hybridoma Search Results


mouse interleukin 6 il 6 elisa kit  (Shanghai Korain Biotech Co Ltd)
94
Shanghai Korain Biotech Co Ltd mouse interleukin 6 il 6 elisa kit
<t>Interleukin-6</t> <t>(IL-6)</t> concentration by (A) RAW 264.7 and (B) MC3T3 cells LPS induction and PDEV (2.5, 5 and 10 μg mL -1 ) treatment released from Gel-Dop hydrogel. Data presented as mean ± standard deviation (*** p < 0.001, **** p <0,0001)
Mouse Interleukin 6 Il 6 Elisa Kit, supplied by Shanghai Korain Biotech Co Ltd, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/hybridoma/Mouse+Interleukin+6/pmc12994586-46-70-75
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hp gi 3 rabbit polyclonal kurt von figura cd m6pr  (Developmental Studies Hybridoma Bank)
94
Developmental Studies Hybridoma Bank hp gi 3 rabbit polyclonal kurt von figura cd m6pr
Fig. 4. Subcellular localization of mannose 6-phosphate receptors in HCMV-infected cells. BJ1 cells were either mock infected (upper panels) or infected with HCMV at an moi of 0.5 (lower panels). After 4 dpi, cells were fixed, permeabilized and stained with <t>anti-CI-M6PR</t> (green in left panels), anti-CD-M6PR (green in right panels) and anti-HCMV glycoprotein gH (red) antibodies. In HCMV-infected cells both M6PRs were located within virus factories. Scale bars, 20 mm.
Hp Gi 3 Rabbit Polyclonal Kurt Von Figura Cd M6pr, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/hybridoma/anti-mannose+6-phosphate+receptor/pm19888988-201-135-156
Average 94 stars, based on 1 article reviews
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tubulin  (Developmental Studies Hybridoma Bank)
98
Developmental Studies Hybridoma Bank tubulin
Spag protein structure and patterns of gene expression and protein accumulation during Drosophila development. A, schematic structure of the Spag protein and evolutionary conservation of the TPR region in Spag orthologs from yeast to human. Filled boxes represent predicted structural domains and lines unstructured domains. The three TPR motifs forming the TPR region are indicated as red, blue, and green boxes. TPR 3′ is a putative helical domain absent in Tah1. Orange box represents a potential α-helical junction between TPR 3 and TPR 3′. The potential monad binding motif (interPro 025986) overlaps with a predicted structured domain (magenta). Below are aligned the amino acid residues characterizing the three TPR motifs in S. cerevisiae (S.c.), D. melanogaster (D.m.), Danio rero (D.r.), Homo sapiens (H.s.), and Xenopus tropicalis (X.t.). Conserved residues identified in Tah1 for Hsp90 binding are indicated in bold red (21). Blue indicates additional residues conserved in TPR domains and important for Hsp70/Hsp90 binding, as defined previously (23). B, expression pattern of spag mRNA (left panel) and pattern of Spag protein accumulation (right panel) during Drosophila development. RNAs and proteins were extracted from (E) embryos with age in hours after egg laying indicated above the lanes (L1, L2, and L3) first, second, and third instar larvae, (P) pupae, (F) female, and (M) male 3-days old imagos. The Northern blot was hybridized with either 32P-labeled spag or <t>β-tubulin</t> cDNA. The Western blot was probed with anti-Spag or anti-ribosomal P40 <t>protein</t> <t>polyclonal</t> antibodies. The β-tubulin cDNA and anti-P40 antibodies were used for loading control.
Tubulin, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/hybridoma/anti-tubulin/pmc03937688-112-65-69
Average 98 stars, based on 1 article reviews
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mouse anti spam antibody  (Developmental Studies Hybridoma Bank)
93
Developmental Studies Hybridoma Bank mouse anti spam antibody
Fig. 2. Overexpression of CrbFLAGintra during late larval and pupal development affects photoreceptor apico-basal polarity. (A–H) Immunostainings on cross-sections of adult wild-type (wt) eyes (A,C,E,G) and eyes overexpressing CrbFLAGintra using elavGal4 (elav.CrbFLAGintra) (B,D,F,H). The sections were stained for Drosophila E-Cad (DE-Cad) <t>(A,B),</t> <t>Rh1</t> (C,D), <t>Spam</t> (Eys) (E,F) or the alpha subunit of Na+/K+-ATPase (G,H) (in red), and Sdt (A–C), Crb (D,E,G,H) or Drosophila PATJ (F) (in green). B-, D-, F- and H- represent higher magnifications of one single ommatidium shown in B0, D0, F0 and G0, respectively. The F-actin stain (blue) shows the rhabdomeres. Sdt (B9), Crb (D9,H9) and Drosophila PATJ (F0) exhibit expanded expression domains and often localise adjacent to ectopic rhabdomeres. Additionally, Sdt localises between split rhabdomeres (B9,B0, white arrowhead). Note ectopic Drosophila E-Cad flanking ectopic rhabdomeres (B, B0,B-, white arrows). Ectopic apical poles are positive for Rh1 (D,D0,D-, arrows) and Spam (Eys) (F, F0, F-, arrows). Na+/K+-ATPase is absent from ectopic rhabdomeres and diminished at membranes with high levels of ectopic Crb (H,H0,H-, the decrease is indicated by a cross). In addition, Na+/K+-ATPase is present at the stalk of cells with ectopic rhabdomeres, which are not R7 (H,H0,H-, some cells other than R7 are indicated by asterisks). Scale bars: 2 mm. (I,J) Schematic summary of the distribution of proteins (as shown in A–H) in wild-type ommatidia (I) and ommatidia overexpressing CrbFLAGintra (J).
Mouse Anti Spam Antibody, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/hybridoma/anti-Eyes+shut+protein/pm22025631-264-67-72
Average 93 stars, based on 1 article reviews
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mouse anti g h2av monoclonal antibody unc93 5 2 1  (Developmental Studies Hybridoma Bank)
95
Developmental Studies Hybridoma Bank mouse anti g h2av monoclonal antibody unc93 5 2 1
Fig. 2. Overexpression of CrbFLAGintra during late larval and pupal development affects photoreceptor apico-basal polarity. (A–H) Immunostainings on cross-sections of adult wild-type (wt) eyes (A,C,E,G) and eyes overexpressing CrbFLAGintra using elavGal4 (elav.CrbFLAGintra) (B,D,F,H). The sections were stained for Drosophila E-Cad (DE-Cad) <t>(A,B),</t> <t>Rh1</t> (C,D), <t>Spam</t> (Eys) (E,F) or the alpha subunit of Na+/K+-ATPase (G,H) (in red), and Sdt (A–C), Crb (D,E,G,H) or Drosophila PATJ (F) (in green). B-, D-, F- and H- represent higher magnifications of one single ommatidium shown in B0, D0, F0 and G0, respectively. The F-actin stain (blue) shows the rhabdomeres. Sdt (B9), Crb (D9,H9) and Drosophila PATJ (F0) exhibit expanded expression domains and often localise adjacent to ectopic rhabdomeres. Additionally, Sdt localises between split rhabdomeres (B9,B0, white arrowhead). Note ectopic Drosophila E-Cad flanking ectopic rhabdomeres (B, B0,B-, white arrows). Ectopic apical poles are positive for Rh1 (D,D0,D-, arrows) and Spam (Eys) (F, F0, F-, arrows). Na+/K+-ATPase is absent from ectopic rhabdomeres and diminished at membranes with high levels of ectopic Crb (H,H0,H-, the decrease is indicated by a cross). In addition, Na+/K+-ATPase is present at the stalk of cells with ectopic rhabdomeres, which are not R7 (H,H0,H-, some cells other than R7 are indicated by asterisks). Scale bars: 2 mm. (I,J) Schematic summary of the distribution of proteins (as shown in A–H) in wild-type ommatidia (I) and ommatidia overexpressing CrbFLAGintra (J).
Mouse Anti G H2av Monoclonal Antibody Unc93 5 2 1, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/hybridoma/anti-histone+2A+gamma+variant/pm25345385-169-82-89
Average 95 stars, based on 1 article reviews
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mouse monoclonal anti titin  (Developmental Studies Hybridoma Bank)
94
Developmental Studies Hybridoma Bank mouse monoclonal anti titin
Fig. 2. Overexpression of CrbFLAGintra during late larval and pupal development affects photoreceptor apico-basal polarity. (A–H) Immunostainings on cross-sections of adult wild-type (wt) eyes (A,C,E,G) and eyes overexpressing CrbFLAGintra using elavGal4 (elav.CrbFLAGintra) (B,D,F,H). The sections were stained for Drosophila E-Cad (DE-Cad) <t>(A,B),</t> <t>Rh1</t> (C,D), <t>Spam</t> (Eys) (E,F) or the alpha subunit of Na+/K+-ATPase (G,H) (in red), and Sdt (A–C), Crb (D,E,G,H) or Drosophila PATJ (F) (in green). B-, D-, F- and H- represent higher magnifications of one single ommatidium shown in B0, D0, F0 and G0, respectively. The F-actin stain (blue) shows the rhabdomeres. Sdt (B9), Crb (D9,H9) and Drosophila PATJ (F0) exhibit expanded expression domains and often localise adjacent to ectopic rhabdomeres. Additionally, Sdt localises between split rhabdomeres (B9,B0, white arrowhead). Note ectopic Drosophila E-Cad flanking ectopic rhabdomeres (B, B0,B-, white arrows). Ectopic apical poles are positive for Rh1 (D,D0,D-, arrows) and Spam (Eys) (F, F0, F-, arrows). Na+/K+-ATPase is absent from ectopic rhabdomeres and diminished at membranes with high levels of ectopic Crb (H,H0,H-, the decrease is indicated by a cross). In addition, Na+/K+-ATPase is present at the stalk of cells with ectopic rhabdomeres, which are not R7 (H,H0,H-, some cells other than R7 are indicated by asterisks). Scale bars: 2 mm. (I,J) Schematic summary of the distribution of proteins (as shown in A–H) in wild-type ommatidia (I) and ommatidia overexpressing CrbFLAGintra (J).
Mouse Monoclonal Anti Titin, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/hybridoma/anti-Titin/pmc03378392-101-21-25
Average 94 stars, based on 1 article reviews
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mouse anti rip  (Developmental Studies Hybridoma Bank)
94
Developmental Studies Hybridoma Bank mouse anti rip
