ) are high-magnified views of outer segment in ( A-C ), respectively.

    Journal: Molecular Vision

    Article Title: A monogenic dominant mutation in Rom1 generated by N-ethyl-N-nitrosourea mutagenesis causes retinal degeneration in mice

    doi:

    Figure Lengend Snippet: Electron micrographs of retinas from 35-week-old Rom1 +/+ , Rom1 Rgsc1156/+ , and Rom1 Rgsc1156/Rgsc1156 mice (n=3 each). The bar represents 10 µm in A, B, and C and 1 µm in D, E, and F . The photoreceptor outer segments were shorter in the heterozygotes ( B ) than in the wild-type mice ( A ). Remnants of the photoreceptor outer segments were observed in 35-week-old homozygotes ( C , arrow). At high magnification the diameters of the discs clearly varied in the heterozygotes ( E ), and the discs of the heterozygotes were more compactly stacked than in the wild-type ( D ). ( D-F ) are high-magnified views of outer segment in ( A-C ), respectively.

    Article Snippet: Sequence analysis demonstrated that the mutation consisted of a single base T→C substitution at position 1,195 in Rom1 (M96760, National Center for Biotechnology Information [NCBI]) and that the mutant allele was expressed.

    Techniques: Mouse Assay

    Immunohistochemical localization of retinal outer segment membrane protein 1 (Rom1) and peripherin/rds (Prph2) in the retinas of 3-week-old Rom1 +/+ , Rom1 Rgsc1156/+ , and Rom1 Rgsc1156/Rgsc1156 mice. Rom1 immunoreactivity was decreased in the outer segments of the heterozygotes ( B ) in comparison with wild-type mice ( A ), and Rom1 immunoreactivity was markedly reduced in the homozygotes ( C ). Prph2 immunoreactivity in the outer segment was also decreased ( E, F, G ). No signals were detected in the retinas of wild-type mice when peptide absorption tests were performed ( D, H ). The bar represents 50 µm. GCL, IPL, INL, OPL, ONL, IS, OS, and RPE refer to ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, inner segment, outer segment, and retinal pigment epithelium.

    Journal: Molecular Vision

    Article Title: A monogenic dominant mutation in Rom1 generated by N-ethyl-N-nitrosourea mutagenesis causes retinal degeneration in mice

    doi:

    Figure Lengend Snippet: Immunohistochemical localization of retinal outer segment membrane protein 1 (Rom1) and peripherin/rds (Prph2) in the retinas of 3-week-old Rom1 +/+ , Rom1 Rgsc1156/+ , and Rom1 Rgsc1156/Rgsc1156 mice. Rom1 immunoreactivity was decreased in the outer segments of the heterozygotes ( B ) in comparison with wild-type mice ( A ), and Rom1 immunoreactivity was markedly reduced in the homozygotes ( C ). Prph2 immunoreactivity in the outer segment was also decreased ( E, F, G ). No signals were detected in the retinas of wild-type mice when peptide absorption tests were performed ( D, H ). The bar represents 50 µm. GCL, IPL, INL, OPL, ONL, IS, OS, and RPE refer to ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, inner segment, outer segment, and retinal pigment epithelium.

    Article Snippet: Sequence analysis demonstrated that the mutation consisted of a single base T→C substitution at position 1,195 in Rom1 (M96760, National Center for Biotechnology Information [NCBI]) and that the mutant allele was expressed.

    Techniques: Immunohistochemistry, Mouse Assay

    Single-flash electroretinograms of Rom1 +/+ , Rom1 Rgsc1156/+ , and Rom1 Rgsc1156/Rgsc1156 mice. A : The amplitude of the a-wave differed significantly among the genotypes at every age examined (* indicates p

    Journal: Molecular Vision

    Article Title: A monogenic dominant mutation in Rom1 generated by N-ethyl-N-nitrosourea mutagenesis causes retinal degeneration in mice

    doi:

    Figure Lengend Snippet: Single-flash electroretinograms of Rom1 +/+ , Rom1 Rgsc1156/+ , and Rom1 Rgsc1156/Rgsc1156 mice. A : The amplitude of the a-wave differed significantly among the genotypes at every age examined (* indicates p

    Article Snippet: Sequence analysis demonstrated that the mutation consisted of a single base T→C substitution at position 1,195 in Rom1 (M96760, National Center for Biotechnology Information [NCBI]) and that the mutant allele was expressed.

    Techniques: Mouse Assay

    Tests for antibody specificity utilizing blocking peptides. 3T3L1 adipocytes were exposed to either 1 µM isoproterenol or 100 nM L-γ-MSH. Prior to labeling, primary antibodies were preincubated with the indicated amounts (µg) of blocking peptides corresponding to pPeri-site 5, pPeri-site 6, or pHSL-serine 660. Anti-phospho-perilipin 1A and anti-phospho-HSL antibodies were visualized in the red and far-red fluorescence channels, respectively. A, Results are shown for cells treated with isoproterenol for 15 minutes in which anti-pPeri-site 5 and anti-pHSLserine 660 were blocked with pPeri-site 5. Upper panels depict cell images. Lower bar graphs depict Tii Pi Pm data for pPeri-site 5 and pHSL-serine 660. B, Results are shown for cells treated with isoproterenol for 10 minutes in which anti-pPeri-site 5 and anti-pHSL-serine 660 were blocked with pHSL-serine 660. C, Results are shown cells treated with L-γ-MSH for 7 minutes in which anti-pPeri-site 6 and anti-pHSLserine563 were blocked with pPeri-site 6. For A, each bar represents a single well; for B and C, each bar represents the mean ± SD for n = 3 wells.

    Journal: PLoS ONE

    Article Title: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis

    doi: 10.1371/journal.pone.0055511

    Figure Lengend Snippet: Tests for antibody specificity utilizing blocking peptides. 3T3L1 adipocytes were exposed to either 1 µM isoproterenol or 100 nM L-γ-MSH. Prior to labeling, primary antibodies were preincubated with the indicated amounts (µg) of blocking peptides corresponding to pPeri-site 5, pPeri-site 6, or pHSL-serine 660. Anti-phospho-perilipin 1A and anti-phospho-HSL antibodies were visualized in the red and far-red fluorescence channels, respectively. A, Results are shown for cells treated with isoproterenol for 15 minutes in which anti-pPeri-site 5 and anti-pHSLserine 660 were blocked with pPeri-site 5. Upper panels depict cell images. Lower bar graphs depict Tii Pi Pm data for pPeri-site 5 and pHSL-serine 660. B, Results are shown for cells treated with isoproterenol for 10 minutes in which anti-pPeri-site 5 and anti-pHSL-serine 660 were blocked with pHSL-serine 660. C, Results are shown cells treated with L-γ-MSH for 7 minutes in which anti-pPeri-site 6 and anti-pHSLserine563 were blocked with pPeri-site 6. For A, each bar represents a single well; for B and C, each bar represents the mean ± SD for n = 3 wells.

    Article Snippet: Murine perilipin 1A (National Center for Biotechnology Information (NCBI) reference sequence NP_783571.2) has six potential PKA phosphorylation sites located at serines 81, 222, 276, 433, 492, and 517, referred to as PKA-sites 1–6, respectively ; splice variants of perilipin 1 (PLIN1b and PLIN1c), which lack PKA sites 4–6, are also less commonly expressed .

