Structured Review

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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1) Product Images from "Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair"

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

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E14-04-0947

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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

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

2) Product Images from "Avian viral surveillance in Victoria, Australia, and detection of two novel avian herpesviruses"

Article Title: Avian viral surveillance in Victoria, Australia, and detection of two novel avian herpesviruses

Journal: PLoS ONE

doi: 10.1371/journal.pone.0194457

PhyML maximum likelihood phylogenetic tree of avian alphaherpesviruses. Generated from a ClustalW2 alignment [ 59 ] of the partial DNA polymerase gene sequences of Podargid alphaherpesvirus 1 and Cacatuid alphaherpesvirus 1 with published avian alphaherpesvirus nucleotide sequences available in GenBank, with the two novel alphaherpesviruses detected in this study highlighted in bold [ 60 ]. The GenBank accession numbers for sequences used are included in the tip labels, and Macropodid alphaherpesvirus 1 (highlighted in italics) is included as an outgroup. Branching with greater than 50% support from 100 bootstrap replicates is indicated at major node points. The distances indicated by black horizontal lines correspond to genetic distances, with the scale bar representing nucleotide substitutions/site.
Figure Legend Snippet: PhyML maximum likelihood phylogenetic tree of avian alphaherpesviruses. Generated from a ClustalW2 alignment [ 59 ] of the partial DNA polymerase gene sequences of Podargid alphaherpesvirus 1 and Cacatuid alphaherpesvirus 1 with published avian alphaherpesvirus nucleotide sequences available in GenBank, with the two novel alphaherpesviruses detected in this study highlighted in bold [ 60 ]. The GenBank accession numbers for sequences used are included in the tip labels, and Macropodid alphaherpesvirus 1 (highlighted in italics) is included as an outgroup. Branching with greater than 50% support from 100 bootstrap replicates is indicated at major node points. The distances indicated by black horizontal lines correspond to genetic distances, with the scale bar representing nucleotide substitutions/site.

Techniques Used: Generated

Phylogenetic tree for the genus avulvirus of the family Paramyxoviridae based on partial RNA-dependent RNA polymerase gene sequences. Maximum likelihood phylogenetic tree constructed using PhyML from a ClustalW2 alignment of the partial RNA-dependent RNA polymerase (large polymerase or L protein) gene sequences of the avian avulavirus detected from a musk lorikeet ( Glossopsitta concinna ) in this study (unclassified avian avulavirus strain musk lorikeet/Melbourne/ML22-141263/2014; highlighted in bold) and published paramyxovirus sequences retrieved from GenBank [ 60 ]. The GenBank accession numbers for sequences used are indicated in brackets in the tip labels. Human rubulavirus 2 (highlighted in italics) is included as an outgroup. Branching with greater than 50% support from 100 bootstrap replicates is indicated at major node points. The distances indicated by black horizontal lines correspond to genetic distances, with the scale bar representing nucleotide substitutions/site.
Figure Legend Snippet: Phylogenetic tree for the genus avulvirus of the family Paramyxoviridae based on partial RNA-dependent RNA polymerase gene sequences. Maximum likelihood phylogenetic tree constructed using PhyML from a ClustalW2 alignment of the partial RNA-dependent RNA polymerase (large polymerase or L protein) gene sequences of the avian avulavirus detected from a musk lorikeet ( Glossopsitta concinna ) in this study (unclassified avian avulavirus strain musk lorikeet/Melbourne/ML22-141263/2014; highlighted in bold) and published paramyxovirus sequences retrieved from GenBank [ 60 ]. The GenBank accession numbers for sequences used are indicated in brackets in the tip labels. Human rubulavirus 2 (highlighted in italics) is included as an outgroup. Branching with greater than 50% support from 100 bootstrap replicates is indicated at major node points. The distances indicated by black horizontal lines correspond to genetic distances, with the scale bar representing nucleotide substitutions/site.

Techniques Used: Construct

Unrooted maximum likelihood phylogenetic tree for the family Herpesviridae . Generated from a ClustalW2 alignment of amino acid translations of partial DNA polymerase gene sequences from 40 representative herpesviruses retrieved from GenBank from the three subfamilies: Alphaherpesvirinae (α), Betaherpesvirinae (β) and Gammaherpesvirinae (γ) from a range of host species, and including the two novel alphaherpesviruses detected in this study (highlighted in bold) [ 60 ]. The GenBank accession numbers for sequences used are as follows: Accipitrid alphaherpesvirus 1 AY571851; Alcelaphine gammaherpesvirus 1 AF005370; Anatid alphaherpesvirus 1 EF643560; Bovine alphaherpesvirus 1 X94677; Bovine alphaherpesvirus 2 AF181249; Bovine gammaherpesvirus 4 AF031811; Bovine gammaherpesvirus 6 AF031808; Cacatuid alphaherpesvirus 1 MF576271 ; Columbid alphaherpesvirus 1 AF141890; Elephantid betaherpesvirus 1 AF322977; Elephantid gammaherpesvirus 3 DQ238845; Equid alphaherpesvirus 1 KF434378; Equid gammaherpesvirus 2 NC001650; Equid alphaherpesvirus 4 KT324743; Felid alphaherpesvirus 1 KR296657; Fregatid alphaherpesvirus 1 EU867220; Gallid alphaherpesvirus 1 NC006623; Gallid alphaherpesvirus 2 AF147806; Gallid alphaherpesvirus 3 HQ840738; Gaviid alphaherpesvirus 1 GU130289; Human alphaherpesvirus 1 HQ123098; Human alphaherpesvirus 3 X04370; Human betaherpesvirus 5 NC006273; Human betaherpesvirus 6 X83413; Macacine gammaherpesvirus 5 AF029302; Macropodid alphaherpesvirus 1 NC029132; Macropodid gammaherpesvirus 3 EF467663; Meleagrid alphaherpesvirus 1 AF291866; Murid betaherpesvirus 2 AY728086; Passerid alphaherpesvirus 1 AF520812; Phascolarctid gammaherpesvirus 2 JQ996387; Phocid alphaherpesvirus 1 PHU92269; Phoenicopterid alphaherpesvirus 1 KP244360; Podargid alphaherpesvirus 1 MF576272 ; Psittacid alphaherpesvirus 1 AY372243; Psittacid alphaherpesvirus 2 AY623124; Psittacid alphaherpesvirus 3 JX028240; Saimiriine gammaherpesvirus 2 AJ410493; Spheniscid alphaherpesvirus 1 KJ720217; Spheniscid alphaherpesvirus 2 LT608135; Suid alphaherpesvirus 1 BK001744; Suid betaherpesvirus 2 AF268042. Percentage support from 100 bootstrap replicates is indicated at major branch points. The scale bar represents amino acid substitutions/site.
Figure Legend Snippet: Unrooted maximum likelihood phylogenetic tree for the family Herpesviridae . Generated from a ClustalW2 alignment of amino acid translations of partial DNA polymerase gene sequences from 40 representative herpesviruses retrieved from GenBank from the three subfamilies: Alphaherpesvirinae (α), Betaherpesvirinae (β) and Gammaherpesvirinae (γ) from a range of host species, and including the two novel alphaherpesviruses detected in this study (highlighted in bold) [ 60 ]. The GenBank accession numbers for sequences used are as follows: Accipitrid alphaherpesvirus 1 AY571851; Alcelaphine gammaherpesvirus 1 AF005370; Anatid alphaherpesvirus 1 EF643560; Bovine alphaherpesvirus 1 X94677; Bovine alphaherpesvirus 2 AF181249; Bovine gammaherpesvirus 4 AF031811; Bovine gammaherpesvirus 6 AF031808; Cacatuid alphaherpesvirus 1 MF576271 ; Columbid alphaherpesvirus 1 AF141890; Elephantid betaherpesvirus 1 AF322977; Elephantid gammaherpesvirus 3 DQ238845; Equid alphaherpesvirus 1 KF434378; Equid gammaherpesvirus 2 NC001650; Equid alphaherpesvirus 4 KT324743; Felid alphaherpesvirus 1 KR296657; Fregatid alphaherpesvirus 1 EU867220; Gallid alphaherpesvirus 1 NC006623; Gallid alphaherpesvirus 2 AF147806; Gallid alphaherpesvirus 3 HQ840738; Gaviid alphaherpesvirus 1 GU130289; Human alphaherpesvirus 1 HQ123098; Human alphaherpesvirus 3 X04370; Human betaherpesvirus 5 NC006273; Human betaherpesvirus 6 X83413; Macacine gammaherpesvirus 5 AF029302; Macropodid alphaherpesvirus 1 NC029132; Macropodid gammaherpesvirus 3 EF467663; Meleagrid alphaherpesvirus 1 AF291866; Murid betaherpesvirus 2 AY728086; Passerid alphaherpesvirus 1 AF520812; Phascolarctid gammaherpesvirus 2 JQ996387; Phocid alphaherpesvirus 1 PHU92269; Phoenicopterid alphaherpesvirus 1 KP244360; Podargid alphaherpesvirus 1 MF576272 ; Psittacid alphaherpesvirus 1 AY372243; Psittacid alphaherpesvirus 2 AY623124; Psittacid alphaherpesvirus 3 JX028240; Saimiriine gammaherpesvirus 2 AJ410493; Spheniscid alphaherpesvirus 1 KJ720217; Spheniscid alphaherpesvirus 2 LT608135; Suid alphaherpesvirus 1 BK001744; Suid betaherpesvirus 2 AF268042. Percentage support from 100 bootstrap replicates is indicated at major branch points. The scale bar represents amino acid substitutions/site.

Techniques Used: Generated

3) Product Images from "Paramecium bursaria Chlorella Virus 1 Proteome Reveals Novel Architectural and Regulatory Features of a Giant Virus"

Article Title: Paramecium bursaria Chlorella Virus 1 Proteome Reveals Novel Architectural and Regulatory Features of a Giant Virus

Journal: Journal of Virology

doi: 10.1128/JVI.00907-12

Expression stage distribution of PBCV-1 CDSs as a quartile analysis. (A) Number of all coding CDSs expressed either during the early, early-late, or late stage or not determined shown as a function of the genome map position. The genome map is divided into four regions, both direct (R genes) and reverse (L genes) on each half of the genome (left-half gene numbers, 001 to 327; right-half gene numbers, 328 to 692). (B) Distribution of virion-associated CDSs with respect to expression stage and genome position.
Figure Legend Snippet: Expression stage distribution of PBCV-1 CDSs as a quartile analysis. (A) Number of all coding CDSs expressed either during the early, early-late, or late stage or not determined shown as a function of the genome map position. The genome map is divided into four regions, both direct (R genes) and reverse (L genes) on each half of the genome (left-half gene numbers, 001 to 327; right-half gene numbers, 328 to 692). (B) Distribution of virion-associated CDSs with respect to expression stage and genome position.

Techniques Used: Expressing

Proteomic methodologies for PBCV-1 virions.
Figure Legend Snippet: Proteomic methodologies for PBCV-1 virions.

Techniques Used:

Mass-versus-pI distribution of PBCV-1 virion CDSs identified by two independent proteomic methods. The virion proteins are displayed as a function of their intrinsic molecular masses and isoelectric points. The results of each method are shown. Note that method 2 was especially useful for discovering a set of low-molecular-mass proteins that were not detected by method 1.
Figure Legend Snippet: Mass-versus-pI distribution of PBCV-1 virion CDSs identified by two independent proteomic methods. The virion proteins are displayed as a function of their intrinsic molecular masses and isoelectric points. The results of each method are shown. Note that method 2 was especially useful for discovering a set of low-molecular-mass proteins that were not detected by method 1.