Fig. 2. Overexpression of CrbFLAGintra during late larval and pupal development affects photoreceptor apico-basal polarity. (A–H) Immunostainings on cross-sections of adult wild-type (wt) eyes (A,C,E,G) and eyes overexpressing CrbFLAGintra using elavGal4 (elav.CrbFLAGintra) (B,D,F,H). The sections were stained for Drosophila E-Cad (DE-Cad) <t>(A,B),</t> <t>Rh1</t> (C,D), <t>Spam</t> (Eys) (E,F) or the alpha subunit of Na+/K+-ATPase (G,H) (in red), and Sdt (A–C), Crb (D,E,G,H) or Drosophila PATJ (F) (in green). B-, D-, F- and H- represent higher magnifications of one single ommatidium shown in B0, D0, F0 and G0, respectively. The F-actin stain (blue) shows the rhabdomeres. Sdt (B9), Crb (D9,H9) and Drosophila PATJ (F0) exhibit expanded expression domains and often localise adjacent to ectopic rhabdomeres. Additionally, Sdt localises between split rhabdomeres (B9,B0, white arrowhead). Note ectopic Drosophila E-Cad flanking ectopic rhabdomeres (B, B0,B-, white arrows). Ectopic apical poles are positive for Rh1 (D,D0,D-, arrows) and Spam (Eys) (F, F0, F-, arrows). Na+/K+-ATPase is absent from ectopic rhabdomeres and diminished at membranes with high levels of ectopic Crb (H,H0,H-, the decrease is indicated by a cross). In addition, Na+/K+-ATPase is present at the stalk of cells with ectopic rhabdomeres, which are not R7 (H,H0,H-, some cells other than R7 are indicated by asterisks). Scale bars: 2 mm. (I,J) Schematic summary of the distribution of proteins (as shown in A–H) in wild-type ommatidia (I) and ommatidia overexpressing CrbFLAGintra (J).
Mouse Anti Rip, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/hybridoma/anti-CNP+%2F+Rip+antigen/pmc03561904-49-63-66
Average 94 stars, based on 1 article reviews
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hb9  (Developmental Studies Hybridoma Bank)
96
Developmental Studies Hybridoma Bank hb9
Fig. 2. Overexpression of CrbFLAGintra during late larval and pupal development affects photoreceptor apico-basal polarity. (A–H) Immunostainings on cross-sections of adult wild-type (wt) eyes (A,C,E,G) and eyes overexpressing CrbFLAGintra using elavGal4 (elav.CrbFLAGintra) (B,D,F,H). The sections were stained for Drosophila E-Cad (DE-Cad) <t>(A,B),</t> <t>Rh1</t> (C,D), <t>Spam</t> (Eys) (E,F) or the alpha subunit of Na+/K+-ATPase (G,H) (in red), and Sdt (A–C), Crb (D,E,G,H) or Drosophila PATJ (F) (in green). B-, D-, F- and H- represent higher magnifications of one single ommatidium shown in B0, D0, F0 and G0, respectively. The F-actin stain (blue) shows the rhabdomeres. Sdt (B9), Crb (D9,H9) and Drosophila PATJ (F0) exhibit expanded expression domains and often localise adjacent to ectopic rhabdomeres. Additionally, Sdt localises between split rhabdomeres (B9,B0, white arrowhead). Note ectopic Drosophila E-Cad flanking ectopic rhabdomeres (B, B0,B-, white arrows). Ectopic apical poles are positive for Rh1 (D,D0,D-, arrows) and Spam (Eys) (F, F0, F-, arrows). Na+/K+-ATPase is absent from ectopic rhabdomeres and diminished at membranes with high levels of ectopic Crb (H,H0,H-, the decrease is indicated by a cross). In addition, Na+/K+-ATPase is present at the stalk of cells with ectopic rhabdomeres, which are not R7 (H,H0,H-, some cells other than R7 are indicated by asterisks). Scale bars: 2 mm. (I,J) Schematic summary of the distribution of proteins (as shown in A–H) in wild-type ommatidia (I) and ommatidia overexpressing CrbFLAGintra (J).
Hb9, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/hybridoma/anti-MNR2%2FHB9%2FMnx1/pmc03567634-49-40-42
Average 96 stars, based on 1 article reviews
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xenopus neurofilament  (Developmental Studies Hybridoma Bank)
96
Developmental Studies Hybridoma Bank xenopus neurofilament
Fig. 1 – RAI1/Rai1 protein organization and induction by retinoic acid. (A) A schematic representation of the Rai1 protein in human, mouse, two frog species (X. laevis and X. tropicalis), and zebrafish. This illustrates that the organization of the primary protein domains, including the Trans-Activation Domain (TAD), Nuclear Localization Signal containing region (NLS; see Supplementary material Fig. S1B for details), and extended plant homeodomain (ePHD/ADD, also known as a PHD finger) are conserved in the species examined (see Supplementary material Fig. S1A for sequence alignment of the PHD finger). A 14 poly-glutamine stretch (Q) is found in humans and reduced glutamine stretches can also be found in a similar region of other species except zebrafish. The NLS region contains the first known nuclear localization sequence (Darvekar et al., 2013). Figure not to scale. (B) Retinoic acid induces expression of rai1 during <t>Xenopus</t> development. Quantitative PCR of rai1 mRNA after all-trans retinoic treatment (ATRA) at stage 23 showing significant 3.91-fold increase (t-test, p = 0.0037) relative to the control housekeeping gene (ef1 alpha). (C) Retinoic acid may directly regulate rai1. A histogram of RAR binding sites in a 2 kb region upstream of the rai1 start site in X. laevis reveals the enrichment of retinoic acid receptor binding sites.
Xenopus Neurofilament, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/hybridoma/anti-neurofilament-associated/pm24878353-240-41-43
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mouse a nestin  (Developmental Studies Hybridoma Bank)
95
Developmental Studies Hybridoma Bank mouse a nestin
Fig. 1 – RAI1/Rai1 protein organization and induction by retinoic acid. (A) A schematic representation of the Rai1 protein in human, mouse, two frog species (X. laevis and X. tropicalis), and zebrafish. This illustrates that the organization of the primary protein domains, including the Trans-Activation Domain (TAD), Nuclear Localization Signal containing region (NLS; see Supplementary material Fig. S1B for details), and extended plant homeodomain (ePHD/ADD, also known as a PHD finger) are conserved in the species examined (see Supplementary material Fig. S1A for sequence alignment of the PHD finger). A 14 poly-glutamine stretch (Q) is found in humans and reduced glutamine stretches can also be found in a similar region of other species except zebrafish. The NLS region contains the first known nuclear localization sequence (Darvekar et al., 2013). Figure not to scale. (B) Retinoic acid induces expression of rai1 during <t>Xenopus</t> development. Quantitative PCR of rai1 mRNA after all-trans retinoic treatment (ATRA) at stage 23 showing significant 3.91-fold increase (t-test, p = 0.0037) relative to the control housekeeping gene (ef1 alpha). (C) Retinoic acid may directly regulate rai1. A histogram of RAR binding sites in a 2 kb region upstream of the rai1 start site in X. laevis reveals the enrichment of retinoic acid receptor binding sites.
Mouse A Nestin, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/hybridoma/anti-Nestin/pm19795519-133-51-54
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anti syntaxin  (Developmental Studies Hybridoma Bank)
94
Developmental Studies Hybridoma Bank anti syntaxin
Fig. 1 – RAI1/Rai1 protein organization and induction by retinoic acid. (A) A schematic representation of the Rai1 protein in human, mouse, two frog species (X. laevis and X. tropicalis), and zebrafish. This illustrates that the organization of the primary protein domains, including the Trans-Activation Domain (TAD), Nuclear Localization Signal containing region (NLS; see Supplementary material Fig. S1B for details), and extended plant homeodomain (ePHD/ADD, also known as a PHD finger) are conserved in the species examined (see Supplementary material Fig. S1A for sequence alignment of the PHD finger). A 14 poly-glutamine stretch (Q) is found in humans and reduced glutamine stretches can also be found in a similar region of other species except zebrafish. The NLS region contains the first known nuclear localization sequence (Darvekar et al., 2013). Figure not to scale. (B) Retinoic acid induces expression of rai1 during <t>Xenopus</t> development. Quantitative PCR of rai1 mRNA after all-trans retinoic treatment (ATRA) at stage 23 showing significant 3.91-fold increase (t-test, p = 0.0037) relative to the control housekeeping gene (ef1 alpha). (C) Retinoic acid may directly regulate rai1. A histogram of RAR binding sites in a 2 kb region upstream of the rai1 start site in X. laevis reveals the enrichment of retinoic acid receptor binding sites.
Anti Syntaxin, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/hybridoma/anti-Syntaxin/pmc08111063-46-9-10
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mouse anti spectrin  (Developmental Studies Hybridoma Bank)
95
Developmental Studies Hybridoma Bank mouse anti spectrin
Fig. 1 – RAI1/Rai1 protein organization and induction by retinoic acid. (A) A schematic representation of the Rai1 protein in human, mouse, two frog species (X. laevis and X. tropicalis), and zebrafish. This illustrates that the organization of the primary protein domains, including the Trans-Activation Domain (TAD), Nuclear Localization Signal containing region (NLS; see Supplementary material Fig. S1B for details), and extended plant homeodomain (ePHD/ADD, also known as a PHD finger) are conserved in the species examined (see Supplementary material Fig. S1A for sequence alignment of the PHD finger). A 14 poly-glutamine stretch (Q) is found in humans and reduced glutamine stretches can also be found in a similar region of other species except zebrafish. The NLS region contains the first known nuclear localization sequence (Darvekar et al., 2013). Figure not to scale. (B) Retinoic acid induces expression of rai1 during <t>Xenopus</t> development. Quantitative PCR of rai1 mRNA after all-trans retinoic treatment (ATRA) at stage 23 showing significant 3.91-fold increase (t-test, p = 0.0037) relative to the control housekeeping gene (ef1 alpha). (C) Retinoic acid may directly regulate rai1. A histogram of RAR binding sites in a 2 kb region upstream of the rai1 start site in X. laevis reveals the enrichment of retinoic acid receptor binding sites.
Mouse Anti Spectrin, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