    Techniques: Blocking Assay, Labeling, Fluorescence

    Phosphorylation of Perilipin 1A and HSL in response to FSK and L-γ-MSH. 3T3L1 adipocytes were exposed to control medium, 6 µM forskolin (F), or 200 nM L-γ-MSH (M) for 1, 5, or 20 minutes, then fixed and co-labeled with either anti-pPeri-site 5+ anti-pHSL-serine 563, or anti-pPeri-site 6+ anti-pHSL-serine 660. Images were obtained with a 20X objective (4 images/well, representing an average of 930 cells/well). A and B represent Area Pm values obtained for pPeri-site 5 and pHSL-serine 563. C and D represent Area Pm values obtained for pPeri-site 6 and pHSL-serine 660, respectively. Each bar represents the mean ± SD for n = 7 to 8 wells. ** p

    Journal: PLoS ONE

    Article Title: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis

    doi: 10.1371/journal.pone.0055511

    Figure Lengend Snippet: Phosphorylation of Perilipin 1A and HSL in response to FSK and L-γ-MSH. 3T3L1 adipocytes were exposed to control medium, 6 µM forskolin (F), or 200 nM L-γ-MSH (M) for 1, 5, or 20 minutes, then fixed and co-labeled with either anti-pPeri-site 5+ anti-pHSL-serine 563, or anti-pPeri-site 6+ anti-pHSL-serine 660. Images were obtained with a 20X objective (4 images/well, representing an average of 930 cells/well). A and B represent Area Pm values obtained for pPeri-site 5 and pHSL-serine 563. C and D represent Area Pm values obtained for pPeri-site 6 and pHSL-serine 660, respectively. Each bar represents the mean ± SD for n = 7 to 8 wells. ** p

    Article Snippet: Murine perilipin 1A (National Center for Biotechnology Information (NCBI) reference sequence NP_783571.2) has six potential PKA phosphorylation sites located at serines 81, 222, 276, 433, 492, and 517, referred to as PKA-sites 1–6, respectively ; splice variants of perilipin 1 (PLIN1b and PLIN1c), which lack PKA sites 4–6, are also less commonly expressed .

    Techniques: Labeling

    Determination of antibody specificity for perilipin 1A PKA-site 5, and PKA-site 6. Hela cells were transfected separately with plasmids encoding wild-type (GFP, green) or mutant (mCherry, red) perilipin 1A ( PLIN1 ) plasmids. Oleic acid was added for 24 h, followed by 7 min treatment with 24 µM forskolin plus 125 µM IBMX and the cells labeled with either anti-pPeri-site 5 or anti-pPeri-site 6. For each condition, images are shown for nuclei (DAPI), fluorescent protein (either GFP or m-Cherry), or for antibody labeling (the far red fluorescence channel). A, Cells expressing GFP-w/t-perilipin 1A labeled with anti-pPeri-site 5. B, Cells expressing mCh-perilipin 1A S497A, which did not label for anti-pPeri-site 5. C, Cells expressing GFP-w/t-perilipin 1A labeled with anti-pPeri-site 6. D, Cells expressing mCh-perilipin 1A S522A, which did not label with anti-pPeri-site 6.

    Journal: PLoS ONE

    Article Title: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis

    doi: 10.1371/journal.pone.0055511

    Figure Lengend Snippet: Determination of antibody specificity for perilipin 1A PKA-site 5, and PKA-site 6. Hela cells were transfected separately with plasmids encoding wild-type (GFP, green) or mutant (mCherry, red) perilipin 1A ( PLIN1 ) plasmids. Oleic acid was added for 24 h, followed by 7 min treatment with 24 µM forskolin plus 125 µM IBMX and the cells labeled with either anti-pPeri-site 5 or anti-pPeri-site 6. For each condition, images are shown for nuclei (DAPI), fluorescent protein (either GFP or m-Cherry), or for antibody labeling (the far red fluorescence channel). A, Cells expressing GFP-w/t-perilipin 1A labeled with anti-pPeri-site 5. B, Cells expressing mCh-perilipin 1A S497A, which did not label for anti-pPeri-site 5. C, Cells expressing GFP-w/t-perilipin 1A labeled with anti-pPeri-site 6. D, Cells expressing mCh-perilipin 1A S522A, which did not label with anti-pPeri-site 6.

    Article Snippet: Murine perilipin 1A (National Center for Biotechnology Information (NCBI) reference sequence NP_783571.2) has six potential PKA phosphorylation sites located at serines 81, 222, 276, 433, 492, and 517, referred to as PKA-sites 1–6, respectively ; splice variants of perilipin 1 (PLIN1b and PLIN1c), which lack PKA sites 4–6, are also less commonly expressed .

    Techniques: Transfection, Mutagenesis, Labeling, Antibody Labeling, Fluorescence, Expressing

    Downregulation of anti-pPeri-site 5, anti-pPeri-site 6, and GP29 labeling by siRNA to perilipin 1A. Preadipocytes were transfected with either control or perilipin 1A siRNA (0 to 50 nM), exposed to differentiation medium for 6 days, treated with 6 µM FSK for 20 minutes, then fixed and labeled for nuclei (blue), lipid (green), and either anti-pPeri-site 5 or anti-pPeri-site 6 (red) plus GP29 (yellow). A, Representative fields of view are shown for cells transfected with 10 nM siRNA and labeled with anti-pPeri-site 5 plus GP29. B, Representative fields of view are shown for cells transfected with 10 nM siRNA and labeled with anti-pPeri-site 6 plus GP29. C, D, and E are mean values for Area of the Protein mask (Area Pm), for GP29, anti-pPeri-site 5, and anti-pPeri-site 6, respectively. Data are normalized to the 0 siRNA control, and each bar represents the mean ± SD, for n = 6 wells. *** p

    Journal: PLoS ONE

    Article Title: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis

    doi: 10.1371/journal.pone.0055511

    Figure Lengend Snippet: Downregulation of anti-pPeri-site 5, anti-pPeri-site 6, and GP29 labeling by siRNA to perilipin 1A. Preadipocytes were transfected with either control or perilipin 1A siRNA (0 to 50 nM), exposed to differentiation medium for 6 days, treated with 6 µM FSK for 20 minutes, then fixed and labeled for nuclei (blue), lipid (green), and either anti-pPeri-site 5 or anti-pPeri-site 6 (red) plus GP29 (yellow). A, Representative fields of view are shown for cells transfected with 10 nM siRNA and labeled with anti-pPeri-site 5 plus GP29. B, Representative fields of view are shown for cells transfected with 10 nM siRNA and labeled with anti-pPeri-site 6 plus GP29. C, D, and E are mean values for Area of the Protein mask (Area Pm), for GP29, anti-pPeri-site 5, and anti-pPeri-site 6, respectively. Data are normalized to the 0 siRNA control, and each bar represents the mean ± SD, for n = 6 wells. *** p

    Article Snippet: Murine perilipin 1A (National Center for Biotechnology Information (NCBI) reference sequence NP_783571.2) has six potential PKA phosphorylation sites located at serines 81, 222, 276, 433, 492, and 517, referred to as PKA-sites 1–6, respectively ; splice variants of perilipin 1 (PLIN1b and PLIN1c), which lack PKA sites 4–6, are also less commonly expressed .

    Techniques: Labeling, Transfection

    Regulation of lipolysis in adipocytes. A, A current model for the hormonal regulation of lipolysis initiation is shown. Proteins depicted include perilipin 1A (Peri), Hormone Sensitive Lipase (HSL), Adipocyte Triglyceride Lipase (ATGL), CGI-58, and PKA. Lipid species depicted include triacylglycerol (TAG), diacylglycerol (DAG), monoacylglycerol (MAG), and fatty acid (FA). Under basal conditions, perilipin and HSL are unphosphorylated and HSL is found throughout the cytoplasm. Stimulation of lipolysis involves activation of PKA, phosphorylation of perilipin 1A and HSL, release of CGI-58 from perilipin, binding of CGI-58 to ATGL, and translocation of HSL to perilipin. TAG is sequentially processed to DAG by ATGL and to MAG by HSL with FA released at each step. B, Amino acid sequences are shown for perilipin 1A PKA site 5, and PKA site 6, and for HSL serine 563 and serine 660. The target serine in each sequence is underlined.