Techniques Used:

4) Product Images from "Adenosine A1 receptor, a target and regulator of ER? action, mediates the proliferative effects of estradiol in breast cancer"

Article Title: Adenosine A1 receptor, a target and regulator of ER? action, mediates the proliferative effects of estradiol in breast cancer

Journal: Oncogene

doi: 10.1038/onc.2009.409

Inhibition of Adora1 results in significantly decreased endogenous ERα in MCF-7 cells. MCF-7 cells were transiently transfected with an Adora1-targeted siRNA or a control siRNA construct; (A) mRNA levels of Adora1 and ERα were measured by real-time PCR; (B) protein levels of Adora1 and ERα were measured in immunoblot analyses using the indicated antibodies; and (C) MCF-7 cells were treated with vehicle or DPCPX at the concentration of 10 3 µM and 10 4 µM for 12 h, protein levels of ERα were measured in immunoblot analyses using ERα antibody. Data are the average of 3 replicates ± SD. *, p
Figure Legend Snippet: Inhibition of Adora1 results in significantly decreased endogenous ERα in MCF-7 cells. MCF-7 cells were transiently transfected with an Adora1-targeted siRNA or a control siRNA construct; (A) mRNA levels of Adora1 and ERα were measured by real-time PCR; (B) protein levels of Adora1 and ERα were measured in immunoblot analyses using the indicated antibodies; and (C) MCF-7 cells were treated with vehicle or DPCPX at the concentration of 10 3 µM and 10 4 µM for 12 h, protein levels of ERα were measured in immunoblot analyses using ERα antibody. Data are the average of 3 replicates ± SD. *, p

Techniques Used: Inhibition, Transfection, Construct, Real-time Polymerase Chain Reaction, Concentration Assay

Silencing of Adora1 in MCF-7 cells leads to reduced binding of ERα to TFF1 promoter, TFF1 promoter driven luferase activity, and mRNA expression of TFF1. (A) Knock-down of Adora1 results in decreased binding of ERa to the TFF1 promoter. B) siRNA knock-down of Adora1 expression decreases ERα-stimulated transcriptional activation of the TFF1 promoter. MCF-7 cells were co-transfected with a TFF1-Luc reporter or pGL4 vector (200ng), pCMVβGal (80ng), and either control or Adora1 siRNA (100 nM) in the presence or absence of E2 (10 −8 M) overnight. C) Adora1 silencing results in significantly decreased TFF1 expression. MCF-7 cells were transfected either with control or Adora1 siRNA (100 nM) and treated with vehicle or E2 (10 −8 M) overnight. Cells were then lysed, total RNA was extracted, and mRNA levels of Adora1 were measured by real-time PCR and normalized to GAPDH. Data represent the average of 3 replicates ± SD (*, p
Figure Legend Snippet: Silencing of Adora1 in MCF-7 cells leads to reduced binding of ERα to TFF1 promoter, TFF1 promoter driven luferase activity, and mRNA expression of TFF1. (A) Knock-down of Adora1 results in decreased binding of ERa to the TFF1 promoter. B) siRNA knock-down of Adora1 expression decreases ERα-stimulated transcriptional activation of the TFF1 promoter. MCF-7 cells were co-transfected with a TFF1-Luc reporter or pGL4 vector (200ng), pCMVβGal (80ng), and either control or Adora1 siRNA (100 nM) in the presence or absence of E2 (10 −8 M) overnight. C) Adora1 silencing results in significantly decreased TFF1 expression. MCF-7 cells were transfected either with control or Adora1 siRNA (100 nM) and treated with vehicle or E2 (10 −8 M) overnight. Cells were then lysed, total RNA was extracted, and mRNA levels of Adora1 were measured by real-time PCR and normalized to GAPDH. Data represent the average of 3 replicates ± SD (*, p

Techniques Used: Binding Assay, Activity Assay, Expressing, Activation Assay, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction

5) Product Images from "First Report on Fusarium Wilt of Zucchini Caused by Fusarium oxysporum, in Korea"

Article Title: First Report on Fusarium Wilt of Zucchini Caused by Fusarium oxysporum, in Korea

Journal: Mycobiology

doi: 10.5941/MYCO.2015.43.2.174

Phylogenetic analysis using the neighbor-joining method comparing the sequence of translation elongation factor 1α of Fusarium oxysporum with that of other Fusarium spp. obtained from GenBank. The numbers above the branches represent the bootstrap value. The fungal strains identified in this study are shown in boldface.
Figure Legend Snippet: Phylogenetic analysis using the neighbor-joining method comparing the sequence of translation elongation factor 1α of Fusarium oxysporum with that of other Fusarium spp. obtained from GenBank. The numbers above the branches represent the bootstrap value. The fungal strains identified in this study are shown in boldface.

Techniques Used: Sequencing

Phylogenetic analysis by the neighbor-joining method comparing the sequence of the internal transcribed spacer ribosomal DNA (rDNA) region from Fusarium oxysporum with that of other Fusarium spp. obtained from GenBank. The numbers above the branches represent the bootstrap value. The fungal strains identified in this study are shown in boldface.
Figure Legend Snippet: Phylogenetic analysis by the neighbor-joining method comparing the sequence of the internal transcribed spacer ribosomal DNA (rDNA) region from Fusarium oxysporum with that of other Fusarium spp. obtained from GenBank. The numbers above the branches represent the bootstrap value. The fungal strains identified in this study are shown in boldface.

Techniques Used: Sequencing

6) Product Images from "A Tale of Two Oxidation States: Bacterial Colonization of Arsenic-Rich Environments"

Article Title: A Tale of Two Oxidation States: Bacterial Colonization of Arsenic-Rich Environments

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.0030053

Phylogenetic Tree of ArsCa Arsenate Reductases in Various Arsenic-Resistant Microorganisms The proteins of loci 1 (HEAR3302), 2 (HEAR0500), and 4 (HEAR3207) in cluster with reductases present in acr3 -type transporter operons. In H. arsenicoxydans, two of them are, however, associated with an ArsB-type transporter . Protein sequences involved in arsenate reduction were retrieved from the National Center for Biotechnology Information GenBank database ( http://www.ncbi.nlm.nih.gov/entrez ) and phylogenetic trees were reconstructed from multiple sequence alignments using the neighbor-joining algorithm implemented in ClustalX. The following sequences were used as references: Roseovarius nubinhibens, Xanthobacter autotrophicus, Rhodospirillum rubrum, Bradyrhizobium japonicum, Rhodopseudomonas palustris, Nitrobacter winogradskyi, Nitrobacter hamburgensis, Xanthobacter autotrophicus, Acidovorax sp., Comamonas sp., Rubrivivax gelatinosus, Delftia acidovorans, Azotobacter vinelandii, Rubrivivax gelatinosus, Burkholderia multivorans, Ralstonia metallidurans, Shigella flexneri, Shigella flexneri, Polaromonas naphthalenivorans, Comamonas testosteroni, Burkholderia vietnamiensis, Burkholderia pseudomallei, Burkholderia mallei, Azoarcus sp., Methylobacillus flagellatus, Alcaligenes faecalis, Rhodoferax ferrireducens, Pseudomonas syringae, Pseudomonas putida, Pseudomonas aeruginosa, Shewanella oneidensis, Wolinella succinogenes, Corynebacterium efficiens, Corynebacterium efficiens, Alkalilimnicola ehrlichei, and Chlorobium phaeobacteroides .
Figure Legend Snippet: Phylogenetic Tree of ArsCa Arsenate Reductases in Various Arsenic-Resistant Microorganisms The proteins of loci 1 (HEAR3302), 2 (HEAR0500), and 4 (HEAR3207) in cluster with reductases present in acr3 -type transporter operons. In H. arsenicoxydans, two of them are, however, associated with an ArsB-type transporter . Protein sequences involved in arsenate reduction were retrieved from the National Center for Biotechnology Information GenBank database ( http://www.ncbi.nlm.nih.gov/entrez ) and phylogenetic trees were reconstructed from multiple sequence alignments using the neighbor-joining algorithm implemented in ClustalX. The following sequences were used as references: Roseovarius nubinhibens, Xanthobacter autotrophicus, Rhodospirillum rubrum, Bradyrhizobium japonicum, Rhodopseudomonas palustris, Nitrobacter winogradskyi, Nitrobacter hamburgensis, Xanthobacter autotrophicus, Acidovorax sp., Comamonas sp., Rubrivivax gelatinosus, Delftia acidovorans, Azotobacter vinelandii, Rubrivivax gelatinosus, Burkholderia multivorans, Ralstonia metallidurans, Shigella flexneri, Shigella flexneri, Polaromonas naphthalenivorans, Comamonas testosteroni, Burkholderia vietnamiensis, Burkholderia pseudomallei, Burkholderia mallei, Azoarcus sp., Methylobacillus flagellatus, Alcaligenes faecalis, Rhodoferax ferrireducens, Pseudomonas syringae, Pseudomonas putida, Pseudomonas aeruginosa, Shewanella oneidensis, Wolinella succinogenes, Corynebacterium efficiens, Corynebacterium efficiens, Alkalilimnicola ehrlichei, and Chlorobium phaeobacteroides .

Techniques Used: Sequencing

Organization of the aox Gene Cluster in H. arsenicoxydans and Various Arsenic-Metabolizing Microorganisms The aoxAB operon is close to arsenic-resistance genes in H. arsenicoxydans , A. faecalis, X. autotrophicus, N. hamburgensis, and C. phaeobacteroides. In the first three bacteria, these genes are associated with an aoxRS two-component regulatory system. In H. arsenicoxydans, the CDS number of aoxABCD, aoxRS, and arsRCBCH are hear 0479–0476, hear 0483–0482, and hear 0499–0503, respectively. Sequence information of other genes was obtained from GenBank database and their localization on the chromosome or the plasmid is given by nucleotide numbering. The following bacterial genomes were used: Alcaligenes faecalis , Agrobacterium tumefaciens , Rhodoferax ferrireducens , Burkholderia multivorans , Xanthobacter autotrophicus , Roseovarius sp217, Nitrobacter hamburgensis , Chlorobium phaerobacteroides , Chloroflexus aurentiacus , Thermus thermophilus HB8, Aeropyrum pernix , Sulfolobus tokodai , Environmental sample 1, and Environmental sample 2.
Figure Legend Snippet: Organization of the aox Gene Cluster in H. arsenicoxydans and Various Arsenic-Metabolizing Microorganisms The aoxAB operon is close to arsenic-resistance genes in H. arsenicoxydans , A. faecalis, X. autotrophicus, N. hamburgensis, and C. phaeobacteroides. In the first three bacteria, these genes are associated with an aoxRS two-component regulatory system. In H. arsenicoxydans, the CDS number of aoxABCD, aoxRS, and arsRCBCH are hear 0479–0476, hear 0483–0482, and hear 0499–0503, respectively. Sequence information of other genes was obtained from GenBank database and their localization on the chromosome or the plasmid is given by nucleotide numbering. The following bacterial genomes were used: Alcaligenes faecalis , Agrobacterium tumefaciens , Rhodoferax ferrireducens , Burkholderia multivorans , Xanthobacter autotrophicus , Roseovarius sp217, Nitrobacter hamburgensis , Chlorobium phaerobacteroides , Chloroflexus aurentiacus , Thermus thermophilus HB8, Aeropyrum pernix , Sulfolobus tokodai , Environmental sample 1, and Environmental sample 2.

Techniques Used: Sequencing, Plasmid Preparation, Environmental Sampling

7) Product Images from "A New Thermophilic Nitrilase from an Antarctic Hyperthermophilic Microorganism"

Article Title: A New Thermophilic Nitrilase from an Antarctic Hyperthermophilic Microorganism

Journal: Frontiers in Bioengineering and Biotechnology

doi: 10.3389/fbioe.2016.00005

Analysis of the amino acid sequence alignment of the nitrilase gene of M24D13 microorganism against GenBank, PDB, SwissProt, PIR, and PRF data bases using Clustal W software . The red marked columns indicate the amino acids that belong to the catalytic triad of the known nitrilase enzymes (E-K-C). The blue marked column corresponds to the consensus sequence present at the N-terminal for nitrilases. The green marked lane corresponds to the sequence of NitM24D13.
Figure Legend Snippet: Analysis of the amino acid sequence alignment of the nitrilase gene of M24D13 microorganism against GenBank, PDB, SwissProt, PIR, and PRF data bases using Clustal W software . The red marked columns indicate the amino acids that belong to the catalytic triad of the known nitrilase enzymes (E-K-C). The blue marked column corresponds to the consensus sequence present at the N-terminal for nitrilases. The green marked lane corresponds to the sequence of NitM24D13.

Techniques Used: Sequencing, Software

Phylogenetic tree of the 16S rRNA gene sequence . The sequence obtained from the isolated microorganism M24D13 was compared with date base sequences available in GenBank to the Archaea domain, using Pyrodictium occultum as outgroup. The phylogenetic tree was made by Neighbor-joining method with 1000 bootstrap replicates.
Figure Legend Snippet: Phylogenetic tree of the 16S rRNA gene sequence . The sequence obtained from the isolated microorganism M24D13 was compared with date base sequences available in GenBank to the Archaea domain, using Pyrodictium occultum as outgroup. The phylogenetic tree was made by Neighbor-joining method with 1000 bootstrap replicates.