Interleukin-6 (IL-6) concentration by (A) RAW 264.7 and (B) MC3T3 cells LPS induction and PDEV (2.5, 5 and 10 μg mL -1 ) treatment released from Gel-Dop hydrogel. Data presented as mean ± standard deviation (*** p < 0.001, **** p <0,0001)

Journal: ADMET & DMPK

Article Title: Anti-inflammatory potential of plant-derived extracellular vesicles from Solanum nigrum L. integrated in gelatine-dopamine hydrogel on RAW 264.7 and MC3T3 cells

doi: 10.5599/admet.3149

Figure Lengend Snippet: Interleukin-6 (IL-6) concentration by (A) RAW 264.7 and (B) MC3T3 cells LPS induction and PDEV (2.5, 5 and 10 μg mL -1 ) treatment released from Gel-Dop hydrogel. Data presented as mean ± standard deviation (*** p < 0.001, **** p <0,0001)

Article Snippet: Other materials used in this study were Dulbecco’s modified eagle medium (DMEM) - high glucose, foetal bovine serum (FBS), Antibiotic-Antimycotic (ABAM), polyethylene glycol (PEG) 6000, trehalose (Sigma-Aldrich; Merck), phosphate-buffered saline (PBS), sodium periodate (NaIO 4 ) (Sigma-Aldrich), bovine serum albumin (BSA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT), 4′,6-diamidino-2-phenylindole (DAPI), dimethyl sulfoxide (DMSO), paraformaldehyde (PFA), kit BCA Assay (PierceTM BCA Protein Assay Kit, Thermo Scientific), PKH67 Green Fluorescent Cell Linker Kits (Sigma-Aldrich; Merck), and Mouse Interleukin-6 (IL-6) ELISA Kit (BT Lab, Cat. No. E0049Mo).

Techniques: Concentration Assay, Standard Deviation

Fig. 4. Subcellular localization of mannose 6-phosphate receptors in HCMV-infected cells. BJ1 cells were either mock infected (upper panels) or infected with HCMV at an moi of 0.5 (lower panels). After 4 dpi, cells were fixed, permeabilized and stained with anti-CI-M6PR (green in left panels), anti-CD-M6PR (green in right panels) and anti-HCMV glycoprotein gH (red) antibodies. In HCMV-infected cells both M6PRs were located within virus factories. Scale bars, 20 mm.

Journal: Cellular microbiology

Article Title: Human cytomegalovirus final envelopment on membranes containing both trans-Golgi network and endosomal markers.

doi: 10.1111/j.1462-5822.2009.01405.x

Figure Lengend Snippet: Fig. 4. Subcellular localization of mannose 6-phosphate receptors in HCMV-infected cells. BJ1 cells were either mock infected (upper panels) or infected with HCMV at an moi of 0.5 (lower panels). After 4 dpi, cells were fixed, permeabilized and stained with anti-CI-M6PR (green in left panels), anti-CD-M6PR (green in right panels) and anti-HCMV glycoprotein gH (red) antibodies. In HCMV-infected cells both M6PRs were located within virus factories. Scale bars, 20 mm.

Article Snippet: Antigen Antibodies name Species and isotype Source HCMV glycoprotein H HCMV 16 Mouse IgG1 Helena Browne HCMV glycoprotein B HCMV 37 Mouse IgG2a Helena Browne HCMV UL33 Anti-HCMV UL33 Rabbit polyclonal Wade Gibson HCMV pp28 Anti-HCMV pp28 Mouse IgG1 Thomas Shenk HSV-1 glycoprotein D AP7 Mouse IgG2a Helena Browne CD63 (lumenal domain) Anti-CD63, 1B5 Mouse IgG2b Mark Marsh TGN46 Anti-TGN46 Sheep polyclonal AbD-Serotec Annexin I Anti-annexin I Mouse IgG1 Claire Isacke Early endosome antigen 1 Anti-EEA1 Mouse IgG1 BD Biosciences Hepatocyte growth factor-regulated Tyr-kinase substrate Anti-HRS 958/3 Rabbit polyclonal Sylvie Urbé Transferrin receptor (lumenal domain) B3/25 Mouse IgG1 Santa Cruz Biotechnology Transferrin receptor (cytoplasmic tail) H68.4 Mouse IgG1 Zymed Laboratories CI-M6PR (lumenal domain) Anti-MPR300 G VII-3 Rabbit polyclonal Kurt von Figura CI-M6PR (cytoplasmic tail) Anti-MPR300 H-CI-II Rabbit polyclonal Kurt von Figura CD-M6PR (lumenal domain) Anti-MPR46 HP-GI-3 Rabbit polyclonal Kurt von Figura CD-M6PR (cytoplasmic tail) Anti-MPR46 MSCI Rabbit polyclonal Kurt von Figura CD-M6PR Anti-MPR46, 22d4 Mouse IgG2b DSHB b-actin Anti-b-actin AC-15 Mouse IgG1 Sigma Aldrich Actin Anti-actin Rabbit polyclonal Sigma Aldrich Clathrin heavy chain Anti-CHC 23 Mouse IgG1 BD Biosciences © 2009 Blackwell Publishing Ltd, Cellular Microbiology, 12, 386–404 0.5 infectious particles per cell.