    Journal: PLoS ONE

    Article Title: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis

    doi: 10.1371/journal.pone.0055511

    Figure Lengend Snippet: Regulation of lipolysis in adipocytes. A, A current model for the hormonal regulation of lipolysis initiation is shown. Proteins depicted include perilipin 1A (Peri), Hormone Sensitive Lipase (HSL), Adipocyte Triglyceride Lipase (ATGL), CGI-58, and PKA. Lipid species depicted include triacylglycerol (TAG), diacylglycerol (DAG), monoacylglycerol (MAG), and fatty acid (FA). Under basal conditions, perilipin and HSL are unphosphorylated and HSL is found throughout the cytoplasm. Stimulation of lipolysis involves activation of PKA, phosphorylation of perilipin 1A and HSL, release of CGI-58 from perilipin, binding of CGI-58 to ATGL, and translocation of HSL to perilipin. TAG is sequentially processed to DAG by ATGL and to MAG by HSL with FA released at each step. B, Amino acid sequences are shown for perilipin 1A PKA site 5, and PKA site 6, and for HSL serine 563 and serine 660. The target serine in each sequence is underlined.

    Article Snippet: Murine perilipin 1A (National Center for Biotechnology Information (NCBI) reference sequence NP_783571.2) has six potential PKA phosphorylation sites located at serines 81, 222, 276, 433, 492, and 517, referred to as PKA-sites 1–6, respectively ; splice variants of perilipin 1 (PLIN1b and PLIN1c), which lack PKA sites 4–6, are also less commonly expressed .

    Techniques: Activation Assay, Binding Assay, Translocation Assay, Sequencing

    Differential phosphorylation of perilipin 1A by forskolin (FSK) and LγMSH. 3T3L1 adipocytes were exposed to either FSK (6 µM) or L-γ-MSH (100 nM) for 5 minutes, then fixed and labeled for nuclei (blue), lipid droplets (green), and phosphorylated perilipin (red). The cells were then imaged (20Xobjective, 4 images/well) and the images analyzed utilizing the Colocalization algorithm. A, B, and C, Images are shown of adipocytes exposed to control, FSK, or LγMSH, respectively, labeled with anti-pPeri-site 5. D, E, and F, Images are shown of adipocytes exposed to control, FSK, LγMSH, respectively, labeled with anti-pPeri-site 6. G and H represent Area Pm for pPeri-site 5 and for pPeri-site 6, respectively. Each bar represents the mean SD for n = 3 wells/condition (an average of 532 cells/well) for G, or n = 8 well/condition (an average of 272 cells/well) for H. ** p

    Journal: PLoS ONE

    Article Title: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis

    doi: 10.1371/journal.pone.0055511

    Figure Lengend Snippet: Differential phosphorylation of perilipin 1A by forskolin (FSK) and LγMSH. 3T3L1 adipocytes were exposed to either FSK (6 µM) or L-γ-MSH (100 nM) for 5 minutes, then fixed and labeled for nuclei (blue), lipid droplets (green), and phosphorylated perilipin (red). The cells were then imaged (20Xobjective, 4 images/well) and the images analyzed utilizing the Colocalization algorithm. A, B, and C, Images are shown of adipocytes exposed to control, FSK, or LγMSH, respectively, labeled with anti-pPeri-site 5. D, E, and F, Images are shown of adipocytes exposed to control, FSK, LγMSH, respectively, labeled with anti-pPeri-site 6. G and H represent Area Pm for pPeri-site 5 and for pPeri-site 6, respectively. Each bar represents the mean SD for n = 3 wells/condition (an average of 532 cells/well) for G, or n = 8 well/condition (an average of 272 cells/well) for H. ** p

    Article Snippet: Murine perilipin 1A (National Center for Biotechnology Information (NCBI) reference sequence NP_783571.2) has six potential PKA phosphorylation sites located at serines 81, 222, 276, 433, 492, and 517, referred to as PKA-sites 1–6, respectively ; splice variants of perilipin 1 (PLIN1b and PLIN1c), which lack PKA sites 4–6, are also less commonly expressed .

    Techniques: Labeling

    Phospho-perilipin 1A visualized via Western blotting. 3T3L1 adipocytes were exposed to lipolytic agonist for 5 minutes; whole cell lysates were then prepared and subjected to SDS-PAGE/Western blotting (20 µg protein/lane) utilizing the anti-pPeri-site 6 antibody. A, Results are shown for cells exposed to either control medium (C) or 6 µM FSK (F). B, Results are shown for a separate experiment in which cells were exposed to control medium, 6 µM FSK, 1 µM isoproterenol (I), or 100 nM L-γ-MSH (M).

    Journal: PLoS ONE

    Article Title: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis

    doi: 10.1371/journal.pone.0055511

    Figure Lengend Snippet: Phospho-perilipin 1A visualized via Western blotting. 3T3L1 adipocytes were exposed to lipolytic agonist for 5 minutes; whole cell lysates were then prepared and subjected to SDS-PAGE/Western blotting (20 µg protein/lane) utilizing the anti-pPeri-site 6 antibody. A, Results are shown for cells exposed to either control medium (C) or 6 µM FSK (F). B, Results are shown for a separate experiment in which cells were exposed to control medium, 6 µM FSK, 1 µM isoproterenol (I), or 100 nM L-γ-MSH (M).

    Article Snippet: Murine perilipin 1A (National Center for Biotechnology Information (NCBI) reference sequence NP_783571.2) has six potential PKA phosphorylation sites located at serines 81, 222, 276, 433, 492, and 517, referred to as PKA-sites 1–6, respectively ; splice variants of perilipin 1 (PLIN1b and PLIN1c), which lack PKA sites 4–6, are also less commonly expressed .

    Techniques: Western Blot, SDS Page

    Differential phosphorylation of perilipin 1A and HSL in response to a panel of lipolytic agents. 3T3L1 adipocytes were exposed to the indicated concentrations (µM) of isoproterenol (ISO), FSK, and L-γ-MSH for 20 minutes, then fixed and labeled either PKA-site 5+ pHSL-serine 563 or PKA-site 6+ pHSL-serine 660. The cells were imaged with a 20X objective, 4 images/well, yielding an average of 360 cells/well. Area Pm values are shown for (A) PKA-site 5 (B), pHSL-serine 563, (C) PKA-site 6, and (D), pHSL-serine 660, respectively. Each bar represents the mean ± SD for n = 8 wells. * p

    Journal: PLoS ONE

    Article Title: Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis

    doi: 10.1371/journal.pone.0055511

    Figure Lengend Snippet: Differential phosphorylation of perilipin 1A and HSL in response to a panel of lipolytic agents. 3T3L1 adipocytes were exposed to the indicated concentrations (µM) of isoproterenol (ISO), FSK, and L-γ-MSH for 20 minutes, then fixed and labeled either PKA-site 5+ pHSL-serine 563 or PKA-site 6+ pHSL-serine 660. The cells were imaged with a 20X objective, 4 images/well, yielding an average of 360 cells/well. Area Pm values are shown for (A) PKA-site 5 (B), pHSL-serine 563, (C) PKA-site 6, and (D), pHSL-serine 660, respectively. Each bar represents the mean ± SD for n = 8 wells. * p

    Article Snippet: Murine perilipin 1A (National Center for Biotechnology Information (NCBI) reference sequence NP_783571.2) has six potential PKA phosphorylation sites located at serines 81, 222, 276, 433, 492, and 517, referred to as PKA-sites 1–6, respectively ; splice variants of perilipin 1 (PLIN1b and PLIN1c), which lack PKA sites 4–6, are also less commonly expressed .