Techniques Used: Sequencing, Isolation

8) Product Images from "Identification and Characterization of Pseudocercospora pyricola Causing Leaf Spots on Aronia melanocarpa"

Article Title: Identification and Characterization of Pseudocercospora pyricola Causing Leaf Spots on Aronia melanocarpa

Journal: Mycobiology

doi: 10.5941/MYCO.2017.45.1.39

Neighbor-joining tree of Pseudocercospora pyricola based on the multigene dataset of translation elongation factor 1-alpha (EF-1α), actin (ACT), internal transcribed spacer (ITS), and large subunit ribosomal DNA (LSU). In the phylogenetic tree, the numbers (bootstrap values) are above or under the nodes obtained from 1,000 replicates. The GenBank numbers are represented in the order of EF-1α, ACT, ITS, and LSU, respectively. The isolate obtained in this study is highlighted in bold.
Figure Legend Snippet: Neighbor-joining tree of Pseudocercospora pyricola based on the multigene dataset of translation elongation factor 1-alpha (EF-1α), actin (ACT), internal transcribed spacer (ITS), and large subunit ribosomal DNA (LSU). In the phylogenetic tree, the numbers (bootstrap values) are above or under the nodes obtained from 1,000 replicates. The GenBank numbers are represented in the order of EF-1α, ACT, ITS, and LSU, respectively. The isolate obtained in this study is highlighted in bold.

Techniques Used: Activated Clotting Time Assay

9) Product Images from "Identification and Characterization of Cercospora malayensis Causing Leaf Spot on Kenaf"

Article Title: Identification and Characterization of Cercospora malayensis Causing Leaf Spot on Kenaf

Journal: Mycobiology

doi: 10.5941/MYCO.2017.45.2.114

Neighbor-joining tree of Cercospora malayensis based on the multigene dataset (internal transcribed spacer [ITS], translation elongation factor 1-alpha [EF-1α], actin [ACT], calmodulin [CAL], and histone 3 [HIS]). The numbers above the nodes are the bootstrap values obtained from 1,000 replicates. The GenBank numbers are represented in order of ITS, EF-1α, ACT, CAL, and HIS, respectively. The isolate obtained in this study is shown in boldface.
Figure Legend Snippet: Neighbor-joining tree of Cercospora malayensis based on the multigene dataset (internal transcribed spacer [ITS], translation elongation factor 1-alpha [EF-1α], actin [ACT], calmodulin [CAL], and histone 3 [HIS]). The numbers above the nodes are the bootstrap values obtained from 1,000 replicates. The GenBank numbers are represented in order of ITS, EF-1α, ACT, CAL, and HIS, respectively. The isolate obtained in this study is shown in boldface.

Techniques Used: Activated Clotting Time Assay

10) Product Images from "Genotype diversity and molecular evolution of noroviruses: A 30-year (1982-2011) comprehensive study with children from Northern Brazil"

Article Title: Genotype diversity and molecular evolution of noroviruses: A 30-year (1982-2011) comprehensive study with children from Northern Brazil

Journal: PLoS ONE

doi: 10.1371/journal.pone.0178909

Maximum likelihood phylogenetic tree based on the P2 region of 83 partial genome sequences from noroviruses of different GII.4 variants detected in infected children during various collection periods over 30 years (1982–2011) in Belém, Brazil. Asterisks in the tree represent bootstrap values greater than 70% with 1000 replicates. Groupings of samples from the present study are in red. A dendrogram was constructed using model test GTR+G4. Variant reference strains used in the analyses were submitted to the GenBank database under the accession numbers CHDC_1970s (JX023286), Tokyo_1980s (AB684720), US_95/96 (DQ078829), Bristol_1993 (X86557), Kaiso_2003 (AB294779), Asia_2003 (AJ844476), Hunter_2004 (HM802544), Yerseke_2006a (EF126963), Den_Haag_2006b “O” (EF126965), Den_Haag_2006b “Y” (JX975571), and New_Orleans_2009 (GU445325).
Figure Legend Snippet: Maximum likelihood phylogenetic tree based on the P2 region of 83 partial genome sequences from noroviruses of different GII.4 variants detected in infected children during various collection periods over 30 years (1982–2011) in Belém, Brazil. Asterisks in the tree represent bootstrap values greater than 70% with 1000 replicates. Groupings of samples from the present study are in red. A dendrogram was constructed using model test GTR+G4. Variant reference strains used in the analyses were submitted to the GenBank database under the accession numbers CHDC_1970s (JX023286), Tokyo_1980s (AB684720), US_95/96 (DQ078829), Bristol_1993 (X86557), Kaiso_2003 (AB294779), Asia_2003 (AJ844476), Hunter_2004 (HM802544), Yerseke_2006a (EF126963), Den_Haag_2006b “O” (EF126965), Den_Haag_2006b “Y” (JX975571), and New_Orleans_2009 (GU445325).

Techniques Used: Infection, Construct, Variant Assay

11) Product Images from "High prevalence of norovirus in children with sporadic acute gastroenteritis in Manaus, Amazon Region, northern Brazil"

Article Title: High prevalence of norovirus in children with sporadic acute gastroenteritis in Manaus, Amazon Region, northern Brazil

Journal: Memórias do Instituto Oswaldo Cruz

doi: 10.1590/0074-02760160357

: phylogenetic analyses of norovirus GII sequences obtained from children with diarrhoea from Manaus, Brazil, between January 2010 and December 2011, based on a 253 bp region within the capsid. (A) Phylogenetic tree of norovirus GII genotypes; (B) phylogenetic tree of GII.4 variants. References strains of NoV genotypes are named according to GenBank with their respective accession numbers. Brazilian strains are marked with a filled diamond. The scale bar at the bottom of the tree indicates distance. Bootstrap values (2000 replicates) are shown at the branch nodes and values lower than 60% are not shown. The codes representing the positive samples are in bold and are organised as follows: study area (Amazonas)/sample code/country of collection (Brazil)/month-year of collection.
Figure Legend Snippet: : phylogenetic analyses of norovirus GII sequences obtained from children with diarrhoea from Manaus, Brazil, between January 2010 and December 2011, based on a 253 bp region within the capsid. (A) Phylogenetic tree of norovirus GII genotypes; (B) phylogenetic tree of GII.4 variants. References strains of NoV genotypes are named according to GenBank with their respective accession numbers. Brazilian strains are marked with a filled diamond. The scale bar at the bottom of the tree indicates distance. Bootstrap values (2000 replicates) are shown at the branch nodes and values lower than 60% are not shown. The codes representing the positive samples are in bold and are organised as follows: study area (Amazonas)/sample code/country of collection (Brazil)/month-year of collection.

Techniques Used:

12) Product Images from "Characterization of a Septobasidium sp. Associated with Felt Disease of Schisandra chinensis"

Article Title: Characterization of a Septobasidium sp. Associated with Felt Disease of Schisandra chinensis

Journal: Mycobiology

doi: 10.5941/MYCO.2016.44.1.58

A neighbor-joining tree based on the internal transcribed spacer rDNA sequences of Septobasidium sp. from Schisandra chinensis , including Septobasidium spp. retrieved from GenBank. Numbers above the branches represent bootstrap values. Isolates obtained in this study are shown in boldface.
Figure Legend Snippet: A neighbor-joining tree based on the internal transcribed spacer rDNA sequences of Septobasidium sp. from Schisandra chinensis , including Septobasidium spp. retrieved from GenBank. Numbers above the branches represent bootstrap values. Isolates obtained in this study are shown in boldface.

Techniques Used:

13) Product Images from "An Efficient PCR-RFLP Method for the Rapid Identification of Korean Pyropia Species"

Article Title: An Efficient PCR-RFLP Method for the Rapid Identification of Korean Pyropia Species

Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

doi: 10.3390/molecules22122182

Sequence alignment of rbcL ( A ) and rps11 – trnG ( B ) regions from 15 Pyropia species obtained from GenBank (National Center for Biotechnology Information (NCBI), Rockville, MD, USA). The bases highlighted in yellow correspond to the Tth 111I, Ava II, Bsr I, Bsa AI, Hind III, Sac II, and Sph I enzyme restriction sites.
Figure Legend Snippet: Sequence alignment of rbcL ( A ) and rps11 – trnG ( B ) regions from 15 Pyropia species obtained from GenBank (National Center for Biotechnology Information (NCBI), Rockville, MD, USA). The bases highlighted in yellow correspond to the Tth 111I, Ava II, Bsr I, Bsa AI, Hind III, Sac II, and Sph I enzyme restriction sites.

Techniques Used: Sequencing, Antiviral Assay

14) Product Images from "FALDO: a semantic standard for describing the location of nucleotide and protein feature annotation"

Article Title: FALDO: a semantic standard for describing the location of nucleotide and protein feature annotation

Journal: Journal of Biomedical Semantics

doi: 10.1186/s13326-016-0067-z

DDBJ record associated with UniProt Q6Q250 showing the related CDS sequence, with coding region outside of the known deposited mRNA sequence
Figure Legend Snippet: DDBJ record associated with UniProt Q6Q250 showing the related CDS sequence, with coding region outside of the known deposited mRNA sequence

Techniques Used: Sequencing

15) Product Images from "Diversity and dynamics of bacterial communities in early life stages of the Caribbean coral Porites astreoides"

Article Title: Diversity and dynamics of bacterial communities in early life stages of the Caribbean coral Porites astreoides

Journal: The ISME Journal

doi: 10.1038/ismej.2011.144

Results of MRPP ordination for bacterial community composition in P. astreoides larvae, based on 16S rRNA gene T-RFLP profiles. Ordination plots of T-RFLP profiles, according to the ( a ) developmental stage, ( b ) collection year and ( c ) collection location.
Figure Legend Snippet: Results of MRPP ordination for bacterial community composition in P. astreoides larvae, based on 16S rRNA gene T-RFLP profiles. Ordination plots of T-RFLP profiles, according to the ( a ) developmental stage, ( b ) collection year and ( c ) collection location.

Techniques Used:

16) Product Images from "Intracrine Regulation of Estrogen and Other Sex Steroid Levels in Endometrium and Non-gynecological Tissues; Pathology, Physiology, and Drug Discovery"

Article Title: Intracrine Regulation of Estrogen and Other Sex Steroid Levels in Endometrium and Non-gynecological Tissues; Pathology, Physiology, and Drug Discovery

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2018.00940

Steroid structure. (A) structure of the C21 steroid progesterone (P, used as an example), with carbon numbering and steroid ring numbering. In the storied graphics in Figures 1B and 2 , the H groups and the relative bonds will be omitted (with the exclusion of the H in 5α-reduced steroids - androstanes and pregnanes). Methyl groups will be indicated by the bonds only without the CH 3 group. (B) structures of C21 pregnene (Δ 4 and Δ 5 , i.e., double bond between C4 and C5 or between C5 and C6, respectively), pregnane (5α-reduced steroid), C19 androstene (Δ 4 , Δ 5 ) and androstane and C18 (A-ring)-aromatic estrogens. Chemical structures were designed with the aid of Sketcher V2.4 (Ihlenfeldt et al., 2009 ), available online at PubChem ( www.ncbi.nlm.nih.gov ; pubchem.ncbi.nlm.nih.gov ) (Kim et al., 2016 ).
Figure Legend Snippet: Steroid structure. (A) structure of the C21 steroid progesterone (P, used as an example), with carbon numbering and steroid ring numbering. In the storied graphics in Figures 1B and 2 , the H groups and the relative bonds will be omitted (with the exclusion of the H in 5α-reduced steroids - androstanes and pregnanes). Methyl groups will be indicated by the bonds only without the CH 3 group. (B) structures of C21 pregnene (Δ 4 and Δ 5 , i.e., double bond between C4 and C5 or between C5 and C6, respectively), pregnane (5α-reduced steroid), C19 androstene (Δ 4 , Δ 5 ) and androstane and C18 (A-ring)-aromatic estrogens. Chemical structures were designed with the aid of Sketcher V2.4 (Ihlenfeldt et al., 2009 ), available online at PubChem ( www.ncbi.nlm.nih.gov ; pubchem.ncbi.nlm.nih.gov ) (Kim et al., 2016 ).