Techniques: Infection, Staining, Virus

Fig. 5. Examination of cellular markers in cell lysates and supernatants of HCMV-infected cells. A. Equal number of BJ1 cells mock infected or HCMV infected for 4 days at an moi of 3 were lysed and viral particles pelleted by centrifugation of the clarified supernatants were mixed with non-reducing SDS-PAGE sample buffer. Lysates were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were cut and incubated, or sequentially incubated, with antibodies against cellular markers and antibodies against clathrin heavy chain or actin to check protein loading in cell lysates, and antiviral tegument protein pp28 antibodies to check HCMV viral particles secreted into the supernatants. Autoradiography films were scanned; images were cropped and assembled with Adobe Photoshop. Molecular weights in kDa are indicated. We found CD63, TGN46, EEA1, TfR and CI-M6PR in the supernatants of HCMV-infected cells. B. Results of cell lysates in (A) were analysed by densitometry, normalized to the expression levels of loading controls and expressed as a percentage of the levels in uninfected cells. We observed a reproducible reduction in the levels of CD63, TGN46 and CD-M6PR together with an increase in the levels of TfR by comparison with uninfected cells.

Journal: Cellular microbiology

Article Title: Human cytomegalovirus final envelopment on membranes containing both trans-Golgi network and endosomal markers.

doi: 10.1111/j.1462-5822.2009.01405.x

Figure Lengend Snippet: Fig. 5. Examination of cellular markers in cell lysates and supernatants of HCMV-infected cells. A. Equal number of BJ1 cells mock infected or HCMV infected for 4 days at an moi of 3 were lysed and viral particles pelleted by centrifugation of the clarified supernatants were mixed with non-reducing SDS-PAGE sample buffer. Lysates were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were cut and incubated, or sequentially incubated, with antibodies against cellular markers and antibodies against clathrin heavy chain or actin to check protein loading in cell lysates, and antiviral tegument protein pp28 antibodies to check HCMV viral particles secreted into the supernatants. Autoradiography films were scanned; images were cropped and assembled with Adobe Photoshop. Molecular weights in kDa are indicated. We found CD63, TGN46, EEA1, TfR and CI-M6PR in the supernatants of HCMV-infected cells. B. Results of cell lysates in (A) were analysed by densitometry, normalized to the expression levels of loading controls and expressed as a percentage of the levels in uninfected cells. We observed a reproducible reduction in the levels of CD63, TGN46 and CD-M6PR together with an increase in the levels of TfR by comparison with uninfected cells.

Article Snippet: Antigen Antibodies name Species and isotype Source HCMV glycoprotein H HCMV 16 Mouse IgG1 Helena Browne HCMV glycoprotein B HCMV 37 Mouse IgG2a Helena Browne HCMV UL33 Anti-HCMV UL33 Rabbit polyclonal Wade Gibson HCMV pp28 Anti-HCMV pp28 Mouse IgG1 Thomas Shenk HSV-1 glycoprotein D AP7 Mouse IgG2a Helena Browne CD63 (lumenal domain) Anti-CD63, 1B5 Mouse IgG2b Mark Marsh TGN46 Anti-TGN46 Sheep polyclonal AbD-Serotec Annexin I Anti-annexin I Mouse IgG1 Claire Isacke Early endosome antigen 1 Anti-EEA1 Mouse IgG1 BD Biosciences Hepatocyte growth factor-regulated Tyr-kinase substrate Anti-HRS 958/3 Rabbit polyclonal Sylvie Urbé Transferrin receptor (lumenal domain) B3/25 Mouse IgG1 Santa Cruz Biotechnology Transferrin receptor (cytoplasmic tail) H68.4 Mouse IgG1 Zymed Laboratories CI-M6PR (lumenal domain) Anti-MPR300 G VII-3 Rabbit polyclonal Kurt von Figura CI-M6PR (cytoplasmic tail) Anti-MPR300 H-CI-II Rabbit polyclonal Kurt von Figura CD-M6PR (lumenal domain) Anti-MPR46 HP-GI-3 Rabbit polyclonal Kurt von Figura CD-M6PR (cytoplasmic tail) Anti-MPR46 MSCI Rabbit polyclonal Kurt von Figura CD-M6PR Anti-MPR46, 22d4 Mouse IgG2b DSHB b-actin Anti-b-actin AC-15 Mouse IgG1 Sigma Aldrich Actin Anti-actin Rabbit polyclonal Sigma Aldrich Clathrin heavy chain Anti-CHC 23 Mouse IgG1 BD Biosciences © 2009 Blackwell Publishing Ltd, Cellular Microbiology, 12, 386–404 0.5 infectious particles per cell.

Techniques: Infection, Centrifugation, SDS Page, Incubation, Autoradiography, Expressing, Comparison

Fig. 6. Examination of cellular markers in purified HCMV virions. A. Electron microscopy of purified HCMV virions negatively stained with 2% uranyl acetate. When viral particles were partially disrupted, uranyl acetate revealed the nucleocapsids. Scale bar, 200 nm. B. 35 ng of purified virions per lane were separated by SDS-PAGE under non-reducing conditions and transferred to PVDF membranes. Membranes were cut and incubated, or sequentially incubated, with antibodies against cellular markers and antiviral tegument protein pp28 antibodies. Autoradiography films were scanned; images were cropped and assembled with Adobe Photoshop. Molecular weights in kDa are indicated. We found CD63, TGN46, TfR and CI-M6PR in purified virions.

Journal: Cellular microbiology

Article Title: Human cytomegalovirus final envelopment on membranes containing both trans-Golgi network and endosomal markers.

doi: 10.1111/j.1462-5822.2009.01405.x

Figure Lengend Snippet: Fig. 6. Examination of cellular markers in purified HCMV virions. A. Electron microscopy of purified HCMV virions negatively stained with 2% uranyl acetate. When viral particles were partially disrupted, uranyl acetate revealed the nucleocapsids. Scale bar, 200 nm. B. 35 ng of purified virions per lane were separated by SDS-PAGE under non-reducing conditions and transferred to PVDF membranes. Membranes were cut and incubated, or sequentially incubated, with antibodies against cellular markers and antiviral tegument protein pp28 antibodies. Autoradiography films were scanned; images were cropped and assembled with Adobe Photoshop. Molecular weights in kDa are indicated. We found CD63, TGN46, TfR and CI-M6PR in purified virions.