    Techniques: Labeling

    Mapping of CRD1 residues critical for ELR1 function by gain-of-function analyses of point mutations converting murine HveA to equine ELR1 amino acids. (A) Schematic representation of the series of point mutations constructed by substituting the indicated

    Journal:

    Article Title: Mapping of Equine Lentivirus Receptor 1 Residues Critical for Equine Infectious Anemia Virus Envelope Binding

    doi: 10.1128/JVI.01393-07

    Figure Lengend Snippet: Mapping of CRD1 residues critical for ELR1 function by gain-of-function analyses of point mutations converting murine HveA to equine ELR1 amino acids. (A) Schematic representation of the series of point mutations constructed by substituting the indicated

    Article Snippet: The amino acid sequences of murine HveA and ELR1 (NCBI PID and , respectively) were obtained from the National Center for Biotechnology Information ( ).

    Techniques: Construct

    The EIAV gp90 binding domain of ELR1 maps to CRD1. The roles of individual CRD segments of ELR1 in gp90 binding and virus infection were evaluated by functional assay of chimeric receptor proteins. (A) Schematic representation of chimeric constructs with

    Journal:

    Article Title: Mapping of Equine Lentivirus Receptor 1 Residues Critical for Equine Infectious Anemia Virus Envelope Binding

    doi: 10.1128/JVI.01393-07

    Figure Lengend Snippet: The EIAV gp90 binding domain of ELR1 maps to CRD1. The roles of individual CRD segments of ELR1 in gp90 binding and virus infection were evaluated by functional assay of chimeric receptor proteins. (A) Schematic representation of chimeric constructs with

    Article Snippet: The amino acid sequences of murine HveA and ELR1 (NCBI PID and , respectively) were obtained from the National Center for Biotechnology Information ( ).

    Techniques: Binding Assay, Infection, Functional Assay, Construct

    Validation of the cell-cell binding assay. Shown are representative flow cytometry analysis data that summarize the specificity of the binding of target Cf2Th/ELR1 to the ligand CHO cells expressing gp90-GFP under standard assay conditions. (A) Cf2Th/ELR1

    Journal:

    Article Title: Mapping of Equine Lentivirus Receptor 1 Residues Critical for Equine Infectious Anemia Virus Envelope Binding

    doi: 10.1128/JVI.01393-07

    Figure Lengend Snippet: Validation of the cell-cell binding assay. Shown are representative flow cytometry analysis data that summarize the specificity of the binding of target Cf2Th/ELR1 to the ligand CHO cells expressing gp90-GFP under standard assay conditions. (A) Cf2Th/ELR1

    Article Snippet: The amino acid sequences of murine HveA and ELR1 (NCBI PID and , respectively) were obtained from the National Center for Biotechnology Information ( ).

    Techniques: Cell Binding Assay, Flow Cytometry, Cytometry, Binding Assay, Expressing

    The C terminus of ELR1 CRD1 is critical for EIAV gp90 binding. Fragments of ELR1 CRD1 were substituted into murine HveA, and the chimeric receptors were tested for gp90 binding and virus infection functions. (A) Schematic representation of the panel of

    Journal:

    Article Title: Mapping of Equine Lentivirus Receptor 1 Residues Critical for Equine Infectious Anemia Virus Envelope Binding

    doi: 10.1128/JVI.01393-07

    Figure Lengend Snippet: The C terminus of ELR1 CRD1 is critical for EIAV gp90 binding. Fragments of ELR1 CRD1 were substituted into murine HveA, and the chimeric receptors were tested for gp90 binding and virus infection functions. (A) Schematic representation of the panel of

    Article Snippet: The amino acid sequences of murine HveA and ELR1 (NCBI PID and , respectively) were obtained from the National Center for Biotechnology Information ( ).

    Techniques: Binding Assay, Infection

    Mapping of CRD1 residues critical for ELR1 function by loss-of-function analyses of point mutations converting ELR1 to murine HveA amino acids. (A) Schematic representation of the series of point mutations constructed by substituting the indicated amino

    Journal:

    Article Title: Mapping of Equine Lentivirus Receptor 1 Residues Critical for Equine Infectious Anemia Virus Envelope Binding

    doi: 10.1128/JVI.01393-07

    Figure Lengend Snippet: Mapping of CRD1 residues critical for ELR1 function by loss-of-function analyses of point mutations converting ELR1 to murine HveA amino acids. (A) Schematic representation of the series of point mutations constructed by substituting the indicated amino

    Article Snippet: The amino acid sequences of murine HveA and ELR1 (NCBI PID and , respectively) were obtained from the National Center for Biotechnology Information ( ).

    Techniques: Construct

    Schematic representation of ELR1, mHveA, and point mutation models. In all figures, the β-turn containing residues 68 to 71 is shown as a CPK model, with overlay of accessible surface area shown as white translucence. In all models, the Leu (A

    Journal:

    Article Title: Mapping of Equine Lentivirus Receptor 1 Residues Critical for Equine Infectious Anemia Virus Envelope Binding

    doi: 10.1128/JVI.01393-07

    Figure Lengend Snippet: Schematic representation of ELR1, mHveA, and point mutation models. In all figures, the β-turn containing residues 68 to 71 is shown as a CPK model, with overlay of accessible surface area shown as white translucence. In all models, the Leu (A

    Article Snippet: The amino acid sequences of murine HveA and ELR1 (NCBI PID and , respectively) were obtained from the National Center for Biotechnology Information ( ).

    Techniques: Mutagenesis

    Multiplex genome editing in zebrafish by Cas9. A mix of five gRNAs ( penta gRNAs: ddx19 gRNA, 55 pg; egfp gRNA, 15 pg; gol gRNA, 25 pg; mitfa gRNA, 60 pg; and tyr gRNA, 25 pg) were coinjected with nls-zCas9-nls RNA (150 pg) into Tg(-5.1mnx1:egfp) transgenic

    Journal:

    Article Title: Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system

    doi: 10.1073/pnas.1308335110

    Figure Lengend Snippet: Multiplex genome editing in zebrafish by Cas9. A mix of five gRNAs ( penta gRNAs: ddx19 gRNA, 55 pg; egfp gRNA, 15 pg; gol gRNA, 25 pg; mitfa gRNA, 60 pg; and tyr gRNA, 25 pg) were coinjected with nls-zCas9-nls RNA (150 pg) into Tg(-5.1mnx1:egfp) transgenic

    Article Snippet: The Cas9 protein sequence (National Center for Biotechnology Information, NCBI RefSeq: ) in this study is derived from a recently emerged strain (MGAS1882) and is slightly different from the ones recently reported ( , , ) ( ).

    Techniques: Multiplex Assay, Transgenic Assay

    Biallelic disruption of ddx19 by Cas9 generates null-like phenotypes. ddx19 gRNA (150 pg) and nls-zCas9-nls RNA (150 pg) were injected into wild-type embryos. ( A – G ) Lateral views of the wild-type ( A and E ), ddx19 -targeted ( B , C , and F ), and homozygous

    Journal:

    Article Title: Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system

    doi: 10.1073/pnas.1308335110

    Figure Lengend Snippet: Biallelic disruption of ddx19 by Cas9 generates null-like phenotypes. ddx19 gRNA (150 pg) and nls-zCas9-nls RNA (150 pg) were injected into wild-type embryos. ( A – G ) Lateral views of the wild-type ( A and E ), ddx19 -targeted ( B , C , and F ), and homozygous

    Article Snippet: The Cas9 protein sequence (National Center for Biotechnology Information, NCBI RefSeq: ) in this study is derived from a recently emerged strain (MGAS1882) and is slightly different from the ones recently reported ( , , ) ( ).

    Techniques: Injection

    Efficient disruption of the Tg(-5.1mnx1:EGFP) transgene by Cas9 results in mosaic EGFP expression in the motoneurons. egfp gRNA (6 or 30 pg) and nls-zCas9-nls RNA (150 pg) were injected into Tg(-5.1mnx1:EGFP)/Tg(-5.1mnx1:TagRFP) double transgenic embryos.