Techniques Used:

17) Product Images from "Detection of Viable Mycobacterium ulcerans in Clinical Samples by a Novel Combined 16S rRNA Reverse Transcriptase/IS2404 Real-Time qPCR Assay"

Article Title: Detection of Viable Mycobacterium ulcerans in Clinical Samples by a Novel Combined 16S rRNA Reverse Transcriptase/IS2404 Real-Time qPCR Assay

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0001756

Standard curve and limit of detection of the 16S rRNA RT-qPCR. Figure 3 shows Ct-values of clinical samples plotted versus quantified 16S rRNA copy numbers. Standards for the 16S rRNA RT-qPCR were generated by conventional PCR amplification ( Table 5 ). Log 10 fold serial dilutions ( n = 5) were prepared ranging from 3E+6 to 300 copies of the 16S rRNA gene (PCR template: 2 µl) and were subjected to the assay in quadruplicate to generate a calibration curve. The regression line was y = −3.4x+41.68 with a coefficient of correlation > 0.99 and the efficiency was E = 0.97. M. ulcerans whole genome extracts were quantified by means of IS 2404 qPCR and the analytical sensitivity was determined as limit of detection (LOD) by subjecting 10 aliquots of a dilution series containing 30, 15, 10, 8, 6, 3, or 2 copies of the 16S rRNA gene to the assay. The LOD was 6 copies of the target sequence. The copy number ( n = 1) of the 16S rRNA gene per M. ulcerans genome was determined by copy number variation assay (unpublished data).
Figure Legend Snippet: Standard curve and limit of detection of the 16S rRNA RT-qPCR. Figure 3 shows Ct-values of clinical samples plotted versus quantified 16S rRNA copy numbers. Standards for the 16S rRNA RT-qPCR were generated by conventional PCR amplification ( Table 5 ). Log 10 fold serial dilutions ( n = 5) were prepared ranging from 3E+6 to 300 copies of the 16S rRNA gene (PCR template: 2 µl) and were subjected to the assay in quadruplicate to generate a calibration curve. The regression line was y = −3.4x+41.68 with a coefficient of correlation > 0.99 and the efficiency was E = 0.97. M. ulcerans whole genome extracts were quantified by means of IS 2404 qPCR and the analytical sensitivity was determined as limit of detection (LOD) by subjecting 10 aliquots of a dilution series containing 30, 15, 10, 8, 6, 3, or 2 copies of the 16S rRNA gene to the assay. The LOD was 6 copies of the target sequence. The copy number ( n = 1) of the 16S rRNA gene per M. ulcerans genome was determined by copy number variation assay (unpublished data).

Techniques Used: Quantitative RT-PCR, Generated, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction, Sequencing

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Co-Immunoprecipitation Assay:

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Article Snippet: .. Following are the column headings: accession number (protein ID on NCBI protein database or TrichDB), protein name, molecular weight of the protein, sequence coverage (percentage coverage of the peptide sequence to the full length protein sequence), peptides (number of peptides identified for a particular protein), unique peptides (number of unique peptides identified for a particular protein), score from the MS identification, intensity of the MS, MS/MS count. (A–D) Intensity from four independent IP experiments in binary logarithmic values; mean: arithmetic mean of intensity from four independent (A–D) IP experiments in binary logarithmic values; n: difference between mean of the test and the control samples; and fold change: actual change in the protein levels between the test and the control samples. coIP, co-immunoprecipitation; HA, human influenza hemagglutinin; LFQ-MS, label-free quantitative mass spectrometry; NCBI, National Center for Biotechnology Information; Sam, sorting and assembly machinery; TOM, translocase of the outer membrane; TrichDB, Trichomonas Genome Resource. (XLSX) (145K, xlsx) ..

Article Title:
Article Snippet: .. Following are the column headings: accession number (protein ID on NCBI protein database or TrichDB), protein name, molecular weight of the protein, sequence coverage (percentage coverage of the peptide sequence to the full length protein sequence), peptides (number of peptides identified for a particular protein), unique peptides (number of unique peptides identified for a particular protein), score from the MS identification, intensity of the MS, MS/MS count. (A–D) Intensity from four independent IP experiments in binary logarithmic values; mean: arithmetic mean of intensity from four independent (A–D) IP experiments in binary logarithmic values; n: difference between mean of the test and the control samples; and fold change: actual change in the protein levels between the test and the control samples. coIP, co-immunoprecipitation; HA, human influenza hemagglutinin; LFQ-MS, label-free quantitative mass spectrometry; NCBI, National Center for Biotechnology Information; Sam, sorting and assembly machinery; TOM, translocase of the outer membrane; TrichDB, Trichomonas Genome Resource. (XLSX) Click here for additional data file. ..

Amplification:

Article Title:
Article Snippet: .. The amplified fragment containing the partial sequence of PaPPCK that showed high sequence similarity to PPCK was identified by using a blastx algorithm in a BLAST search against the NCBI database (National Center for Biotechnology Information, GenBank). .. The internal gene-specific primers were designed from the partial sequence of PaPPCK for 5′ and 3′ rapid amplified cDNA ends (RACE) using SMARTTM RACE cDNA amplification kit (Clontech, Palo Alto, CA, United States).

Mass Spectrometry:

Article Title:
Article Snippet: .. Following are the column headings: accession number (protein ID on NCBI protein database or TrichDB), protein name, molecular weight of the protein, sequence coverage (percentage coverage of the peptide sequence to the full length protein sequence), peptides (number of peptides identified for a particular protein), unique peptides (number of unique peptides identified for a particular protein), score from the MS identification, intensity of the MS, MS/MS count. (A–D) Intensity from four independent IP experiments in binary logarithmic values; mean: arithmetic mean of intensity from four independent (A–D) IP experiments in binary logarithmic values; n: difference between mean of the test and the control samples; and fold change: actual change in the protein levels between the test and the control samples. coIP, co-immunoprecipitation; HA, human influenza hemagglutinin; LFQ-MS, label-free quantitative mass spectrometry; NCBI, National Center for Biotechnology Information; Sam, sorting and assembly machinery; TOM, translocase of the outer membrane; TrichDB, Trichomonas Genome Resource. (XLSX) (145K, xlsx) ..

Article Title:
Article Snippet: .. Abbreviations ABySS: Assembly by short sequences software; bcftools: Binary call format tools; CDS: Coding sequences; H: Housekeeping proteins; MS: Mass spectrometry; MS/MS: Tandem MS; MW: Molecular weight; NCBI: National Center for Biotechnology Information; NGS: Next-generation sequencing; NISC: NIH Intramural Sequencing Center; S: Secreted proteins; samtools: Sequence alignment/map tools; SG: Salivary gland; SMase: Sphingomyelin phosphodiesterase; SNP: Single-nucleotide polymorphism; SRA: Sequence read archives; U: Proteins of unknown function; WRP: W-rich protein. ..

Article Title:
Article Snippet: .. Following are the column headings: accession number (protein ID on NCBI protein database or TrichDB), protein name, molecular weight of the protein, sequence coverage (percentage coverage of the peptide sequence to the full length protein sequence), peptides (number of peptides identified for a particular protein), unique peptides (number of unique peptides identified for a particular protein), score from the MS identification, intensity of the MS, MS/MS count. (A–D) Intensity from four independent IP experiments in binary logarithmic values; mean: arithmetic mean of intensity from four independent (A–D) IP experiments in binary logarithmic values; n: difference between mean of the test and the control samples; and fold change: actual change in the protein levels between the test and the control samples. coIP, co-immunoprecipitation; HA, human influenza hemagglutinin; LFQ-MS, label-free quantitative mass spectrometry; NCBI, National Center for Biotechnology Information; Sam, sorting and assembly machinery; TOM, translocase of the outer membrane; TrichDB, Trichomonas Genome Resource. (XLSX) Click here for additional data file. ..

Synthesized:

Article Title:
Article Snippet: The synthesized cDNA fragments and the PaPPCK degenerate primers designed from the conserved amino acid sequences in the expressed sequence tag (EST) library of Phalaenopsis (FC Chen, unpublished) were used in PCR experiments. .. The amplified fragment containing the partial sequence of PaPPCK that showed high sequence similarity to PPCK was identified by using a blastx algorithm in a BLAST search against the NCBI database (National Center for Biotechnology Information, GenBank).

Isolation:

Article Title:
Article Snippet: Paragraph title: Supporting information Comparison of the conserved β-motif of TvTom40-like proteins (TvTom40-1-7) with Tom40s and VDACs of other eukaryotes. Conservation of TOM complex-forming residues. Sequence alignment of Tom22-like protein from T . vaginalis against Tom22 from other eukaryotes. Expression of His-tagged Tom36cd and Tom46cd in E . coli BL21 (DE3) strains. EM analysis of the isolated TvTOM complex. Enlarged version of the phylogenetic tree shown in . HHpred search with each TvTom40 homologue against the NCBI conserved domains database (version 3.16) and S . cerevisiae proteome. Pairwise comparison of HMM profiles for the seven TvTom40 homologues against PDB database using the HHpred tool. TOM subunit orthologues identified in selected eukaryotic lineages. List of oligonucleotides. A list of 24 well-annotated Tom40 sequences that were used to build Tom40 HMM. A data set of proteins identified from TvTom40-2-HA, Tom36-HA, and Sam50-HA coIPs both under crosslinking and native conditions using LFQ-MS analysis. Protein sequences of the TOM subunit orthologues listed in . A set of 1,114 proteins with their coordinates used for CLANS that were obtained from two iterations of PSI–BLAST with Tom36 and ATOM69 as queries. An alignment of 418 TPR proteins from CLANS that were selected for the phylogenetic analysis. A list of 447 Tom22 sequences that were used to build Tom22 HMM. A list of 349 Tom7 sequences that were used to build Tom7 HMM. Sequence alignments for TOM subunits that were used to identify orthologues in different eukaryotic lineages. A list of Tom40 and VDAC sequences that were used for TvTom40-2 modelling. ... Following are the column headings: accession number (protein ID on NCBI protein database or TrichDB), protein name, molecular weight of the protein, sequence coverage (percentage coverage of the peptide sequence to the full length protein sequence), peptides (number of peptides identified for a particular protein), unique peptides (number of unique peptides identified for a particular protein), score from the MS identification, intensity of the MS, MS/MS count. (A–D) Intensity from four independent IP experiments in binary logarithmic values; mean: arithmetic mean of intensity from four independent (A–D) IP experiments in binary logarithmic values; n: difference between mean of the test and the control samples; and fold change: actual change in the protein levels between the test and the control samples. coIP, co-immunoprecipitation; HA, human influenza hemagglutinin; LFQ-MS, label-free quantitative mass spectrometry; NCBI, National Center for Biotechnology Information; Sam, sorting and assembly machinery; TOM, translocase of the outer membrane; TrichDB, Trichomonas Genome Resource. (XLSX) Click here for additional data file.

Next-Generation Sequencing:

Article Title:
Article Snippet: .. Abbreviations ABySS: Assembly by short sequences software; bcftools: Binary call format tools; CDS: Coding sequences; H: Housekeeping proteins; MS: Mass spectrometry; MS/MS: Tandem MS; MW: Molecular weight; NCBI: National Center for Biotechnology Information; NGS: Next-generation sequencing; NISC: NIH Intramural Sequencing Center; S: Secreted proteins; samtools: Sequence alignment/map tools; SG: Salivary gland; SMase: Sphingomyelin phosphodiesterase; SNP: Single-nucleotide polymorphism; SRA: Sequence read archives; U: Proteins of unknown function; WRP: W-rich protein. ..

Sequencing:

Article Title:
Article Snippet: .. Following are the column headings: accession number (protein ID on NCBI protein database or TrichDB), protein name, molecular weight of the protein, sequence coverage (percentage coverage of the peptide sequence to the full length protein sequence), peptides (number of peptides identified for a particular protein), unique peptides (number of unique peptides identified for a particular protein), score from the MS identification, intensity of the MS, MS/MS count. (A–D) Intensity from four independent IP experiments in binary logarithmic values; mean: arithmetic mean of intensity from four independent (A–D) IP experiments in binary logarithmic values; n: difference between mean of the test and the control samples; and fold change: actual change in the protein levels between the test and the control samples. coIP, co-immunoprecipitation; HA, human influenza hemagglutinin; LFQ-MS, label-free quantitative mass spectrometry; NCBI, National Center for Biotechnology Information; Sam, sorting and assembly machinery; TOM, translocase of the outer membrane; TrichDB, Trichomonas Genome Resource. (XLSX) (145K, xlsx) ..

Article Title:
Article Snippet: .. Abbreviations ABySS: Assembly by short sequences software; bcftools: Binary call format tools; CDS: Coding sequences; H: Housekeeping proteins; MS: Mass spectrometry; MS/MS: Tandem MS; MW: Molecular weight; NCBI: National Center for Biotechnology Information; NGS: Next-generation sequencing; NISC: NIH Intramural Sequencing Center; S: Secreted proteins; samtools: Sequence alignment/map tools; SG: Salivary gland; SMase: Sphingomyelin phosphodiesterase; SNP: Single-nucleotide polymorphism; SRA: Sequence read archives; U: Proteins of unknown function; WRP: W-rich protein. ..

Article Title:
Article Snippet: .. The amplified fragment containing the partial sequence of PaPPCK that showed high sequence similarity to PPCK was identified by using a blastx algorithm in a BLAST search against the NCBI database (National Center for Biotechnology Information, GenBank). .. The internal gene-specific primers were designed from the partial sequence of PaPPCK for 5′ and 3′ rapid amplified cDNA ends (RACE) using SMARTTM RACE cDNA amplification kit (Clontech, Palo Alto, CA, United States).