Article Snippet: Antigen Antibodies name Species and isotype Source HCMV glycoprotein H HCMV 16 Mouse IgG1 Helena Browne HCMV glycoprotein B HCMV 37 Mouse IgG2a Helena Browne HCMV UL33 Anti-HCMV UL33 Rabbit polyclonal Wade Gibson HCMV pp28 Anti-HCMV pp28 Mouse IgG1 Thomas Shenk HSV-1 glycoprotein D AP7 Mouse IgG2a Helena Browne CD63 (lumenal domain) Anti-CD63, 1B5 Mouse IgG2b Mark Marsh TGN46 Anti-TGN46 Sheep polyclonal AbD-Serotec Annexin I Anti-annexin I Mouse IgG1 Claire Isacke Early endosome antigen 1 Anti-EEA1 Mouse IgG1 BD Biosciences Hepatocyte growth factor-regulated Tyr-kinase substrate Anti-HRS 958/3 Rabbit polyclonal Sylvie Urbé Transferrin receptor (lumenal domain) B3/25 Mouse IgG1 Santa Cruz Biotechnology Transferrin receptor (cytoplasmic tail) H68.4 Mouse IgG1 Zymed Laboratories CI-M6PR (lumenal domain) Anti-MPR300 G VII-3 Rabbit polyclonal Kurt von Figura CI-M6PR (cytoplasmic tail) Anti-MPR300 H-CI-II Rabbit polyclonal Kurt von Figura CD-M6PR (lumenal domain) Anti-MPR46 HP-GI-3 Rabbit polyclonal Kurt von Figura CD-M6PR (cytoplasmic tail) Anti-MPR46 MSCI Rabbit polyclonal Kurt von Figura CD-M6PR Anti-MPR46, 22d4 Mouse IgG2b DSHB b-actin Anti-b-actin AC-15 Mouse IgG1 Sigma Aldrich Actin Anti-actin Rabbit polyclonal Sigma Aldrich Clathrin heavy chain Anti-CHC 23 Mouse IgG1 BD Biosciences © 2009 Blackwell Publishing Ltd, Cellular Microbiology, 12, 386–404 0.5 infectious particles per cell.

Techniques: Electron Microscopy, Staining, SDS Page, Incubation, Autoradiography

Fig. 7. Transcriptional regulation of cellular markers during HCMV infection. Equal number of BJ1 cells were mock infected or infected with HCMV at an moi of 3, and RNA was extracted at the indicated times. Gene expression of cellular markers and HCMV UL83 (viral protein pp65) was measured by qPCR, normalized to 18S rRNA expression and calibrated to the levels in uninfected cells. CD63, TGN46, HRS and annexin I transcripts were downregulated over the time-course of HCMV infection, while EEA1 and CI-M6PR transcripts levels changed slightly. TfR and CD-M6PR transcripts were transiently upregulated at 1 dpi and then downregulated with the mRNA levels of TfR increasing at 5 dpi and of CD-M6PR unaltered after 3 dpi. HCMV pp65 transcripts levels increased during the infection. Data corresponding to three biological replicates were averaged, bars represent the average normalized fold increase, and error bars represent standard errors of the means.

Journal: Cellular microbiology

Article Title: Human cytomegalovirus final envelopment on membranes containing both trans-Golgi network and endosomal markers.

doi: 10.1111/j.1462-5822.2009.01405.x

Figure Lengend Snippet: Fig. 7. Transcriptional regulation of cellular markers during HCMV infection. Equal number of BJ1 cells were mock infected or infected with HCMV at an moi of 3, and RNA was extracted at the indicated times. Gene expression of cellular markers and HCMV UL83 (viral protein pp65) was measured by qPCR, normalized to 18S rRNA expression and calibrated to the levels in uninfected cells. CD63, TGN46, HRS and annexin I transcripts were downregulated over the time-course of HCMV infection, while EEA1 and CI-M6PR transcripts levels changed slightly. TfR and CD-M6PR transcripts were transiently upregulated at 1 dpi and then downregulated with the mRNA levels of TfR increasing at 5 dpi and of CD-M6PR unaltered after 3 dpi. HCMV pp65 transcripts levels increased during the infection. Data corresponding to three biological replicates were averaged, bars represent the average normalized fold increase, and error bars represent standard errors of the means.

Article Snippet: Antigen Antibodies name Species and isotype Source HCMV glycoprotein H HCMV 16 Mouse IgG1 Helena Browne HCMV glycoprotein B HCMV 37 Mouse IgG2a Helena Browne HCMV UL33 Anti-HCMV UL33 Rabbit polyclonal Wade Gibson HCMV pp28 Anti-HCMV pp28 Mouse IgG1 Thomas Shenk HSV-1 glycoprotein D AP7 Mouse IgG2a Helena Browne CD63 (lumenal domain) Anti-CD63, 1B5 Mouse IgG2b Mark Marsh TGN46 Anti-TGN46 Sheep polyclonal AbD-Serotec Annexin I Anti-annexin I Mouse IgG1 Claire Isacke Early endosome antigen 1 Anti-EEA1 Mouse IgG1 BD Biosciences Hepatocyte growth factor-regulated Tyr-kinase substrate Anti-HRS 958/3 Rabbit polyclonal Sylvie Urbé Transferrin receptor (lumenal domain) B3/25 Mouse IgG1 Santa Cruz Biotechnology Transferrin receptor (cytoplasmic tail) H68.4 Mouse IgG1 Zymed Laboratories CI-M6PR (lumenal domain) Anti-MPR300 G VII-3 Rabbit polyclonal Kurt von Figura CI-M6PR (cytoplasmic tail) Anti-MPR300 H-CI-II Rabbit polyclonal Kurt von Figura CD-M6PR (lumenal domain) Anti-MPR46 HP-GI-3 Rabbit polyclonal Kurt von Figura CD-M6PR (cytoplasmic tail) Anti-MPR46 MSCI Rabbit polyclonal Kurt von Figura CD-M6PR Anti-MPR46, 22d4 Mouse IgG2b DSHB b-actin Anti-b-actin AC-15 Mouse IgG1 Sigma Aldrich Actin Anti-actin Rabbit polyclonal Sigma Aldrich Clathrin heavy chain Anti-CHC 23 Mouse IgG1 BD Biosciences © 2009 Blackwell Publishing Ltd, Cellular Microbiology, 12, 386–404 0.5 infectious particles per cell.

Techniques: Infection, Gene Expression, Expressing

Fig. 8. Immuno-gold localization of viral and cellular proteins on the viral envelope of isolated HCMV viral particles. Isolated viral particles were labelled with antibodies against HCMV viral glycoprotein gH (A, PAG10) and viral chemokine receptor-like protein UL33 (E, PAG15), TGN46 (B, PAG15), CD63 (C, PAG10), EEA1 (F, PAG10), annexin1 (G, PAG10), TfR lumenal domain (H, PAG10), CI-M6PR lumenal domain (I, PAG10) either without (A–D, H–I) or after permeabilization with saponin (E–G). (D) Double-labelling with antibodies against CD63 (PAG10) and TGN46 (PAG15). When viral particles were partially disrupted, uranyl acetate revealed the nucleocapsids (A, C, E–G). Scale bar, 50 nm.

Journal: Cellular microbiology

Article Title: Human cytomegalovirus final envelopment on membranes containing both trans-Golgi network and endosomal markers.

doi: 10.1111/j.1462-5822.2009.01405.x

Figure Lengend Snippet: Fig. 8. Immuno-gold localization of viral and cellular proteins on the viral envelope of isolated HCMV viral particles. Isolated viral particles were labelled with antibodies against HCMV viral glycoprotein gH (A, PAG10) and viral chemokine receptor-like protein UL33 (E, PAG15), TGN46 (B, PAG15), CD63 (C, PAG10), EEA1 (F, PAG10), annexin1 (G, PAG10), TfR lumenal domain (H, PAG10), CI-M6PR lumenal domain (I, PAG10) either without (A–D, H–I) or after permeabilization with saponin (E–G). (D) Double-labelling with antibodies against CD63 (PAG10) and TGN46 (PAG15). When viral particles were partially disrupted, uranyl acetate revealed the nucleocapsids (A, C, E–G). Scale bar, 50 nm.