    Journal:

    Article Title: Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system

    doi: 10.1073/pnas.1308335110

    Figure Lengend Snippet: Efficient disruption of the Tg(-5.1mnx1:EGFP) transgene by Cas9 results in mosaic EGFP expression in the motoneurons. egfp gRNA (6 or 30 pg) and nls-zCas9-nls RNA (150 pg) were injected into Tg(-5.1mnx1:EGFP)/Tg(-5.1mnx1:TagRFP) double transgenic embryos.

    Article Snippet: The Cas9 protein sequence (National Center for Biotechnology Information, NCBI RefSeq: ) in this study is derived from a recently emerged strain (MGAS1882) and is slightly different from the ones recently reported ( , , ) ( ).

    Techniques: Expressing, Injection, Transgenic Assay

    Genome editing in zebrafish using a CRISPR/Cas9 system. The system consists of two components, a dual NLS-tagged zebrafish codon-optimized Cas9 protein and a single crRNA:tracrRNA chimeric gRNA, comprising a 20-bp target sequence (dark red) complementary

    Journal:

    Article Title: Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system

    doi: 10.1073/pnas.1308335110

    Figure Lengend Snippet: Genome editing in zebrafish using a CRISPR/Cas9 system. The system consists of two components, a dual NLS-tagged zebrafish codon-optimized Cas9 protein and a single crRNA:tracrRNA chimeric gRNA, comprising a 20-bp target sequence (dark red) complementary

    Article Snippet: The Cas9 protein sequence (National Center for Biotechnology Information, NCBI RefSeq: ) in this study is derived from a recently emerged strain (MGAS1882) and is slightly different from the ones recently reported ( , , ) ( ).

    Techniques: CRISPR, Sequencing

    Biallelic disruption of golden ( gol ) by Cas9 generates mosaic pigmentation phenotypes. gol gRNA (50 pg) and nls-zCas9-nls RNA (150 pg) were injected into wild-type embryos. ( A – F ) Lateral views of wild-type ( A and E ) and gol -targeted embryos (

    Journal:

    Article Title: Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system

    doi: 10.1073/pnas.1308335110

    Figure Lengend Snippet: Biallelic disruption of golden ( gol ) by Cas9 generates mosaic pigmentation phenotypes. gol gRNA (50 pg) and nls-zCas9-nls RNA (150 pg) were injected into wild-type embryos. ( A – F ) Lateral views of wild-type ( A and E ) and gol -targeted embryos (

    Article Snippet: The Cas9 protein sequence (National Center for Biotechnology Information, NCBI RefSeq: ) in this study is derived from a recently emerged strain (MGAS1882) and is slightly different from the ones recently reported ( , , ) ( ).

    Techniques: Injection

    Biallelic disruption of tyrosinase ( tyr ) by Cas9 generates mosaic pigmentation phenotypes. tyr gRNA (30 pg) and nls-zCas9-nls RNA (150 pg) were injected into wild-type embryos. ( A – E ) Lateral views of wild-type ( A ) and tyr -targeted embryos ( B –

    Journal:

    Article Title: Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system

    doi: 10.1073/pnas.1308335110

    Figure Lengend Snippet: Biallelic disruption of tyrosinase ( tyr ) by Cas9 generates mosaic pigmentation phenotypes. tyr gRNA (30 pg) and nls-zCas9-nls RNA (150 pg) were injected into wild-type embryos. ( A – E ) Lateral views of wild-type ( A ) and tyr -targeted embryos ( B –

    Article Snippet: The Cas9 protein sequence (National Center for Biotechnology Information, NCBI RefSeq: ) in this study is derived from a recently emerged strain (MGAS1882) and is slightly different from the ones recently reported ( , , ) ( ).

    Techniques: Injection

    Effects of overexpressing CaMsrB2 on the Phytophthora blight disease. Leaves of CaMsrB2 -transgenic tomato were inoculated with P. capsici zoospores (5 × 10 5 zoospores mL −1 ). A, Cell death in CaMsrB2 -silenced plants. Cell death on the leaves

    Journal:

    Article Title: CaMsrB2, Pepper Methionine Sulfoxide Reductase B2, Is a Novel Defense Regulator against Oxidative Stress and Pathogen Attack

    doi: 10.1104/pp.110.162339

    Figure Lengend Snippet: Effects of overexpressing CaMsrB2 on the Phytophthora blight disease. Leaves of CaMsrB2 -transgenic tomato were inoculated with P. capsici zoospores (5 × 10 5 zoospores mL −1 ). A, Cell death in CaMsrB2 -silenced plants. Cell death on the leaves

    Article Snippet: The isolated CaMsrB2 (National Center for Biotechnology Information [NCBI] accession no. ) open reading frame encodes a full-length protein of 185 amino acid residues.

    Techniques: Transgenic Assay

    Ectopic expression of CaMsrB2 and tolerance levels to oxidative stresses. A, Electrolyte leakage of CaMsrB2 transgenic tomato leaves following treatment with MV. Electrolyte leakage was measured at 2 d after treatment of 0.005 m m MV. Photographs were

    Journal:

    Article Title: CaMsrB2, Pepper Methionine Sulfoxide Reductase B2, Is a Novel Defense Regulator against Oxidative Stress and Pathogen Attack

    doi: 10.1104/pp.110.162339

    Figure Lengend Snippet: Ectopic expression of CaMsrB2 and tolerance levels to oxidative stresses. A, Electrolyte leakage of CaMsrB2 transgenic tomato leaves following treatment with MV. Electrolyte leakage was measured at 2 d after treatment of 0.005 m m MV. Photographs were

    Article Snippet: The isolated CaMsrB2 (National Center for Biotechnology Information [NCBI] accession no. ) open reading frame encodes a full-length protein of 185 amino acid residues.

    Techniques: Expressing, Transgenic Assay

    Subcellular localization of CaMsrB2. A, Construct map of the CaMsrB2-smGFP fusion protein. The CaMsrB2 coding region fused in-frame to the smGFP was inserted in pUC19 for in planta expression. ORF, Open reading frame. B, Location of the CaMsrB2-smGFP

    Journal:

    Article Title: CaMsrB2, Pepper Methionine Sulfoxide Reductase B2, Is a Novel Defense Regulator against Oxidative Stress and Pathogen Attack

    doi: 10.1104/pp.110.162339

    Figure Lengend Snippet: Subcellular localization of CaMsrB2. A, Construct map of the CaMsrB2-smGFP fusion protein. The CaMsrB2 coding region fused in-frame to the smGFP was inserted in pUC19 for in planta expression. ORF, Open reading frame. B, Location of the CaMsrB2-smGFP

    Article Snippet: The isolated CaMsrB2 (National Center for Biotechnology Information [NCBI] accession no. ) open reading frame encodes a full-length protein of 185 amino acid residues.

    Techniques: Construct, Expressing

    CaMsrB2 expression in tomato is more resistant to Phytophthora . A, Typical disease symptoms after inoculation of tomato leaves with P. capsici . Four days after inoculation, leaves were trypan blue stained to visualize disease development. B, Leaf area

    Journal:

    Article Title: CaMsrB2, Pepper Methionine Sulfoxide Reductase B2, Is a Novel Defense Regulator against Oxidative Stress and Pathogen Attack

    doi: 10.1104/pp.110.162339

    Figure Lengend Snippet: CaMsrB2 expression in tomato is more resistant to Phytophthora . A, Typical disease symptoms after inoculation of tomato leaves with P. capsici . Four days after inoculation, leaves were trypan blue stained to visualize disease development. B, Leaf area

    Article Snippet: The isolated CaMsrB2 (National Center for Biotechnology Information [NCBI] accession no. ) open reading frame encodes a full-length protein of 185 amino acid residues.