Article Title:
Article Snippet: .. Following are the column headings: accession number (protein ID on NCBI protein database or TrichDB), protein name, molecular weight of the protein, sequence coverage (percentage coverage of the peptide sequence to the full length protein sequence), peptides (number of peptides identified for a particular protein), unique peptides (number of unique peptides identified for a particular protein), score from the MS identification, intensity of the MS, MS/MS count. (A–D) Intensity from four independent IP experiments in binary logarithmic values; mean: arithmetic mean of intensity from four independent (A–D) IP experiments in binary logarithmic values; n: difference between mean of the test and the control samples; and fold change: actual change in the protein levels between the test and the control samples. coIP, co-immunoprecipitation; HA, human influenza hemagglutinin; LFQ-MS, label-free quantitative mass spectrometry; NCBI, National Center for Biotechnology Information; Sam, sorting and assembly machinery; TOM, translocase of the outer membrane; TrichDB, Trichomonas Genome Resource. (XLSX) Click here for additional data file. ..

Polymerase Chain Reaction:

Article Title:
Article Snippet: The synthesized cDNA fragments and the PaPPCK degenerate primers designed from the conserved amino acid sequences in the expressed sequence tag (EST) library of Phalaenopsis (FC Chen, unpublished) were used in PCR experiments. .. The amplified fragment containing the partial sequence of PaPPCK that showed high sequence similarity to PPCK was identified by using a blastx algorithm in a BLAST search against the NCBI database (National Center for Biotechnology Information, GenBank).

other:

Article Title:
Article Snippet: Sequence information corresponding to genes or proteins identified in other genomes and presented in , , and 5, respectively, was obtained from the National Center for Biotechnology Information GenBank database ( http://www.ncbi.nlm.nih.gov/entrez ) under the following accession numbers: accession numbers: Azoarcus sp. EbN1 (NC_006513), Burkholderia xenovorans LB400 (NC_007951), Pseudomonas fluorescens Pf-5 (NC_004129), Ralstonia metallidurans CH34 (NC_007973), and Xanthomonas campestris 85–10 (NC_007508). accession numbers: Aeropyrum pernix (NC_000854), Agrobacterium tumefaciens (DQ151549), Alcaligenes faecalis (AY297781), Burkholderia multivorans (NZ_AAVB01000013), Chlorobium phaero-bacteroides (NZ_AAIC01000001), Chloroflexus aurantiacus (NZ_AAAH02000003), Environmental sample 1(AACY01058630), Environmental sample 2 (AACY01082423), Nitrobacter hamburgensis (NC_007960.1), Rhodoferax ferrireducens (NC_007908), Roseovarius sp217 (NZ_AAMV01000002.1), Sulfolobus tokodai (BA000023.2), Thermus thermophilus HB8 (NC_006462), and Xanthobacter autotrophicus (NZ_AAPC01000006). accession numbers: Acidovorax sp. (YP_987260.1), Alcaligenes faecalis (AAS45115.1), Alkalilimnicola ehrlichei (YP_743542.1), Azoarcus sp. (YP_159866.1), Azotobacter vinelandii (ZP_00416369.1), Bradyrhizobium japonicum (NP_769726.1), Burkholderia mallei (YP_105320.1), Burkholderia multivorans (ZP_01573333.1), Burkholderia pseudomallei (ZP_01333850.1), Burkholderia vietnamiensis (ZP_00422769.1), Chlorobium phaeobacteroides (YP_910947.1), Comamonas sp. (ABI49647.1), Comamonas testosteroni (ZP_01519258.1), Corynebacterium efficiens (NP_737487.1), Corynebacterium efficiens (NP_738111.1), Delftia acidovorans (ZP_01580151.1), Methylobacillus flagellatus (YP_545602.1), Nitrobacter hamburgensis (YP_571847.1), Nitrobacter winogradskyi (YP_319723.1), Polaromonas naphthalenivorans (YP_982812.1), Pseudomonas aeruginosa (NP_250969.1), Pseudomonas putida (NP_744860.1), Pseudomonas syringae (YP_234590.1), Ralstonia metallidurans (YP_582486.1), Rhodoferax ferrireducens (YP_524897.1), Rhodopseudomonas palustris (NP_948893.1), Rhodospirillum rubrum (YP_426538.1), Roseovarius nubinhibens (ZP_00960539.1), Rubrivivax gelatinosus (ZP_00242261.1), Rubrivivax gelatinosus (ZP_00243884.1), Shewanella oneidensis (NP_716169.1), Shigella flexneri (AAP17870.1), Shigella flexneri (NP_838060.1), Wolinella succinogenes (NP_906976.1), Xanthobacter autotrophicus (ZP_01198809.1), and Xanthobacter autotrophicus (ZP_01199778.1)

Article Title:
Article Snippet: Specific primers for human CCL18, CD204, and GapDH were designed using Primer3 software (Whitehead Institute for Biomedical Research, Cambridge, USA; http://www.broad.mit.edu/genome_ software/other/primer3.html), Amplify1.2 software (University of Wisconsin, USA; http://engels.genetics.wisc. edu/amplify) using LocusLink and GenBank databases (National Centre for Biotechnology Information; http://www.ncbi.nlm.nih.gov/ LocusLink/index.html).

Article Title:
Article Snippet: Phylogenetic analyses Sequences were identified (Additional file : Table S1) using a BLAST search against the NCBI sequence database (National Center for Biotechnology Information, GenBank) to find the closest sequence matches in the database.

Article Title:
Article Snippet: After analyze of nucleotide sequence (made as described below), the identity of the isolated was confirmed by comparison between the obtained sequence and the available GenBank sequences (National Center for Biotechnology Information, NCBI).

Article Title:
Article Snippet: The sRNA reads were annotated using the GenBank database (National Center for Biotechnology Information [NCBI], USA) and the Rfam RNA database (Sanger Institute, UK).

Article Title:
Article Snippet: Materials and Methods PubChem is a public molecular repository established by the National Center for Biotechnology Information (NCBI), as a division of the National Institutes of Health (NIH).

Article Title:
Article Snippet: Ancient Aboriginal mitochondrial data have been deposited in the GenBank database ( http://ncbi.nlm.nih.gov/genbank ), which is hosted by the National Center for Biotechnology Information, under accession numbers MK165665 to MK165690.

Article Title:
Article Snippet: ) using a BLAST search against the NCBI sequence database (National Center for Biotechnology Information, GenBank) to find the closest sequence matches in the database.

Article Title:
Article Snippet: Most of us in molecular biology use the excellent resources of NCBI (the US National Center for Biotechnology Information) on a daily basis, courtesy of the US taxpayer, and partner organizations.

Article Title:
Article Snippet: Primers and Probes Primers and a hydrolysis probe (TibMolBiol, Berlin, Germany) for specific amplification of M . ulcerans 16S rRNA were designed using DNAsis Max (MiraiBio, San Francisco, USA) by alignment of 16S rRNA gene sequences (GenBank, National Center for Biotechnology Information [NCBI]) from closely related mycobacteria and other bacteria potentially contaminating the human skin ( ).

Article Title:
Article Snippet: Each primer pair was verified for gene specificity using Nucleotide Basic Local Alignment Search Tool from the GenBank non-redundant nucleotide sequence database (National Centre for Biotechnology Information, 2009, http://www.ncbi.nlm.nih.gov/ ).

Article Title:
Article Snippet: The DNA sequence data for six6b - and six7 -expressing plasmids are available from the DNA Data Bank of Japan (DDBJ)/European Molecular Biology Laboratory (EMBL)/National Center for Biotechnology Information (NCBI) [accession nos.

Article Title:
Article Snippet: Though > 96 products were cloned and sequenced from each stage, many of the sequenced clones matched most closely to eukaryotic 18S rRNA or chloroplast 16S rRNA gene sequence (GenBank/NCBI(National Center for Biotechnology Information)).

Article Title:
Article Snippet: The obtained sequences (DWV—388 bp, SBV—417 bp, ABPV—435 bp and BQCV—486 bp) were deposited in the GenBank database National Biotechnology Information Center (NCBI) under accession numbers MG599458 – MG599464 and MG649495 – MG649502 .

Article Title:
Article Snippet: Related sequences were obtained from GenBank database [National Center for Biotechnology Information (NCBI), Bethesda, MD, USA] PDB, SwissProt, PIR, and PRF using the BLAST search program.

Article Title:
Article Snippet: Discussion and prototyping with representatives from major sequence databases such as UniProt [ ], DDBJ (DNA Data Bank of Japan) [ ] (part of the INSDC partnership with the National Center for Biotechnology Information (NCBI)-GenBank [ ] and European Molecular Biology Laboratory (EMBL)-Bank [ ]), and a number of glycomics databases (BCSDB [ ], GlycomeDB [ ], GLYCOSCIENCES.de [ ], JCGGDB, RINGS [ ] and UniCarbKB [ ]) and assorted open source developers during these meetings led to the development of the Feature Annotation Location Description Ontology (FALDO).

Article Title:
Article Snippet: Sequences identified in this study were submitted to the GenBank database [National Center for Biotechnology Information, US (www.ncbi.nlm.nih.gov)] under accession numbers: KX232361-KX232423.

Article Title:
Article Snippet: Collation of published sequences Genbank (the National Centre for Biotechnology Information (NCBI) ( http://www.ncbi.nlm.nih.gov/ )) was searched in various ways for NNV sequences.

Article Title:
Article Snippet: Using ‘Spirodela polyrhiza ’ as the search phrase, a total of 401 whole‐genome shotgun sequences were downloaded from the National Center for Biotechnology Information’s GenBank database ( http://www.ncbi.nlm.nih.gov/ ).

Article Title:
Article Snippet: Constructs 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).

Article Title:
Article Snippet: Hepatic mRNA expression level Hepatic RNA was isolated using the GF-TR-100 RNA Isolation Kit (Vivantis, Malaysia) according to the kit protocol, and primers were designed in the GenomeLabeXpress Profiler software using Rattus norvegicus sequence adopted from the National Center for Biotechnology Information GenBank Database ( http://www.ncbi.nlm.nih.gov/nucleotide/ ).

Article Title:
Article Snippet: To verify that these oligonucleotide sequences in the Adora1 siRNA pool specifically targeted Adora1 but not ERα mRNA, we aligned the four Adora1 siRNA oligonucleotide sequences with ERα mRNA (Locus number: NM_000125 from NCBI DNA database) by Blast alignment from National Center for Biotechnology Information (NCBI).

Article Title:
Article Snippet: For candidate proteins, amino acid sequences from the National Center for Biotechnology Information (NCBI) were used to calculate the theoretical pI , molecular weight, intramolecular disulfide bonds and number of aromatic amino acids ( ).

Article Title:
Article Snippet: The cloned products were sequenced and screened against the GenBank database (National Center for Biotechnology Information [NCBI]) to verify the identity and orientation of the DWNN fragment within the vector.

Article Title:
Article Snippet: The sequences of targeted genes were obtained from the National Center for Biotechnology Information GenBank sequence database ( https://www.ncbi.nlm.nih.gov/ ).

Article Title:
Article Snippet: Data Availability The following information was supplied regarding data availability: The obtained sequences (DWV - 388 bp, SBV - 417 bp, ABPV - 435 bp and BQCV - 486 bp) were deposited in the GenBank database National Biotechnology Information Center (NCBI) under accession numbers MG599458 – MG599464 and MG649495 – MG649502 .

Article Title:
Article Snippet: Availability of data and materials The Whole Genome Shotgun projects of 95 C. diphtheriae strains sequenced in this study have been deposited at DDBJ/EMBL/GenBank (National Center for Biotechnology Information, GenBank Database: http://www.ncbi.nlm.nih.gov/nuccore/ ) under the accession numbers LSVL00000000-LSYP00000000, LSZF00000000, LTAR00000000-LTAS00000000 and MSIH00000000- MSIR00000000 and are publicly available.

Article Title:
Article Snippet: Homology of obtained sequences was confirmed in NCBI GenBank database (National Center for Biotechnology Information, Bethesda, MD, http://www.ncbi.nlm.nih.gov/ ) using BLASTn and BLASTp algorithm [ ].

Article Title:
Article Snippet: The primers sequence for COX2, iNOS and housekeeping gene 18srRNA for human cell lines and rat were designed from the sequence list of GenBank database (National Centre for Biotechnology Information – NCBI) by using Beacon designer 8 software and then blasted against GenBank database sequences.

Article Title:
Article Snippet: Based on the partial sequences of rbcL and trnC –trnP for 15 Pyropia species available in the GenBank public database (National Center for Biotechnology Information (NCBI), Rockville, MD, USA), we found that six restriction enzymes were predicted to show species-specific RFLP patterns and could be used to identify the six Pyropia species using rbcL in P. yezoensis and rps11 –sdh3 in P. seriata , P. pseudolinearis , P. dentate , P. suborbiculata , and P. haitanensis .