Article Snippet: Antigen Antibodies name Species and isotype Source HCMV glycoprotein H HCMV 16 Mouse IgG1 Helena Browne HCMV glycoprotein B HCMV 37 Mouse IgG2a Helena Browne HCMV UL33 Anti-HCMV UL33 Rabbit polyclonal Wade Gibson HCMV pp28 Anti-HCMV pp28 Mouse IgG1 Thomas Shenk HSV-1 glycoprotein D AP7 Mouse IgG2a Helena Browne CD63 (lumenal domain) Anti-CD63, 1B5 Mouse IgG2b Mark Marsh TGN46 Anti-TGN46 Sheep polyclonal AbD-Serotec Annexin I Anti-annexin I Mouse IgG1 Claire Isacke Early endosome antigen 1 Anti-EEA1 Mouse IgG1 BD Biosciences Hepatocyte growth factor-regulated Tyr-kinase substrate Anti-HRS 958/3 Rabbit polyclonal Sylvie Urbé Transferrin receptor (lumenal domain) B3/25 Mouse IgG1 Santa Cruz Biotechnology Transferrin receptor (cytoplasmic tail) H68.4 Mouse IgG1 Zymed Laboratories CI-M6PR (lumenal domain) Anti-MPR300 G VII-3 Rabbit polyclonal Kurt von Figura CI-M6PR (cytoplasmic tail) Anti-MPR300 H-CI-II Rabbit polyclonal Kurt von Figura CD-M6PR (lumenal domain) Anti-MPR46 HP-GI-3 Rabbit polyclonal Kurt von Figura CD-M6PR (cytoplasmic tail) Anti-MPR46 MSCI Rabbit polyclonal Kurt von Figura CD-M6PR Anti-MPR46, 22d4 Mouse IgG2b DSHB b-actin Anti-b-actin AC-15 Mouse IgG1 Sigma Aldrich Actin Anti-actin Rabbit polyclonal Sigma Aldrich Clathrin heavy chain Anti-CHC 23 Mouse IgG1 BD Biosciences © 2009 Blackwell Publishing Ltd, Cellular Microbiology, 12, 386–404 0.5 infectious particles per cell.

Techniques: Isolation

Fig. 10. Model of HCMV envelopment and secretion. Nucleocapsids assembled in the nucleus are released into the cytoplasm through the nuclear membranes. In the perinuclear region, where virus factories are established, nucleocapsids acquire tegument before completing their final envelopment in a hybrid compartment or in the transport vesicles between the TGN and endosomes. During envelopment viral glycoproteins and TGN46, CD63, annexin 1, EEA1, TfR and CI-M6PR are incorporated into the viral membrane of numerous particles. Mature virions are transported inside a vacuole towards the plasma membrane to be secreted into the extracellular environment. N: nucleus, INM: inner nuclear membrane, ONM: outer nuclear membrane, ER, endoplasmic reticulum; TGN, trans-Golgi network; MVB, multivesicular body; EE, early endosome; V, vacuole; PM, plasma membrane.

Journal: Cellular microbiology

Article Title: Human cytomegalovirus final envelopment on membranes containing both trans-Golgi network and endosomal markers.

doi: 10.1111/j.1462-5822.2009.01405.x

Figure Lengend Snippet: Fig. 10. Model of HCMV envelopment and secretion. Nucleocapsids assembled in the nucleus are released into the cytoplasm through the nuclear membranes. In the perinuclear region, where virus factories are established, nucleocapsids acquire tegument before completing their final envelopment in a hybrid compartment or in the transport vesicles between the TGN and endosomes. During envelopment viral glycoproteins and TGN46, CD63, annexin 1, EEA1, TfR and CI-M6PR are incorporated into the viral membrane of numerous particles. Mature virions are transported inside a vacuole towards the plasma membrane to be secreted into the extracellular environment. N: nucleus, INM: inner nuclear membrane, ONM: outer nuclear membrane, ER, endoplasmic reticulum; TGN, trans-Golgi network; MVB, multivesicular body; EE, early endosome; V, vacuole; PM, plasma membrane.

Article Snippet: Antigen Antibodies name Species and isotype Source HCMV glycoprotein H HCMV 16 Mouse IgG1 Helena Browne HCMV glycoprotein B HCMV 37 Mouse IgG2a Helena Browne HCMV UL33 Anti-HCMV UL33 Rabbit polyclonal Wade Gibson HCMV pp28 Anti-HCMV pp28 Mouse IgG1 Thomas Shenk HSV-1 glycoprotein D AP7 Mouse IgG2a Helena Browne CD63 (lumenal domain) Anti-CD63, 1B5 Mouse IgG2b Mark Marsh TGN46 Anti-TGN46 Sheep polyclonal AbD-Serotec Annexin I Anti-annexin I Mouse IgG1 Claire Isacke Early endosome antigen 1 Anti-EEA1 Mouse IgG1 BD Biosciences Hepatocyte growth factor-regulated Tyr-kinase substrate Anti-HRS 958/3 Rabbit polyclonal Sylvie Urbé Transferrin receptor (lumenal domain) B3/25 Mouse IgG1 Santa Cruz Biotechnology Transferrin receptor (cytoplasmic tail) H68.4 Mouse IgG1 Zymed Laboratories CI-M6PR (lumenal domain) Anti-MPR300 G VII-3 Rabbit polyclonal Kurt von Figura CI-M6PR (cytoplasmic tail) Anti-MPR300 H-CI-II Rabbit polyclonal Kurt von Figura CD-M6PR (lumenal domain) Anti-MPR46 HP-GI-3 Rabbit polyclonal Kurt von Figura CD-M6PR (cytoplasmic tail) Anti-MPR46 MSCI Rabbit polyclonal Kurt von Figura CD-M6PR Anti-MPR46, 22d4 Mouse IgG2b DSHB b-actin Anti-b-actin AC-15 Mouse IgG1 Sigma Aldrich Actin Anti-actin Rabbit polyclonal Sigma Aldrich Clathrin heavy chain Anti-CHC 23 Mouse IgG1 BD Biosciences © 2009 Blackwell Publishing Ltd, Cellular Microbiology, 12, 386–404 0.5 infectious particles per cell.

Techniques: Virus, Membrane, Clinical Proteomics

Spag protein structure and patterns of gene expression and protein accumulation during Drosophila development. A, schematic structure of the Spag protein and evolutionary conservation of the TPR region in Spag orthologs from yeast to human. Filled boxes represent predicted structural domains and lines unstructured domains. The three TPR motifs forming the TPR region are indicated as red, blue, and green boxes. TPR 3′ is a putative helical domain absent in Tah1. Orange box represents a potential α-helical junction between TPR 3 and TPR 3′. The potential monad binding motif (interPro 025986) overlaps with a predicted structured domain (magenta). Below are aligned the amino acid residues characterizing the three TPR motifs in S. cerevisiae (S.c.), D. melanogaster (D.m.), Danio rero (D.r.), Homo sapiens (H.s.), and Xenopus tropicalis (X.t.). Conserved residues identified in Tah1 for Hsp90 binding are indicated in bold red (21). Blue indicates additional residues conserved in TPR domains and important for Hsp70/Hsp90 binding, as defined previously (23). B, expression pattern of spag mRNA (left panel) and pattern of Spag protein accumulation (right panel) during Drosophila development. RNAs and proteins were extracted from (E) embryos with age in hours after egg laying indicated above the lanes (L1, L2, and L3) first, second, and third instar larvae, (P) pupae, (F) female, and (M) male 3-days old imagos. The Northern blot was hybridized with either 32P-labeled spag or β-tubulin cDNA. The Western blot was probed with anti-Spag or anti-ribosomal P40 protein polyclonal antibodies. The β-tubulin cDNA and anti-P40 antibodies were used for loading control.

Journal: The Journal of Biological Chemistry

Article Title: Drosophila Spag Is the Homolog of RNA Polymerase II-associated Protein 3 (RPAP3) and Recruits the Heat Shock Proteins 70 and 90 (Hsp70 and Hsp90) during the Assembly of Cellular Machineries * ♦

doi: 10.1074/jbc.M113.499608

Figure Lengend Snippet: Spag protein structure and patterns of gene expression and protein accumulation during Drosophila development. A, schematic structure of the Spag protein and evolutionary conservation of the TPR region in Spag orthologs from yeast to human. Filled boxes represent predicted structural domains and lines unstructured domains. The three TPR motifs forming the TPR region are indicated as red, blue, and green boxes. TPR 3′ is a putative helical domain absent in Tah1. Orange box represents a potential α-helical junction between TPR 3 and TPR 3′. The potential monad binding motif (interPro 025986) overlaps with a predicted structured domain (magenta). Below are aligned the amino acid residues characterizing the three TPR motifs in S. cerevisiae (S.c.), D. melanogaster (D.m.), Danio rero (D.r.), Homo sapiens (H.s.), and Xenopus tropicalis (X.t.). Conserved residues identified in Tah1 for Hsp90 binding are indicated in bold red (21). Blue indicates additional residues conserved in TPR domains and important for Hsp70/Hsp90 binding, as defined previously (23). B, expression pattern of spag mRNA (left panel) and pattern of Spag protein accumulation (right panel) during Drosophila development. RNAs and proteins were extracted from (E) embryos with age in hours after egg laying indicated above the lanes (L1, L2, and L3) first, second, and third instar larvae, (P) pupae, (F) female, and (M) male 3-days old imagos. The Northern blot was hybridized with either 32P-labeled spag or β-tubulin cDNA. The Western blot was probed with anti-Spag or anti-ribosomal P40 protein polyclonal antibodies. The β-tubulin cDNA and anti-P40 antibodies were used for loading control.