    Techniques: Expressing, Staining

    Effects of silencing CaMsrB2 on HR. A, Cell death in the silenced plants. Leaves silenced with GFP (control), CaMsrB2 -N, or CaMsrB2 -C were infiltrated with Xav race 1. B, Cell death on the leaves was verified visually and by trypan blue staining. Dark

    Journal:

    Article Title: CaMsrB2, Pepper Methionine Sulfoxide Reductase B2, Is a Novel Defense Regulator against Oxidative Stress and Pathogen Attack

    doi: 10.1104/pp.110.162339

    Figure Lengend Snippet: Effects of silencing CaMsrB2 on HR. A, Cell death in the silenced plants. Leaves silenced with GFP (control), CaMsrB2 -N, or CaMsrB2 -C were infiltrated with Xav race 1. B, Cell death on the leaves was verified visually and by trypan blue staining. Dark

    Article Snippet: The isolated CaMsrB2 (National Center for Biotechnology Information [NCBI] accession no. ) open reading frame encodes a full-length protein of 185 amino acid residues.

    Techniques: Staining

    Analysis of the expression patterns of CaMsrB2 and enzymatic activity of CaMsrB2. A, Expression of CaMsrB2 in ECW-30R pepper inoculated with Xav race 1 (incompatible) or race 3 (compatible) or with the nonhost pathogen Xag 8ra. Total RNA was extracted

    Journal:

    Article Title: CaMsrB2, Pepper Methionine Sulfoxide Reductase B2, Is a Novel Defense Regulator against Oxidative Stress and Pathogen Attack

    doi: 10.1104/pp.110.162339

    Figure Lengend Snippet: Analysis of the expression patterns of CaMsrB2 and enzymatic activity of CaMsrB2. A, Expression of CaMsrB2 in ECW-30R pepper inoculated with Xav race 1 (incompatible) or race 3 (compatible) or with the nonhost pathogen Xag 8ra. Total RNA was extracted

    Article Snippet: The isolated CaMsrB2 (National Center for Biotechnology Information [NCBI] accession no. ) open reading frame encodes a full-length protein of 185 amino acid residues.

    Techniques: Expressing, Activity Assay

    VIGS of the CaMsrB2 gene in ECW-30R pepper plant. A, Diagram of silencing constructs CaMsrB2 -N (targeting the N terminus of CaMsrB2 ) and CaMsrB2 -C (targeting the C terminus of CaMsrB2 ). The CaMsrB2 -N and -C fragments were designed not to include the SelR-like

    Journal:

    Article Title: CaMsrB2, Pepper Methionine Sulfoxide Reductase B2, Is a Novel Defense Regulator against Oxidative Stress and Pathogen Attack

    doi: 10.1104/pp.110.162339

    Figure Lengend Snippet: VIGS of the CaMsrB2 gene in ECW-30R pepper plant. A, Diagram of silencing constructs CaMsrB2 -N (targeting the N terminus of CaMsrB2 ) and CaMsrB2 -C (targeting the C terminus of CaMsrB2 ). The CaMsrB2 -N and -C fragments were designed not to include the SelR-like

    Article Snippet: The isolated CaMsrB2 (National Center for Biotechnology Information [NCBI] accession no. ) open reading frame encodes a full-length protein of 185 amino acid residues.

    Techniques: Construct

    Characterization of the CaMsrB2 ), Arabidopsis (AtCaMsrB2;

    Journal:

    Article Title: CaMsrB2, Pepper Methionine Sulfoxide Reductase B2, Is a Novel Defense Regulator against Oxidative Stress and Pathogen Attack

    doi: 10.1104/pp.110.162339

    Figure Lengend Snippet: Characterization of the CaMsrB2 ), Arabidopsis (AtCaMsrB2;

    Article Snippet: The isolated CaMsrB2 (National Center for Biotechnology Information [NCBI] accession no. ) open reading frame encodes a full-length protein of 185 amino acid residues.

    Techniques:

    Enhanced susceptibility to Xav race 3 in CaMsrB2 -silenced pepper plant. A, Bacterial growth in the silenced plants. Leaves of GFP (control), CaMsrB2 -N-, or CaMsrB2 -C-silenced plants were infiltrated with Xav race 3 (approximately 6 × 10 4 cfu mL

    Journal:

    Article Title: CaMsrB2, Pepper Methionine Sulfoxide Reductase B2, Is a Novel Defense Regulator against Oxidative Stress and Pathogen Attack

    doi: 10.1104/pp.110.162339

    Figure Lengend Snippet: Enhanced susceptibility to Xav race 3 in CaMsrB2 -silenced pepper plant. A, Bacterial growth in the silenced plants. Leaves of GFP (control), CaMsrB2 -N-, or CaMsrB2 -C-silenced plants were infiltrated with Xav race 3 (approximately 6 × 10 4 cfu mL

    Article Snippet: The isolated CaMsrB2 (National Center for Biotechnology Information [NCBI] accession no. ) open reading frame encodes a full-length protein of 185 amino acid residues.

    Techniques:

    CSP comparison of cofilin variants. A , changes in the WT cofilin structure caused by reduction ( red ) superimposed on the three-dimensional surface model of the protein (PDB ID 1Q8G ), as in Fig. 1 C. B , overlay of CSPs > 0.1 ppm between mutant C39A and the nonreduced WT ( white ) and the changes shown in A ( red ). The overlap of both is colored pink. C , comparison of mutant C139A and nonreduced WT cofilin with the same colors as in B. D , comparison of mutant C147A and nonreduced WT cofilin with the same colors as in B .

    Journal: The Journal of Biological Chemistry

    Article Title: A Reducing Milieu Renders Cofilin Insensitive to Phosphatidylinositol 4,5-Bisphosphate (PIP

    doi: 10.1074/jbc.M113.479766

    Figure Lengend Snippet: CSP comparison of cofilin variants. A , changes in the WT cofilin structure caused by reduction ( red ) superimposed on the three-dimensional surface model of the protein (PDB ID 1Q8G ), as in Fig. 1 C. B , overlay of CSPs > 0.1 ppm between mutant C39A and the nonreduced WT ( white ) and the changes shown in A ( red ). The overlap of both is colored pink. C , comparison of mutant C139A and nonreduced WT cofilin with the same colors as in B. D , comparison of mutant C147A and nonreduced WT cofilin with the same colors as in B .

    Article Snippet: The human cofilin gene (National Center for Biotechnology Information (NCBI) Gene ID 1072) was cloned into expression vector pETM11 (European Molecular Biology Laboratory (EMBL) Vector Collection) via NcoI and BamHI restriction sites.

    Techniques: Mutagenesis

    Analysis of the cofilin redox state and its influence on cofilin structure. A , mass spectrometry analysis of recombinant WT cofilin purified under nonreducing conditions. The His-tagged untreated molecule ( left spectrum ) shows a main mass peak at 21,831 atomic mass units ( amu ). Treatment with IAA ( right spectrum ) resulted in an increase in size by 114 atomic mass units (to 21,945 atomic mass units), corresponding to the addition of two IAA molecules. The accuracy of the spectrometer was ±1 atomic mass unit. B , Western blot of Mal-PEG cysteine modification assay performed with PBT lysate with and without reducing treatment. The membrane was probed for total cofilin; note that although the same amount of lysate was loaded on each lane, antibody detection decreased in Mal-PEGylated samples, probably due to epitope masking by Mal-PEG molecules. M , molecular mass markers shown in kilodaltons. C , the 15 N- 1 H heteronuclear single quantum coherence spectrum of untreated cofilin ( black ) is overlaid with that of DTT-treated cofilin ( red ). Amino acids that exhibit distinct CSPs ( > 0.1 ppm) are labeled with their amino acid position. D , all CSPs occurring upon cofilin reduction. Again, all CSPs > 0.1 ppm are labeled; additionally, the four cysteines are marked in red . CSPs involving the complete disappearance of an amino acid peak in one of the spectra are marked by a negative bar. E , a three-dimensional rendering of the molecular surface of cofilin (PDB ID 1Q8G ), with CSPs from the WT with/without DTT comparison colored red . Four views from around the molecule are shown by a perspective shift of 90° from one image to the next.