Article Title:
Article Snippet: Annotation of putative UGT genes was carried using both the nucleotide and translated protein sequences in a BLAST comparison with the GenBank sequence database (National Center for Biotechnology Information, NCBI) and by similarity comparison to the UDPGT (UDP-glucuronosyl and UDP-glucosyltransferase) Pfam protein family (PF00201) .

Article Title:
Article Snippet: Mutations were detected using Clone Manager software (v. 8.0, Scientific & Educational Software, USA) by comparing the obtained sequences with the M. tuberculosis reference strain H37Rv sequences of respective loci, deposited in the GenBank database (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/ ).

Article Title:
Article Snippet: To conduct phylogenetic analysis of the sequences obtained, sequences for selected taxa based on recent phylogenetic studies of Lecanicillium ( , ) and Cordycipitaceae ( , , ) were downloaded from the National Center for Biotechnology Information GenBank database ( https://www.ncbi.nlm.nih.gov/genbank/ ).

Article Title:
Article Snippet: 2.7.2 Primer design Primers were designed on the GenomeLab Xpress Profiler software with Homo sapiens sequence from National Centre for Biotechnology Information GenBank Database ( http://www.ncbi.nlm.nih.gov/nucleotide/ ).

Article Title:
Article Snippet: This yielded five distinct nrITS sequences which were verified using a BLAST search against the NCBI sequence database (National Center for Biotechnology Information, GenBank).

Article Title:
Article Snippet: The possible identity of the isolates was established by comparing their ITS sequences with those in the GenBank database (National Center for Biotechnology Information [NCBI] US National Institute of Health, Bethesda, MD, USA; http://www.ncbi.nlm.nih.gov/BLAST ).

Article Title:
Article Snippet: Molecular Genetic Analysis The sequencing results were aligned to the National Center for Biotechnology Information (NCBI) human reference genome assembly (GRCh37/hg19), and PCR duplicates were removed using the SAM tools software package (version 0.1.16) [ ].

Article Title:
Article Snippet: GenBank sequences In addition to the 311 locations we physically sampled or were sampled for us, we also searched the National Center for Biotechnology Information's GenBank database http://www.ncbi.nlm.nih.gov/genbank/ for ND2 sequences of cutthroat trout.

Article Title:
Article Snippet: The sequences characterized in this study were submitted to the GenBank database (National Center for Biotechnology Information, USA-[ www.ncbi.nlm.nih.gov ]) and are provided in .

Article Title:
Article Snippet: Molecular Analysis We used National Center for Biotechnology Information (NCBI) accession numbers, including NG_031875.1, NM_001252127.1 and NP_001239056.1 for the number of AP4E1 genomic DNA (gDNA), mRNA and protein sequences, respectively.

Article Title:
Article Snippet: The sequences were checked for chimeras using Bellerophon (Huber et al., ) and then compared to the GenBank nucleotide database [National Center for Biotechnology Information, (NCBI)] using BLAST.

Article Title:
Article Snippet: The resulting sequences were aligned against reference sequences downloaded from the National Center for Biotechnology Information GenBank database ( https://www.ncbi.nlm.nih.gov/ ) using the ClustalX 2.1 program to determine the assemblages of G. duodenalis in each specimen.

Article Title:
Article Snippet: The identity of the isolates was established by comparing their ITS and TEF sequences with those in the GenBank database (National Center for Biotechnology Information [NCBI] US National Institute of Health, Bethesda, MD, USA; http://www.ncbi.nlm.nih.gov/BLAST ) and FUSARIUM ID ( http://isolate.fusariumdb.org/blast.php ), respectively.

Article Title:
Article Snippet: Sequence data We obtained all full-length haemagglutinin (HA) segment nucleotide sequences of avian-origin H9N2 from Asian countries and regions that were available in the GenBank Influenza Virus Database ( http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html ) hosted by the National Center for Biotechnology Information (NCBI).

Article Title:
Article Snippet: We also would like to thank the management and curator of the online databases we made use of, namely: the database of chemical molecules PubChem ( www.ncbi.nlm.nih.gov ; pubchem.ncbi.nlm.nih.gov ) maintained by the National Center for Biotechnology Information (NCBI; National Library of Medicine/United States National Institutes of Health - NIH); Chemical Abstracts Service (CAS), maintained by the American Chemical Society ( www.cas.org ); Human Metabolome Data Base (HMDB, www.hmdb.ca ), funded and maintained by Genome Canada; Chemical Book ( www.chemicalbook.com ), funded by industrial partners; Chemical Entities of Biological Interest (ChEBI; www.ebi.ac.uk/chebi/init.do ), curated by the European Bioinformatics Institute of the European Molecular Biology Laboratory (EMBL); drug and drug target database Drugbank ( www.drugbank.ca/drugs ), University of Alberta and The Metabolomics Innovation Centre; GeneCards ( www.genecards.org ), developed and maintained by the Crown Human Genome Center at the Weizmann Institute of Science; Online Mendelian Inheritance in Man® (OMIM®, https://omim.org/ ), McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, MD); Mouse Genome Informatics (MGI; www.informatics.jax.org ), Mouse Genome Database at the Mouse Genome Informatics website, The Jackson Laboratory, Bar Harbor, Maine.

Article Title:
Article Snippet: Data accessibility The full 16S rDNA sequences of the genetically analyzed strains are available at the National Center for Biotechnology Information GenBank database ( http://www.ncbi.nlm.nih.gov/genbank/ ) under the following accession numbers: KM052277, KM052278, KM052279, KM052280, KM052281, KM052282, KM052283, KM052284, KM052285, KM052286, KM052287, KM052288, KM052289, KM052290, KM052291, KM052292, KM052293, KM052294, KM052295, KM052296, KM052297, KM052298, KM052299, KM052300, KM052301, KM052302, KM052303, KM052304, KM052305, KM052306, KM052307, KM052308, KM052309, KM052310, KM052311, KM052312, KM052313, KM052314, KM052315, KM052316, and KM052317.

Article Title:
Article Snippet: Availability of supporting data The Whole Genome Shotgun projects for C. diphtheriae strains ISS 3319, ISS 4060, ISS 4746 and ISS 4749 have been deposited at DDBJ/EMBL/GenBank (National Center for Biotechnology Information, GenBank Database: http://www.ncbi.nlm.nih.gov/nuccore/ ) under the accession numbers JAQO00000000, JAQN00000000, JAQP00000000 and JAQQ00000000, respectively and are publicly available.

Article Title:
Article Snippet: Molecular cloning of the human Endothelial and Smooth muscle Derived Neuropilin like (DCBLD2/ESDN/CLCP1) promoter The upstream regulatory region of the DCBLD2/ESDN/CLCP1 (discoidin, CUB and LCCL domain containing 2/Endothelial and Smooth muscle cell Derived Neuropin-like molecule/CUB, LCCL-homology, coagulation factor V/VIII homology domains protein) gene was identified using the National Center for Biotechnology Information (NCBI) gene bank database (accession number NM_080927).

Article Title:
Article Snippet: Nucleotide Sequence Accession Numbers The nucleotide sequences of the rpsL , rrs , and gidB genes containing novel mutations were deposited in the GenBank database (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/ ) under the following accession numbers: KF740612, KF740621, and KF740622 for the rpsL gene mutants, KF796660 to KF796662, and KF796665 for the rrs gene mutants, and KF740589, KF740590, KF740598 to KF740601, KF740603 to KF740605, KF740607, KF740609, KF740611, KF796668, and KF796669 for the gidB gene mutants.

Article Title:
Article Snippet: The genetic identity of each strain was determined by comparison with reference strains available in GenBank database (US National Center for Biotechnology Information, NCBI).

Article Title:
Article Snippet: The identified N-terminal amino acid sequence of purified rhPA was compared with the theoretical sequence data retrieved from the GenBank database ( http://www.ncbi.nlm.nih.gov/genbank/ ) of the National Center for Biotechnology Information.

Article Title:
Article Snippet: In addition, as previously noted, the hypothetical accessory protein did not exhibit significant similarity to any known FV proteins [ ] or indeed any molecular sequences in the National Center for Biotechnology Information (NCBI) non-redundant (nr) nucleotide collection database.

Article Title:
Article Snippet: Nucleotide sequences were compared with publicly available sequences in the GenBank® database (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/genbank/ ) using the NCBI Nucleotide Basic Local Alignment Search Tool (BLASTN® ) online algorithm ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ), and ClustalW2 alignments with their closest matches were generated within Geneious R9 to determine nucleotide identities for the region of available sequence.

Article Title:
Article Snippet: Inferences on the Structure of the Enzyme In order to evaluate the potential novelty of the enzyme structure, the primary protein sequence was compared using the BlastP algorithm [ ] to the non-redundant Genbank protein database (National Center for Biotechnology Information—NCBI).

Expressing:

Article Title:
Article Snippet: Paragraph title: Supporting information Comparison of the conserved β-motif of TvTom40-like proteins (TvTom40-1-7) with Tom40s and VDACs of other eukaryotes. Conservation of TOM complex-forming residues. Sequence alignment of Tom22-like protein from T . vaginalis against Tom22 from other eukaryotes. Expression of His-tagged Tom36cd and Tom46cd in E . coli BL21 (DE3) strains. EM analysis of the isolated TvTOM complex. Enlarged version of the phylogenetic tree shown in . HHpred search with each TvTom40 homologue against the NCBI conserved domains database (version 3.16) and S . cerevisiae proteome. Pairwise comparison of HMM profiles for the seven TvTom40 homologues against PDB database using the HHpred tool. TOM subunit orthologues identified in selected eukaryotic lineages. List of oligonucleotides. A list of 24 well-annotated Tom40 sequences that were used to build Tom40 HMM. A data set of proteins identified from TvTom40-2-HA, Tom36-HA, and Sam50-HA coIPs both under crosslinking and native conditions using LFQ-MS analysis. Protein sequences of the TOM subunit orthologues listed in . A set of 1,114 proteins with their coordinates used for CLANS that were obtained from two iterations of PSI–BLAST with Tom36 and ATOM69 as queries. An alignment of 418 TPR proteins from CLANS that were selected for the phylogenetic analysis. A list of 447 Tom22 sequences that were used to build Tom22 HMM. A list of 349 Tom7 sequences that were used to build Tom7 HMM. Sequence alignments for TOM subunits that were used to identify orthologues in different eukaryotic lineages. A list of Tom40 and VDAC sequences that were used for TvTom40-2 modelling. ... Following are the column headings: accession number (protein ID on NCBI protein database or TrichDB), protein name, molecular weight of the protein, sequence coverage (percentage coverage of the peptide sequence to the full length protein sequence), peptides (number of peptides identified for a particular protein), unique peptides (number of unique peptides identified for a particular protein), score from the MS identification, intensity of the MS, MS/MS count. (A–D) Intensity from four independent IP experiments in binary logarithmic values; mean: arithmetic mean of intensity from four independent (A–D) IP experiments in binary logarithmic values; n: difference between mean of the test and the control samples; and fold change: actual change in the protein levels between the test and the control samples. coIP, co-immunoprecipitation; HA, human influenza hemagglutinin; LFQ-MS, label-free quantitative mass spectrometry; NCBI, National Center for Biotechnology Information; Sam, sorting and assembly machinery; TOM, translocase of the outer membrane; TrichDB, Trichomonas Genome Resource. (XLSX) Click here for additional data file.

Tandem Mass Spectroscopy:

Article Title:
Article Snippet: .. Following are the column headings: accession number (protein ID on NCBI protein database or TrichDB), protein name, molecular weight of the protein, sequence coverage (percentage coverage of the peptide sequence to the full length protein sequence), peptides (number of peptides identified for a particular protein), unique peptides (number of unique peptides identified for a particular protein), score from the MS identification, intensity of the MS, MS/MS count. (A–D) Intensity from four independent IP experiments in binary logarithmic values; mean: arithmetic mean of intensity from four independent (A–D) IP experiments in binary logarithmic values; n: difference between mean of the test and the control samples; and fold change: actual change in the protein levels between the test and the control samples. coIP, co-immunoprecipitation; HA, human influenza hemagglutinin; LFQ-MS, label-free quantitative mass spectrometry; NCBI, National Center for Biotechnology Information; Sam, sorting and assembly machinery; TOM, translocase of the outer membrane; TrichDB, Trichomonas Genome Resource. (XLSX) (145K, xlsx) ..

Article Title:
Article Snippet: .. Abbreviations ABySS: Assembly by short sequences software; bcftools: Binary call format tools; CDS: Coding sequences; H: Housekeeping proteins; MS: Mass spectrometry; MS/MS: Tandem MS; MW: Molecular weight; NCBI: National Center for Biotechnology Information; NGS: Next-generation sequencing; NISC: NIH Intramural Sequencing Center; S: Secreted proteins; samtools: Sequence alignment/map tools; SG: Salivary gland; SMase: Sphingomyelin phosphodiesterase; SNP: Single-nucleotide polymorphism; SRA: Sequence read archives; U: Proteins of unknown function; WRP: W-rich protein. ..