Article Snippet: Proteins were detected as follows: Rpb1 detected with mouse monoclonal PB7-C2 antibody; Rpb2 with goat S20 from Santa Cruz Biotechnology; Nop58 with polyclonal antibodies generated from rabbits immunized with an KKLQEVDSLWKEFETPEK peptide ( 14 ); p70 S6K with monoclonal antibody SC-9027 from Santa Cruz Biotechnology; phospho-Thr-398 p70 S6K with monoclonal antibody provided by Cell Signaling Technology (reference 9209); fibrillarin with monoclonal antibody 5821 from Abcam; tubulin with monoclonal 12G10 (Developmental Studies Hybridoma Bank, Iowa City, IA); polyclonal anti-15.5K antibodies from Santa Cruz Biotechnology (SC-86760); polyclonal anti-P40 antibodies have been described previously ( 15 ), and anti-FLAG M2 monoclonal antibody was from Sigma.

Techniques: Gene Expression, Binding Assay, Expressing, Northern Blot, Labeling, Western Blot, Control

Spag and Nufip are required for box C/D sno core protein stabilization. A, Western blot analysis of protein extracts from third instar larvae spagk12101, as compared with wild-type w1118 (Ct), showed a significant diminution in the content of Nop58 (Nop5) and 15.5K (Hoip) but not fibrillarin. Tubulin was used as a loading control. B, this phenomenon was also observed in pupae extracts from animals in which RNAi was induced by Gal4act5C to knock down spag (Gal4act5c/spagRNAi as compared with Gal4act5c/+). C, similar results were observed in pupae extracts from animals in which RNAi was induced against Nufip (C, Gal4act5c/nufipRNAi compared with w1118).

Journal: The Journal of Biological Chemistry

Article Title: Drosophila Spag Is the Homolog of RNA Polymerase II-associated Protein 3 (RPAP3) and Recruits the Heat Shock Proteins 70 and 90 (Hsp70 and Hsp90) during the Assembly of Cellular Machineries * ♦

doi: 10.1074/jbc.M113.499608

Figure Lengend Snippet: Spag and Nufip are required for box C/D sno core protein stabilization. A, Western blot analysis of protein extracts from third instar larvae spagk12101, as compared with wild-type w1118 (Ct), showed a significant diminution in the content of Nop58 (Nop5) and 15.5K (Hoip) but not fibrillarin. Tubulin was used as a loading control. B, this phenomenon was also observed in pupae extracts from animals in which RNAi was induced by Gal4act5C to knock down spag (Gal4act5c/spagRNAi as compared with Gal4act5c/+). C, similar results were observed in pupae extracts from animals in which RNAi was induced against Nufip (C, Gal4act5c/nufipRNAi compared with w1118).

Article Snippet: Proteins were detected as follows: Rpb1 detected with mouse monoclonal PB7-C2 antibody; Rpb2 with goat S20 from Santa Cruz Biotechnology; Nop58 with polyclonal antibodies generated from rabbits immunized with an KKLQEVDSLWKEFETPEK peptide ( 14 ); p70 S6K with monoclonal antibody SC-9027 from Santa Cruz Biotechnology; phospho-Thr-398 p70 S6K with monoclonal antibody provided by Cell Signaling Technology (reference 9209); fibrillarin with monoclonal antibody 5821 from Abcam; tubulin with monoclonal 12G10 (Developmental Studies Hybridoma Bank, Iowa City, IA); polyclonal anti-15.5K antibodies from Santa Cruz Biotechnology (SC-86760); polyclonal anti-P40 antibodies have been described previously ( 15 ), and anti-FLAG M2 monoclonal antibody was from Sigma.

Techniques: Western Blot, Control, Knockdown

Fig. 2. Overexpression of CrbFLAGintra during late larval and pupal development affects photoreceptor apico-basal polarity. (A–H) Immunostainings on cross-sections of adult wild-type (wt) eyes (A,C,E,G) and eyes overexpressing CrbFLAGintra using elavGal4 (elav.CrbFLAGintra) (B,D,F,H). The sections were stained for Drosophila E-Cad (DE-Cad) (A,B), Rh1 (C,D), Spam (Eys) (E,F) or the alpha subunit of Na+/K+-ATPase (G,H) (in red), and Sdt (A–C), Crb (D,E,G,H) or Drosophila PATJ (F) (in green). B-, D-, F- and H- represent higher magnifications of one single ommatidium shown in B0, D0, F0 and G0, respectively. The F-actin stain (blue) shows the rhabdomeres. Sdt (B9), Crb (D9,H9) and Drosophila PATJ (F0) exhibit expanded expression domains and often localise adjacent to ectopic rhabdomeres. Additionally, Sdt localises between split rhabdomeres (B9,B0, white arrowhead). Note ectopic Drosophila E-Cad flanking ectopic rhabdomeres (B, B0,B-, white arrows). Ectopic apical poles are positive for Rh1 (D,D0,D-, arrows) and Spam (Eys) (F, F0, F-, arrows). Na+/K+-ATPase is absent from ectopic rhabdomeres and diminished at membranes with high levels of ectopic Crb (H,H0,H-, the decrease is indicated by a cross). In addition, Na+/K+-ATPase is present at the stalk of cells with ectopic rhabdomeres, which are not R7 (H,H0,H-, some cells other than R7 are indicated by asterisks). Scale bars: 2 mm. (I,J) Schematic summary of the distribution of proteins (as shown in A–H) in wild-type ommatidia (I) and ommatidia overexpressing CrbFLAGintra (J).

Journal: Journal of cell science

Article Title: Increased levels of the cytoplasmic domain of Crumbs repolarise developing Drosophila photoreceptors.

doi: 10.1242/jcs.091223

Figure Lengend Snippet: Fig. 2. Overexpression of CrbFLAGintra during late larval and pupal development affects photoreceptor apico-basal polarity. (A–H) Immunostainings on cross-sections of adult wild-type (wt) eyes (A,C,E,G) and eyes overexpressing CrbFLAGintra using elavGal4 (elav.CrbFLAGintra) (B,D,F,H). The sections were stained for Drosophila E-Cad (DE-Cad) (A,B), Rh1 (C,D), Spam (Eys) (E,F) or the alpha subunit of Na+/K+-ATPase (G,H) (in red), and Sdt (A–C), Crb (D,E,G,H) or Drosophila PATJ (F) (in green). B-, D-, F- and H- represent higher magnifications of one single ommatidium shown in B0, D0, F0 and G0, respectively. The F-actin stain (blue) shows the rhabdomeres. Sdt (B9), Crb (D9,H9) and Drosophila PATJ (F0) exhibit expanded expression domains and often localise adjacent to ectopic rhabdomeres. Additionally, Sdt localises between split rhabdomeres (B9,B0, white arrowhead). Note ectopic Drosophila E-Cad flanking ectopic rhabdomeres (B, B0,B-, white arrows). Ectopic apical poles are positive for Rh1 (D,D0,D-, arrows) and Spam (Eys) (F, F0, F-, arrows). Na+/K+-ATPase is absent from ectopic rhabdomeres and diminished at membranes with high levels of ectopic Crb (H,H0,H-, the decrease is indicated by a cross). In addition, Na+/K+-ATPase is present at the stalk of cells with ectopic rhabdomeres, which are not R7 (H,H0,H-, some cells other than R7 are indicated by asterisks). Scale bars: 2 mm. (I,J) Schematic summary of the distribution of proteins (as shown in A–H) in wild-type ommatidia (I) and ommatidia overexpressing CrbFLAGintra (J).