    Journal: The Journal of Biological Chemistry

    Article Title: A Reducing Milieu Renders Cofilin Insensitive to Phosphatidylinositol 4,5-Bisphosphate (PIP

    doi: 10.1074/jbc.M113.479766

    Figure Lengend Snippet: Analysis of the cofilin redox state and its influence on cofilin structure. A , mass spectrometry analysis of recombinant WT cofilin purified under nonreducing conditions. The His-tagged untreated molecule ( left spectrum ) shows a main mass peak at 21,831 atomic mass units ( amu ). Treatment with IAA ( right spectrum ) resulted in an increase in size by 114 atomic mass units (to 21,945 atomic mass units), corresponding to the addition of two IAA molecules. The accuracy of the spectrometer was ±1 atomic mass unit. B , Western blot of Mal-PEG cysteine modification assay performed with PBT lysate with and without reducing treatment. The membrane was probed for total cofilin; note that although the same amount of lysate was loaded on each lane, antibody detection decreased in Mal-PEGylated samples, probably due to epitope masking by Mal-PEG molecules. M , molecular mass markers shown in kilodaltons. C , the 15 N- 1 H heteronuclear single quantum coherence spectrum of untreated cofilin ( black ) is overlaid with that of DTT-treated cofilin ( red ). Amino acids that exhibit distinct CSPs ( > 0.1 ppm) are labeled with their amino acid position. D , all CSPs occurring upon cofilin reduction. Again, all CSPs > 0.1 ppm are labeled; additionally, the four cysteines are marked in red . CSPs involving the complete disappearance of an amino acid peak in one of the spectra are marked by a negative bar. E , a three-dimensional rendering of the molecular surface of cofilin (PDB ID 1Q8G ), with CSPs from the WT with/without DTT comparison colored red . Four views from around the molecule are shown by a perspective shift of 90° from one image to the next.

    Article Snippet: The human cofilin gene (National Center for Biotechnology Information (NCBI) Gene ID 1072) was cloned into expression vector pETM11 (European Molecular Biology Laboratory (EMBL) Vector Collection) via NcoI and BamHI restriction sites.

    Techniques: Mass Spectrometry, Recombinant, Purification, Western Blot, Modification, Labeling

    Analysis of PBT-APC contacts for F-actin enrichment in the immune synapse after reducing treatment. A , flow cytometry analysis of T cell-APC conjugates formed with untreated or 1 m m β-ME-treated PBTs. The extent of contact formation was determined by the percentage of CD3/CD19 double-positive events. B , quantification of conjugation assay after β-ME treatment. The data were statistically analyzed with a two-tailed paired t test ( n = 3). C , cell contacts imaged by multispectral imaging flow cytometry divided into contacts with high, medium, and low F-actin enrichment in the T cell part of the synapse. Numbers in the gate designate the percentage of cells in the entire population of contacts. D , exemplary in-flow microscopy images of T cell-APC contacts with high ( upper panel ), medium ( middle panel ), or low ( lower panel ) F-actin-enriched T cells. trans , transmitted light microscopic image. E , quantification of T cells with high F-actin enrichment in the contact zone. F , quantification of T cells with high cofilin ( left panel ) or CD3 ( right panel ). G , comparison of the F-actin enrichment between contacts in the absence or presence of superantigen (staphylococcal enterotoxin B ( SEB )). *, p

    Journal: The Journal of Biological Chemistry

    Article Title: A Reducing Milieu Renders Cofilin Insensitive to Phosphatidylinositol 4,5-Bisphosphate (PIP

    doi: 10.1074/jbc.M113.479766

    Figure Lengend Snippet: Analysis of PBT-APC contacts for F-actin enrichment in the immune synapse after reducing treatment. A , flow cytometry analysis of T cell-APC conjugates formed with untreated or 1 m m β-ME-treated PBTs. The extent of contact formation was determined by the percentage of CD3/CD19 double-positive events. B , quantification of conjugation assay after β-ME treatment. The data were statistically analyzed with a two-tailed paired t test ( n = 3). C , cell contacts imaged by multispectral imaging flow cytometry divided into contacts with high, medium, and low F-actin enrichment in the T cell part of the synapse. Numbers in the gate designate the percentage of cells in the entire population of contacts. D , exemplary in-flow microscopy images of T cell-APC contacts with high ( upper panel ), medium ( middle panel ), or low ( lower panel ) F-actin-enriched T cells. trans , transmitted light microscopic image. E , quantification of T cells with high F-actin enrichment in the contact zone. F , quantification of T cells with high cofilin ( left panel ) or CD3 ( right panel ). G , comparison of the F-actin enrichment between contacts in the absence or presence of superantigen (staphylococcal enterotoxin B ( SEB )). *, p

    Article Snippet: The human cofilin gene (National Center for Biotechnology Information (NCBI) Gene ID 1072) was cloned into expression vector pETM11 (European Molecular Biology Laboratory (EMBL) Vector Collection) via NcoI and BamHI restriction sites.

    Techniques: Flow Cytometry, Cytometry, Conjugation Assay, Two Tailed Test, Imaging, Microscopy

    Analysis of the actin-depolymerizing capacity of reduced WT cofilin and cysteine mutants in the presence of PIP 2 . A , cofilin residues affected by PIP 2 binding (as reported by Gorbatyuk et al. ( 24 )) are shown in blue , changes upon reduction are shown in red , and changes caused by both PIP 2 binding and DTT are shown in purple. B , F-actin-depolymerizing capacity of WT cofilin under nonreducing/reducing conditions and in the presence or absence of PIP 2 . The treatment combinations are shown above the lanes. Left panels , pretreated cofilin and actin; right panels , actin alone underwent the corresponding treatments and the assay. S , supernatant; P , pellet of the depolymerization assay. White dividers were inserted for clarity. C , a magnified detail from the gel indicated displays the upward shifts of cofilin following binding to PIP 2 (1 kDa per PIP 2 molecule). The image was enhanced by optimizing contrast for better visibility. D , results of a paired t test from actin sedimentation assay of WT cofilin and cysteine mutants with PIP 2 . Statistical significances (*, p

    Journal: The Journal of Biological Chemistry

    Article Title: A Reducing Milieu Renders Cofilin Insensitive to Phosphatidylinositol 4,5-Bisphosphate (PIP

    doi: 10.1074/jbc.M113.479766

    Figure Lengend Snippet: Analysis of the actin-depolymerizing capacity of reduced WT cofilin and cysteine mutants in the presence of PIP 2 . A , cofilin residues affected by PIP 2 binding (as reported by Gorbatyuk et al. ( 24 )) are shown in blue , changes upon reduction are shown in red , and changes caused by both PIP 2 binding and DTT are shown in purple. B , F-actin-depolymerizing capacity of WT cofilin under nonreducing/reducing conditions and in the presence or absence of PIP 2 . The treatment combinations are shown above the lanes. Left panels , pretreated cofilin and actin; right panels , actin alone underwent the corresponding treatments and the assay. S , supernatant; P , pellet of the depolymerization assay. White dividers were inserted for clarity. C , a magnified detail from the gel indicated displays the upward shifts of cofilin following binding to PIP 2 (1 kDa per PIP 2 molecule). The image was enhanced by optimizing contrast for better visibility. D , results of a paired t test from actin sedimentation assay of WT cofilin and cysteine mutants with PIP 2 . Statistical significances (*, p

    Article Snippet: The human cofilin gene (National Center for Biotechnology Information (NCBI) Gene ID 1072) was cloned into expression vector pETM11 (European Molecular Biology Laboratory (EMBL) Vector Collection) via NcoI and BamHI restriction sites.