Article Title:
Article Snippet: .. Following are the column headings: accession number (protein ID on NCBI protein database or TrichDB), protein name, molecular weight of the protein, sequence coverage (percentage coverage of the peptide sequence to the full length protein sequence), peptides (number of peptides identified for a particular protein), unique peptides (number of unique peptides identified for a particular protein), score from the MS identification, intensity of the MS, MS/MS count. (A–D) Intensity from four independent IP experiments in binary logarithmic values; mean: arithmetic mean of intensity from four independent (A–D) IP experiments in binary logarithmic values; n: difference between mean of the test and the control samples; and fold change: actual change in the protein levels between the test and the control samples. coIP, co-immunoprecipitation; HA, human influenza hemagglutinin; LFQ-MS, label-free quantitative mass spectrometry; NCBI, National Center for Biotechnology Information; Sam, sorting and assembly machinery; TOM, translocase of the outer membrane; TrichDB, Trichomonas Genome Resource. (XLSX) Click here for additional data file. ..

Modification:

Article Title:
Article Snippet: Cloning of cDNAs of PaPPCK of P. aphrodite subsp. formosana Total RNA was extracted from the leaves of seedlings at stage 5 using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and modified as . .. The amplified fragment containing the partial sequence of PaPPCK that showed high sequence similarity to PPCK was identified by using a blastx algorithm in a BLAST search against the NCBI database (National Center for Biotechnology Information, GenBank).

Molecular Weight:

Article Title:
Article Snippet: .. Following are the column headings: accession number (protein ID on NCBI protein database or TrichDB), protein name, molecular weight of the protein, sequence coverage (percentage coverage of the peptide sequence to the full length protein sequence), peptides (number of peptides identified for a particular protein), unique peptides (number of unique peptides identified for a particular protein), score from the MS identification, intensity of the MS, MS/MS count. (A–D) Intensity from four independent IP experiments in binary logarithmic values; mean: arithmetic mean of intensity from four independent (A–D) IP experiments in binary logarithmic values; n: difference between mean of the test and the control samples; and fold change: actual change in the protein levels between the test and the control samples. coIP, co-immunoprecipitation; HA, human influenza hemagglutinin; LFQ-MS, label-free quantitative mass spectrometry; NCBI, National Center for Biotechnology Information; Sam, sorting and assembly machinery; TOM, translocase of the outer membrane; TrichDB, Trichomonas Genome Resource. (XLSX) (145K, xlsx) ..

Article Title:
Article Snippet: .. Abbreviations ABySS: Assembly by short sequences software; bcftools: Binary call format tools; CDS: Coding sequences; H: Housekeeping proteins; MS: Mass spectrometry; MS/MS: Tandem MS; MW: Molecular weight; NCBI: National Center for Biotechnology Information; NGS: Next-generation sequencing; NISC: NIH Intramural Sequencing Center; S: Secreted proteins; samtools: Sequence alignment/map tools; SG: Salivary gland; SMase: Sphingomyelin phosphodiesterase; SNP: Single-nucleotide polymorphism; SRA: Sequence read archives; U: Proteins of unknown function; WRP: W-rich protein. ..

Article Title:
Article Snippet: .. Following are the column headings: accession number (protein ID on NCBI protein database or TrichDB), protein name, molecular weight of the protein, sequence coverage (percentage coverage of the peptide sequence to the full length protein sequence), peptides (number of peptides identified for a particular protein), unique peptides (number of unique peptides identified for a particular protein), score from the MS identification, intensity of the MS, MS/MS count. (A–D) Intensity from four independent IP experiments in binary logarithmic values; mean: arithmetic mean of intensity from four independent (A–D) IP experiments in binary logarithmic values; n: difference between mean of the test and the control samples; and fold change: actual change in the protein levels between the test and the control samples. coIP, co-immunoprecipitation; HA, human influenza hemagglutinin; LFQ-MS, label-free quantitative mass spectrometry; NCBI, National Center for Biotechnology Information; Sam, sorting and assembly machinery; TOM, translocase of the outer membrane; TrichDB, Trichomonas Genome Resource. (XLSX) Click here for additional data file. ..

Software:

Article Title:
Article Snippet: .. Abbreviations ABySS: Assembly by short sequences software; bcftools: Binary call format tools; CDS: Coding sequences; H: Housekeeping proteins; MS: Mass spectrometry; MS/MS: Tandem MS; MW: Molecular weight; NCBI: National Center for Biotechnology Information; NGS: Next-generation sequencing; NISC: NIH Intramural Sequencing Center; S: Secreted proteins; samtools: Sequence alignment/map tools; SG: Salivary gland; SMase: Sphingomyelin phosphodiesterase; SNP: Single-nucleotide polymorphism; SRA: Sequence read archives; U: Proteins of unknown function; WRP: W-rich protein. ..

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    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.
    Dysferlin Construct, supplied by Biotechnology Information, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biotechnology Information genbank database
    PhyML maximum likelihood phylogenetic tree of avian alphaherpesviruses. Generated from a ClustalW2 alignment [ 59 ] of the partial DNA polymerase gene sequences of Podargid alphaherpesvirus 1 and Cacatuid alphaherpesvirus 1 with published avian alphaherpesvirus nucleotide sequences available in <t>GenBank,</t> with the two novel alphaherpesviruses detected in this study highlighted in bold [ 60 ]. The GenBank accession numbers for sequences used are included in the tip labels, and Macropodid alphaherpesvirus 1 (highlighted in italics) is included as an outgroup. Branching with greater than 50% support from 100 bootstrap replicates is indicated at major node points. The distances indicated by black horizontal lines correspond to genetic distances, with the scale bar representing nucleotide substitutions/site.
    Genbank Database, supplied by Biotechnology Information, used in various techniques. Bioz Stars score: 99/100, based on 509 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biotechnology Information pbcv 1 gene annotations
    Expression stage distribution of <t>PBCV-1</t> CDSs as a quartile analysis. (A) Number of all coding CDSs expressed either during the early, early-late, or late stage or not determined shown as a function of the genome map position. The genome map is divided into four regions, both direct (R genes) and reverse (L genes) on each half of the genome (left-half gene numbers, 001 to 327; right-half gene numbers, 328 to 692). (B) Distribution of virion-associated CDSs with respect to expression stage and genome position.
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    Biotechnology Information erα mrna
    Inhibition of Adora1 results in significantly decreased endogenous <t>ERα</t> in MCF-7 cells. MCF-7 cells were transiently transfected with an Adora1-targeted siRNA or a control siRNA construct; (A) <t>mRNA</t> levels of Adora1 and ERα were measured by real-time PCR; (B) protein levels of Adora1 and ERα were measured in immunoblot analyses using the indicated antibodies; and (C) MCF-7 cells were treated with vehicle or DPCPX at the concentration of 10 3 µM and 10 4 µM for 12 h, protein levels of ERα were measured in immunoblot analyses using ERα antibody. Data are the average of 3 replicates ± SD. *, p
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    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: Constructs 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: Constructs 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: Constructs 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: Constructs 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: Constructs 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: Constructs 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: Constructs 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

    PhyML maximum likelihood phylogenetic tree of avian alphaherpesviruses. Generated from a ClustalW2 alignment [ 59 ] of the partial DNA polymerase gene sequences of Podargid alphaherpesvirus 1 and Cacatuid alphaherpesvirus 1 with published avian alphaherpesvirus nucleotide sequences available in GenBank, with the two novel alphaherpesviruses detected in this study highlighted in bold [ 60 ]. The GenBank accession numbers for sequences used are included in the tip labels, and Macropodid alphaherpesvirus 1 (highlighted in italics) is included as an outgroup. Branching with greater than 50% support from 100 bootstrap replicates is indicated at major node points. The distances indicated by black horizontal lines correspond to genetic distances, with the scale bar representing nucleotide substitutions/site.

    Journal: PLoS ONE

    Article Title: Avian viral surveillance in Victoria, Australia, and detection of two novel avian herpesviruses

    doi: 10.1371/journal.pone.0194457

    Figure Lengend Snippet: PhyML maximum likelihood phylogenetic tree of avian alphaherpesviruses. Generated from a ClustalW2 alignment [ 59 ] of the partial DNA polymerase gene sequences of Podargid alphaherpesvirus 1 and Cacatuid alphaherpesvirus 1 with published avian alphaherpesvirus nucleotide sequences available in GenBank, with the two novel alphaherpesviruses detected in this study highlighted in bold [ 60 ]. The GenBank accession numbers for sequences used are included in the tip labels, and Macropodid alphaherpesvirus 1 (highlighted in italics) is included as an outgroup. Branching with greater than 50% support from 100 bootstrap replicates is indicated at major node points. The distances indicated by black horizontal lines correspond to genetic distances, with the scale bar representing nucleotide substitutions/site.

    Article Snippet: Nucleotide sequences were compared with publicly available sequences in the GenBank® database (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/genbank/ ) using the NCBI Nucleotide Basic Local Alignment Search Tool (BLASTN® ) online algorithm ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ), and ClustalW2 alignments with their closest matches were generated within Geneious R9 to determine nucleotide identities for the region of available sequence.

    Techniques: Generated

    Phylogenetic tree for the genus avulvirus of the family Paramyxoviridae based on partial RNA-dependent RNA polymerase gene sequences. Maximum likelihood phylogenetic tree constructed using PhyML from a ClustalW2 alignment of the partial RNA-dependent RNA polymerase (large polymerase or L protein) gene sequences of the avian avulavirus detected from a musk lorikeet ( Glossopsitta concinna ) in this study (unclassified avian avulavirus strain musk lorikeet/Melbourne/ML22-141263/2014; highlighted in bold) and published paramyxovirus sequences retrieved from GenBank [ 60 ]. The GenBank accession numbers for sequences used are indicated in brackets in the tip labels. Human rubulavirus 2 (highlighted in italics) is included as an outgroup. Branching with greater than 50% support from 100 bootstrap replicates is indicated at major node points. The distances indicated by black horizontal lines correspond to genetic distances, with the scale bar representing nucleotide substitutions/site.

    Journal: PLoS ONE

    Article Title: Avian viral surveillance in Victoria, Australia, and detection of two novel avian herpesviruses

    doi: 10.1371/journal.pone.0194457

    Figure Lengend Snippet: Phylogenetic tree for the genus avulvirus of the family Paramyxoviridae based on partial RNA-dependent RNA polymerase gene sequences. Maximum likelihood phylogenetic tree constructed using PhyML from a ClustalW2 alignment of the partial RNA-dependent RNA polymerase (large polymerase or L protein) gene sequences of the avian avulavirus detected from a musk lorikeet ( Glossopsitta concinna ) in this study (unclassified avian avulavirus strain musk lorikeet/Melbourne/ML22-141263/2014; highlighted in bold) and published paramyxovirus sequences retrieved from GenBank [ 60 ]. The GenBank accession numbers for sequences used are indicated in brackets in the tip labels. Human rubulavirus 2 (highlighted in italics) is included as an outgroup. Branching with greater than 50% support from 100 bootstrap replicates is indicated at major node points. The distances indicated by black horizontal lines correspond to genetic distances, with the scale bar representing nucleotide substitutions/site.

    Article Snippet: Nucleotide sequences were compared with publicly available sequences in the GenBank® database (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/genbank/ ) using the NCBI Nucleotide Basic Local Alignment Search Tool (BLASTN® ) online algorithm ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ), and ClustalW2 alignments with their closest matches were generated within Geneious R9 to determine nucleotide identities for the region of available sequence.