Article Snippet: The following primary antibodies were used: rat anti-Crb2.8 antibody (1:1000) (Richard et al., 2006), rabbit anti-Sdt-PDZ antibody (1:500) (Berger et al., 2007), mouse anti-Sdt-PDZ antibody (B1-8, 1:200) (Bulgakova et al., 2010), rabbit antiDrosophila-PATJ antibody (1:500) (Richard et al., 2006), rat anti-DE-Cad antibody (DCAD2, 1:50, DSHB), mouse anti-Na+/K+-ATPase antibody (a5, alpha subunit, 1:500 concentrate, DSHB), rabbit anti-Rh1 antibody (1:1000) (Satoh et al., 2005), mouse anti-Rh1 (4C5, 1:100, DSHB), mouse anti-Spam antibody (21A6, 1:100, DSHB) (Zelhof et al., 2006), mouse anti-FLAG M2 antibody (1:1000, Sigma), rabbit anti-FLAG antibody (1:200, Sigma, F7425), rabbit anti-Baz antibody (1:400, kindly provided by Andreas Wodarz) (Wodarz et al., 1999), guinea pig anti-Par-6 antibody (1:1000, kindly provided by Andreas Wodarz) (Kim et al., 2009), mouse anti-Arm antibody (N2 7A1, 1:50, DSHB), rabbit anti-PKC f C20 (1:1000, Santa Cruz Biotechnology), rabbit anti-phosphorylated-Ezrin/Radixin/Moesin (1:100, Cell Signalling Technology), and rabbit anti-bH-spec (1:500, kindly provided by Graham Thomas) (Thomas and Kiehart, 1994).

Techniques: Over Expression, Staining, Expressing

Fig. 1 – RAI1/Rai1 protein organization and induction by retinoic acid. (A) A schematic representation of the Rai1 protein in human, mouse, two frog species (X. laevis and X. tropicalis), and zebrafish. This illustrates that the organization of the primary protein domains, including the Trans-Activation Domain (TAD), Nuclear Localization Signal containing region (NLS; see Supplementary material Fig. S1B for details), and extended plant homeodomain (ePHD/ADD, also known as a PHD finger) are conserved in the species examined (see Supplementary material Fig. S1A for sequence alignment of the PHD finger). A 14 poly-glutamine stretch (Q) is found in humans and reduced glutamine stretches can also be found in a similar region of other species except zebrafish. The NLS region contains the first known nuclear localization sequence (Darvekar et al., 2013). Figure not to scale. (B) Retinoic acid induces expression of rai1 during Xenopus development. Quantitative PCR of rai1 mRNA after all-trans retinoic treatment (ATRA) at stage 23 showing significant 3.91-fold increase (t-test, p = 0.0037) relative to the control housekeeping gene (ef1 alpha). (C) Retinoic acid may directly regulate rai1. A histogram of RAR binding sites in a 2 kb region upstream of the rai1 start site in X. laevis reveals the enrichment of retinoic acid receptor binding sites.

Journal: Mechanisms of development

Article Title: Retinoic acid induced-1 (Rai1) regulates craniofacial and brain development in Xenopus.

doi: 10.1016/j.mod.2014.05.004

Figure Lengend Snippet: Fig. 1 – RAI1/Rai1 protein organization and induction by retinoic acid. (A) A schematic representation of the Rai1 protein in human, mouse, two frog species (X. laevis and X. tropicalis), and zebrafish. This illustrates that the organization of the primary protein domains, including the Trans-Activation Domain (TAD), Nuclear Localization Signal containing region (NLS; see Supplementary material Fig. S1B for details), and extended plant homeodomain (ePHD/ADD, also known as a PHD finger) are conserved in the species examined (see Supplementary material Fig. S1A for sequence alignment of the PHD finger). A 14 poly-glutamine stretch (Q) is found in humans and reduced glutamine stretches can also be found in a similar region of other species except zebrafish. The NLS region contains the first known nuclear localization sequence (Darvekar et al., 2013). Figure not to scale. (B) Retinoic acid induces expression of rai1 during Xenopus development. Quantitative PCR of rai1 mRNA after all-trans retinoic treatment (ATRA) at stage 23 showing significant 3.91-fold increase (t-test, p = 0.0037) relative to the control housekeeping gene (ef1 alpha). (C) Retinoic acid may directly regulate rai1. A histogram of RAR binding sites in a 2 kb region upstream of the rai1 start site in X. laevis reveals the enrichment of retinoic acid receptor binding sites.

Article Snippet: Primary antibodies included a rabbit polyclonal anti-cleaved caspase-3 (Cell Signaling, 9661S, diluted 1:1000), rabbit polyclonal human RAI1 (generated by Alpha Diagnostic International for the Elsea lab, diluted 1:100 for further details see Supplementary material) and Xenopus monoclonal antibody that detects a Xenopus neurofilament (Developmental Studies Hybridoma Bank, 3A10, diluted 1:100).

Techniques: Activation Assay, Sequencing, Expressing, Real-time Polymerase Chain Reaction, Control, Binding Assay

Fig. 5 – Decreased Rai1 results in defects in axon patterns, brain morphology, increased forebrain apoptosis and decreased bdnf expression. Rai1 morphants injected with 5 ng of morpholino were used for each analysis. (A, B) Dorsal view of representative tadpoles (stage 42) labeled with a neurofilament marker (3A10) showing aberrant axon patterns and axonal beading in Rai1 morphants (arrowheads). The brain is outlined with white dots to provide context. (C, D) Dorsal view of representative tadpoles (stage 42) where the ventricles were injected with Texas Red labeled dextran. Fluorescent images were superimposed onto the light micrographs to provide context. Results show that Rai1 morphants have dramatically decreased ventricle space in the forebrain. (E, F) A marker of apoptosis, cleaved caspase-3 (green) reveals increased cell death in the forebrain of Rai1 morphants. Shows a compressed z-stack of a midline sagital section at stage 33–34. Counterstained with nuclear marker (propidium iodide; red). The brain is outlined with white dots and the position of the developing embryonic mouth is indicated by a white arrow for context. (G) Bar graph of the average number of cleaved caspase-3 positive cells showing a significant difference (t tests, brain; p = 2.48E-10, face; p = 0.713) in the brain (star) but not in the face. (H) qPCR analysis of bdnf expression in Rai1 morphants at stage 25–26 (28 hpf). Expression was normalized to ef1alpha and presented as a fold change compared to control. Results indicate a statistically significant negative 1.5-fold change (t test, p = 0.0049) in Rai1 morphants.

Journal: Mechanisms of development

Article Title: Retinoic acid induced-1 (Rai1) regulates craniofacial and brain development in Xenopus.

doi: 10.1016/j.mod.2014.05.004

Figure Lengend Snippet: Fig. 5 – Decreased Rai1 results in defects in axon patterns, brain morphology, increased forebrain apoptosis and decreased bdnf expression. Rai1 morphants injected with 5 ng of morpholino were used for each analysis. (A, B) Dorsal view of representative tadpoles (stage 42) labeled with a neurofilament marker (3A10) showing aberrant axon patterns and axonal beading in Rai1 morphants (arrowheads). The brain is outlined with white dots to provide context. (C, D) Dorsal view of representative tadpoles (stage 42) where the ventricles were injected with Texas Red labeled dextran. Fluorescent images were superimposed onto the light micrographs to provide context. Results show that Rai1 morphants have dramatically decreased ventricle space in the forebrain. (E, F) A marker of apoptosis, cleaved caspase-3 (green) reveals increased cell death in the forebrain of Rai1 morphants. Shows a compressed z-stack of a midline sagital section at stage 33–34. Counterstained with nuclear marker (propidium iodide; red). The brain is outlined with white dots and the position of the developing embryonic mouth is indicated by a white arrow for context. (G) Bar graph of the average number of cleaved caspase-3 positive cells showing a significant difference (t tests, brain; p = 2.48E-10, face; p = 0.713) in the brain (star) but not in the face. (H) qPCR analysis of bdnf expression in Rai1 morphants at stage 25–26 (28 hpf). Expression was normalized to ef1alpha and presented as a fold change compared to control. Results indicate a statistically significant negative 1.5-fold change (t test, p = 0.0049) in Rai1 morphants.

Article Snippet: Primary antibodies included a rabbit polyclonal anti-cleaved caspase-3 (Cell Signaling, 9661S, diluted 1:1000), rabbit polyclonal human RAI1 (generated by Alpha Diagnostic International for the Elsea lab, diluted 1:100 for further details see Supplementary material) and Xenopus monoclonal antibody that detects a Xenopus neurofilament (Developmental Studies Hybridoma Bank, 3A10, diluted 1:100).

Techniques: Expressing, Injection, Labeling, Marker, Control