    Techniques: Binding Assay, Sedimentation

    F-actin-depolymerizing capacity of cofilin variants under nonreducing and reducing conditions. A , the changes caused in cofilin by reduction are compared with the changes caused by actin binding to cofilin (summarized by Gorbatyuk et al. ( 24 )). Amino acids influenced by actin binding are shown in yellow , and changes caused by reduction are shown in red , as in Fig. 1 C. B , F-actin depolymerization assay of WT cofilin and the cysteine mutants showed the independence of F-actin-depolymerizing activity from DTT treatment. S , supernatant fraction after sedimentation (ultracentrifugation); P , pellet fraction after sedimentation. All bands shown are from one and the same experiment and partly from the same SDS gel; for clarity, white dividers have been put between each fraction couple. C , statistical evaluation of the F-actin-depolymerizing capacity of cofilin variants. ***, p

    Journal: The Journal of Biological Chemistry

    Article Title: A Reducing Milieu Renders Cofilin Insensitive to Phosphatidylinositol 4,5-Bisphosphate (PIP

    doi: 10.1074/jbc.M113.479766

    Figure Lengend Snippet: F-actin-depolymerizing capacity of cofilin variants under nonreducing and reducing conditions. A , the changes caused in cofilin by reduction are compared with the changes caused by actin binding to cofilin (summarized by Gorbatyuk et al. ( 24 )). Amino acids influenced by actin binding are shown in yellow , and changes caused by reduction are shown in red , as in Fig. 1 C. B , F-actin depolymerization assay of WT cofilin and the cysteine mutants showed the independence of F-actin-depolymerizing activity from DTT treatment. S , supernatant fraction after sedimentation (ultracentrifugation); P , pellet fraction after sedimentation. All bands shown are from one and the same experiment and partly from the same SDS gel; for clarity, white dividers have been put between each fraction couple. C , statistical evaluation of the F-actin-depolymerizing capacity of cofilin variants. ***, p

    Article Snippet: The human cofilin gene (National Center for Biotechnology Information (NCBI) Gene ID 1072) was cloned into expression vector pETM11 (European Molecular Biology Laboratory (EMBL) Vector Collection) via NcoI and BamHI restriction sites.

    Techniques: Binding Assay, Activity Assay, Sedimentation, SDS-Gel

    FAdV-4 viral DNA concentration in visceral tissues samples at different dpi ( A : oral group; B : subcutaneous group).

    Journal: Frontiers in Microbiology

    Article Title: Pathogenic, Phylogenetic, and Serological Analysis of Group I Fowl Adenovirus Serotype 4 SDSX Isolated From Shandong, China

    doi: 10.3389/fmicb.2018.02772

    Figure Lengend Snippet: FAdV-4 viral DNA concentration in visceral tissues samples at different dpi ( A : oral group; B : subcutaneous group).

    Article Snippet: Duck-origin FAdV-4 [SDSX, National Center for Biotechnology Information (NCBI) GenBank Accession No. KT899325 ] was isolated from dead ducks in 2015 in Shenxian County, Shandong Province, China, and stored.

    Techniques: Concentration Assay

    Gross lesions and postmortem changes in ducks infected with FAdV-4 (12 days post-infection [dpi]). (A,B) severe hydropericardium accompanied by heart yellow coronary fat; (D,E) enlarged, crisp livers with a pale yellow appearance; (G,H) swelling and spotted bleeding in spleen; (J, K, M, N) edema with severe congestion in lungs and kidney; (P,Q) swelling in bursa of Fabricius; (S,T) proliferation in thymus; (V,W) edema with hemorrhage in brain. No significant clinical signs or gross lesions were found in the control (C, F, I, L, O, R, U, X) .

    Journal: Frontiers in Microbiology

    Article Title: Pathogenic, Phylogenetic, and Serological Analysis of Group I Fowl Adenovirus Serotype 4 SDSX Isolated From Shandong, China

    doi: 10.3389/fmicb.2018.02772

    Figure Lengend Snippet: Gross lesions and postmortem changes in ducks infected with FAdV-4 (12 days post-infection [dpi]). (A,B) severe hydropericardium accompanied by heart yellow coronary fat; (D,E) enlarged, crisp livers with a pale yellow appearance; (G,H) swelling and spotted bleeding in spleen; (J, K, M, N) edema with severe congestion in lungs and kidney; (P,Q) swelling in bursa of Fabricius; (S,T) proliferation in thymus; (V,W) edema with hemorrhage in brain. No significant clinical signs or gross lesions were found in the control (C, F, I, L, O, R, U, X) .

    Article Snippet: Duck-origin FAdV-4 [SDSX, National Center for Biotechnology Information (NCBI) GenBank Accession No. KT899325 ] was isolated from dead ducks in 2015 in Shenxian County, Shandong Province, China, and stored.

    Techniques: Infection

    Histopathologic changes in viscera tissue of meat ducks infected with FAdV-4 (12 days post-infection [dpi]). (A,B) heart, slight granular degeneration, congestion, and dilated intercellular space; (D,E) liver, obvious fatty degeneration and basophilic inclusion bodies presented in hepatocytes, tissue structural disorders; (G,H) spleen, various degrees of hemorrhage; (J,K) lung, blood capillary congestion of pulmonary alveoli, multifocal areas with lymphocytes gathering; (M,N) kidney, enlargement and degeneration of glomerulus, narrowed glomerular sac, severe congestion accompanied by excessive lymphocyte infiltration; (P,Q) bursa, lymphocyte reduction and vacuole formation; (S,T) thymus, lymphocyte depletion; (V,W) brain, hemorrhage and edema. No pathological changes were found in the control group (C, F, I, L, O, R, U, X) . Scale bar = 100 μm.

    Journal: Frontiers in Microbiology

    Article Title: Pathogenic, Phylogenetic, and Serological Analysis of Group I Fowl Adenovirus Serotype 4 SDSX Isolated From Shandong, China

    doi: 10.3389/fmicb.2018.02772

    Figure Lengend Snippet: Histopathologic changes in viscera tissue of meat ducks infected with FAdV-4 (12 days post-infection [dpi]). (A,B) heart, slight granular degeneration, congestion, and dilated intercellular space; (D,E) liver, obvious fatty degeneration and basophilic inclusion bodies presented in hepatocytes, tissue structural disorders; (G,H) spleen, various degrees of hemorrhage; (J,K) lung, blood capillary congestion of pulmonary alveoli, multifocal areas with lymphocytes gathering; (M,N) kidney, enlargement and degeneration of glomerulus, narrowed glomerular sac, severe congestion accompanied by excessive lymphocyte infiltration; (P,Q) bursa, lymphocyte reduction and vacuole formation; (S,T) thymus, lymphocyte depletion; (V,W) brain, hemorrhage and edema. No pathological changes were found in the control group (C, F, I, L, O, R, U, X) . Scale bar = 100 μm.

    Article Snippet: Duck-origin FAdV-4 [SDSX, National Center for Biotechnology Information (NCBI) GenBank Accession No. KT899325 ] was isolated from dead ducks in 2015 in Shenxian County, Shandong Province, China, and stored.

    Techniques: Infection

    FAdV-4 antibody levels in the different groups. The comparison was between the infected groups (i.e., oral or subcutaneous group) and the control group at the same number of days post-infection (dpi). * P

    Journal: Frontiers in Microbiology

    Article Title: Pathogenic, Phylogenetic, and Serological Analysis of Group I Fowl Adenovirus Serotype 4 SDSX Isolated From Shandong, China

    doi: 10.3389/fmicb.2018.02772

    Figure Lengend Snippet: FAdV-4 antibody levels in the different groups. The comparison was between the infected groups (i.e., oral or subcutaneous group) and the control group at the same number of days post-infection (dpi). * P

    Article Snippet: Duck-origin FAdV-4 [SDSX, National Center for Biotechnology Information (NCBI) GenBank Accession No. KT899325 ] was isolated from dead ducks in 2015 in Shenxian County, Shandong Province, China, and stored.

    Techniques: Infection