    Techniques: Construct

    Unrooted maximum likelihood phylogenetic tree for the family Herpesviridae . Generated from a ClustalW2 alignment of amino acid translations of partial DNA polymerase gene sequences from 40 representative herpesviruses retrieved from GenBank from the three subfamilies: Alphaherpesvirinae (α), Betaherpesvirinae (β) and Gammaherpesvirinae (γ) from a range of host species, and including the two novel alphaherpesviruses detected in this study (highlighted in bold) [ 60 ]. The GenBank accession numbers for sequences used are as follows: Accipitrid alphaherpesvirus 1 AY571851; Alcelaphine gammaherpesvirus 1 AF005370; Anatid alphaherpesvirus 1 EF643560; Bovine alphaherpesvirus 1 X94677; Bovine alphaherpesvirus 2 AF181249; Bovine gammaherpesvirus 4 AF031811; Bovine gammaherpesvirus 6 AF031808; Cacatuid alphaherpesvirus 1 MF576271 ; Columbid alphaherpesvirus 1 AF141890; Elephantid betaherpesvirus 1 AF322977; Elephantid gammaherpesvirus 3 DQ238845; Equid alphaherpesvirus 1 KF434378; Equid gammaherpesvirus 2 NC001650; Equid alphaherpesvirus 4 KT324743; Felid alphaherpesvirus 1 KR296657; Fregatid alphaherpesvirus 1 EU867220; Gallid alphaherpesvirus 1 NC006623; Gallid alphaherpesvirus 2 AF147806; Gallid alphaherpesvirus 3 HQ840738; Gaviid alphaherpesvirus 1 GU130289; Human alphaherpesvirus 1 HQ123098; Human alphaherpesvirus 3 X04370; Human betaherpesvirus 5 NC006273; Human betaherpesvirus 6 X83413; Macacine gammaherpesvirus 5 AF029302; Macropodid alphaherpesvirus 1 NC029132; Macropodid gammaherpesvirus 3 EF467663; Meleagrid alphaherpesvirus 1 AF291866; Murid betaherpesvirus 2 AY728086; Passerid alphaherpesvirus 1 AF520812; Phascolarctid gammaherpesvirus 2 JQ996387; Phocid alphaherpesvirus 1 PHU92269; Phoenicopterid alphaherpesvirus 1 KP244360; Podargid alphaherpesvirus 1 MF576272 ; Psittacid alphaherpesvirus 1 AY372243; Psittacid alphaherpesvirus 2 AY623124; Psittacid alphaherpesvirus 3 JX028240; Saimiriine gammaherpesvirus 2 AJ410493; Spheniscid alphaherpesvirus 1 KJ720217; Spheniscid alphaherpesvirus 2 LT608135; Suid alphaherpesvirus 1 BK001744; Suid betaherpesvirus 2 AF268042. Percentage support from 100 bootstrap replicates is indicated at major branch points. The scale bar represents amino acid substitutions/site.

    Journal: PLoS ONE

    Article Title: Avian viral surveillance in Victoria, Australia, and detection of two novel avian herpesviruses

    doi: 10.1371/journal.pone.0194457

    Figure Lengend Snippet: Unrooted maximum likelihood phylogenetic tree for the family Herpesviridae . Generated from a ClustalW2 alignment of amino acid translations of partial DNA polymerase gene sequences from 40 representative herpesviruses retrieved from GenBank from the three subfamilies: Alphaherpesvirinae (α), Betaherpesvirinae (β) and Gammaherpesvirinae (γ) from a range of host species, and including the two novel alphaherpesviruses detected in this study (highlighted in bold) [ 60 ]. The GenBank accession numbers for sequences used are as follows: Accipitrid alphaherpesvirus 1 AY571851; Alcelaphine gammaherpesvirus 1 AF005370; Anatid alphaherpesvirus 1 EF643560; Bovine alphaherpesvirus 1 X94677; Bovine alphaherpesvirus 2 AF181249; Bovine gammaherpesvirus 4 AF031811; Bovine gammaherpesvirus 6 AF031808; Cacatuid alphaherpesvirus 1 MF576271 ; Columbid alphaherpesvirus 1 AF141890; Elephantid betaherpesvirus 1 AF322977; Elephantid gammaherpesvirus 3 DQ238845; Equid alphaherpesvirus 1 KF434378; Equid gammaherpesvirus 2 NC001650; Equid alphaherpesvirus 4 KT324743; Felid alphaherpesvirus 1 KR296657; Fregatid alphaherpesvirus 1 EU867220; Gallid alphaherpesvirus 1 NC006623; Gallid alphaherpesvirus 2 AF147806; Gallid alphaherpesvirus 3 HQ840738; Gaviid alphaherpesvirus 1 GU130289; Human alphaherpesvirus 1 HQ123098; Human alphaherpesvirus 3 X04370; Human betaherpesvirus 5 NC006273; Human betaherpesvirus 6 X83413; Macacine gammaherpesvirus 5 AF029302; Macropodid alphaherpesvirus 1 NC029132; Macropodid gammaherpesvirus 3 EF467663; Meleagrid alphaherpesvirus 1 AF291866; Murid betaherpesvirus 2 AY728086; Passerid alphaherpesvirus 1 AF520812; Phascolarctid gammaherpesvirus 2 JQ996387; Phocid alphaherpesvirus 1 PHU92269; Phoenicopterid alphaherpesvirus 1 KP244360; Podargid alphaherpesvirus 1 MF576272 ; Psittacid alphaherpesvirus 1 AY372243; Psittacid alphaherpesvirus 2 AY623124; Psittacid alphaherpesvirus 3 JX028240; Saimiriine gammaherpesvirus 2 AJ410493; Spheniscid alphaherpesvirus 1 KJ720217; Spheniscid alphaherpesvirus 2 LT608135; Suid alphaherpesvirus 1 BK001744; Suid betaherpesvirus 2 AF268042. Percentage support from 100 bootstrap replicates is indicated at major branch points. The scale bar represents amino acid substitutions/site.

    Article Snippet: Nucleotide sequences were compared with publicly available sequences in the GenBank® database (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/genbank/ ) using the NCBI Nucleotide Basic Local Alignment Search Tool (BLASTN® ) online algorithm ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ), and ClustalW2 alignments with their closest matches were generated within Geneious R9 to determine nucleotide identities for the region of available sequence.

    Techniques: Generated

    Expression stage distribution of PBCV-1 CDSs as a quartile analysis. (A) Number of all coding CDSs expressed either during the early, early-late, or late stage or not determined shown as a function of the genome map position. The genome map is divided into four regions, both direct (R genes) and reverse (L genes) on each half of the genome (left-half gene numbers, 001 to 327; right-half gene numbers, 328 to 692). (B) Distribution of virion-associated CDSs with respect to expression stage and genome position.

    Journal: Journal of Virology

    Article Title: Paramecium bursaria Chlorella Virus 1 Proteome Reveals Novel Architectural and Regulatory Features of a Giant Virus

    doi: 10.1128/JVI.00907-12

    Figure Lengend Snippet: Expression stage distribution of PBCV-1 CDSs as a quartile analysis. (A) Number of all coding CDSs expressed either during the early, early-late, or late stage or not determined shown as a function of the genome map position. The genome map is divided into four regions, both direct (R genes) and reverse (L genes) on each half of the genome (left-half gene numbers, 001 to 327; right-half gene numbers, 328 to 692). (B) Distribution of virion-associated CDSs with respect to expression stage and genome position.

    Article Snippet: Preliminary proteomic analyses using the existing PBCV-1 gene annotations (National Center for Biotechnology Information [NCBI] Refseq, ) revealed possible errors in the genome sequence, which prompted us to resequence the PBCV-1 genome.

    Techniques: Expressing

    Proteomic methodologies for PBCV-1 virions.

    Journal: Journal of Virology

    Article Title: Paramecium bursaria Chlorella Virus 1 Proteome Reveals Novel Architectural and Regulatory Features of a Giant Virus

    doi: 10.1128/JVI.00907-12

    Figure Lengend Snippet: Proteomic methodologies for PBCV-1 virions.

    Article Snippet: Preliminary proteomic analyses using the existing PBCV-1 gene annotations (National Center for Biotechnology Information [NCBI] Refseq, ) revealed possible errors in the genome sequence, which prompted us to resequence the PBCV-1 genome.

    Techniques:

    Mass-versus-pI distribution of PBCV-1 virion CDSs identified by two independent proteomic methods. The virion proteins are displayed as a function of their intrinsic molecular masses and isoelectric points. The results of each method are shown. Note that method 2 was especially useful for discovering a set of low-molecular-mass proteins that were not detected by method 1.

    Journal: Journal of Virology

    Article Title: Paramecium bursaria Chlorella Virus 1 Proteome Reveals Novel Architectural and Regulatory Features of a Giant Virus

    doi: 10.1128/JVI.00907-12

    Figure Lengend Snippet: Mass-versus-pI distribution of PBCV-1 virion CDSs identified by two independent proteomic methods. The virion proteins are displayed as a function of their intrinsic molecular masses and isoelectric points. The results of each method are shown. Note that method 2 was especially useful for discovering a set of low-molecular-mass proteins that were not detected by method 1.

    Article Snippet: Preliminary proteomic analyses using the existing PBCV-1 gene annotations (National Center for Biotechnology Information [NCBI] Refseq, ) revealed possible errors in the genome sequence, which prompted us to resequence the PBCV-1 genome.

    Techniques:

    Inhibition of Adora1 results in significantly decreased endogenous ERα in MCF-7 cells. MCF-7 cells were transiently transfected with an Adora1-targeted siRNA or a control siRNA construct; (A) mRNA levels of Adora1 and ERα were measured by real-time PCR; (B) protein levels of Adora1 and ERα were measured in immunoblot analyses using the indicated antibodies; and (C) MCF-7 cells were treated with vehicle or DPCPX at the concentration of 10 3 µM and 10 4 µM for 12 h, protein levels of ERα were measured in immunoblot analyses using ERα antibody. Data are the average of 3 replicates ± SD. *, p

    Journal: Oncogene

    Article Title: Adenosine A1 receptor, a target and regulator of ER? action, mediates the proliferative effects of estradiol in breast cancer

    doi: 10.1038/onc.2009.409

    Figure Lengend Snippet: Inhibition of Adora1 results in significantly decreased endogenous ERα in MCF-7 cells. MCF-7 cells were transiently transfected with an Adora1-targeted siRNA or a control siRNA construct; (A) mRNA levels of Adora1 and ERα were measured by real-time PCR; (B) protein levels of Adora1 and ERα were measured in immunoblot analyses using the indicated antibodies; and (C) MCF-7 cells were treated with vehicle or DPCPX at the concentration of 10 3 µM and 10 4 µM for 12 h, protein levels of ERα were measured in immunoblot analyses using ERα antibody. Data are the average of 3 replicates ± SD. *, p

    Article Snippet: To verify that these oligonucleotide sequences in the Adora1 siRNA pool specifically targeted Adora1 but not ERα mRNA, we aligned the four Adora1 siRNA oligonucleotide sequences with ERα mRNA (Locus number: NM_000125 from NCBI DNA database) by Blast alignment from National Center for Biotechnology Information (NCBI).

    Techniques: Inhibition, Transfection, Construct, Real-time Polymerase Chain Reaction, Concentration Assay

    Silencing of Adora1 in MCF-7 cells leads to reduced binding of ERα to TFF1 promoter, TFF1 promoter driven luferase activity, and mRNA expression of TFF1. (A) Knock-down of Adora1 results in decreased binding of ERa to the TFF1 promoter. B) siRNA knock-down of Adora1 expression decreases ERα-stimulated transcriptional activation of the TFF1 promoter. MCF-7 cells were co-transfected with a TFF1-Luc reporter or pGL4 vector (200ng), pCMVβGal (80ng), and either control or Adora1 siRNA (100 nM) in the presence or absence of E2 (10 −8 M) overnight. C) Adora1 silencing results in significantly decreased TFF1 expression. MCF-7 cells were transfected either with control or Adora1 siRNA (100 nM) and treated with vehicle or E2 (10 −8 M) overnight. Cells were then lysed, total RNA was extracted, and mRNA levels of Adora1 were measured by real-time PCR and normalized to GAPDH. Data represent the average of 3 replicates ± SD (*, p

    Journal: Oncogene

    Article Title: Adenosine A1 receptor, a target and regulator of ER? action, mediates the proliferative effects of estradiol in breast cancer

    doi: 10.1038/onc.2009.409

    Figure Lengend Snippet: Silencing of Adora1 in MCF-7 cells leads to reduced binding of ERα to TFF1 promoter, TFF1 promoter driven luferase activity, and mRNA expression of TFF1. (A) Knock-down of Adora1 results in decreased binding of ERa to the TFF1 promoter. B) siRNA knock-down of Adora1 expression decreases ERα-stimulated transcriptional activation of the TFF1 promoter. MCF-7 cells were co-transfected with a TFF1-Luc reporter or pGL4 vector (200ng), pCMVβGal (80ng), and either control or Adora1 siRNA (100 nM) in the presence or absence of E2 (10 −8 M) overnight. C) Adora1 silencing results in significantly decreased TFF1 expression. MCF-7 cells were transfected either with control or Adora1 siRNA (100 nM) and treated with vehicle or E2 (10 −8 M) overnight. Cells were then lysed, total RNA was extracted, and mRNA levels of Adora1 were measured by real-time PCR and normalized to GAPDH. Data represent the average of 3 replicates ± SD (*, p

    Article Snippet: To verify that these oligonucleotide sequences in the Adora1 siRNA pool specifically targeted Adora1 but not ERα mRNA, we aligned the four Adora1 siRNA oligonucleotide sequences with ERα mRNA (Locus number: NM_000125 from NCBI DNA database) by Blast alignment from National Center for Biotechnology Information (NCBI).

    Techniques: Binding Assay, Activity Assay, Expressing, Activation Assay, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction