hek 293t cells Search Results


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  • 99
    ATCC hek 293t cells
    Comprehensive analysis of protein N -glycosylation in human cells. a Comparison of unique protein N -glycosylation sites identified in MCF7 cells in duplicate experiments. b Comparison of unique glycosylation sites and glycoproteins identified with the boronic acid derivative magnetic beads (designated as BA, shown in green) and with the dendrimer beads conjugated with the boronic acid derivative (DBA) (blue). c Abundance distributions of N -glycoproteins identified with the BA (green) or DBA (blue) beads. d Overlap of N -glycoproteins in three different types of cells (MCF7, <t>HEK</t> <t>293T,</t> and Jurkat). e Protein-clustering results for 180 N -glycoproteins identified exclusively in Jurkat cells. f Distribution of membrane proteins (type I, II, III, and IV, and multi-pass transmembrane (TM)) among all identified N -glycoproteins. g N -glycosylation site locations on 301 receptors with X -axis as the TM domain. Each glycoprotein sequence was aligned against the transmembrane domain, and the glycosylation sites are indicated as yellow dots. All sites are located in the extracellular space. h Domain analysis of N -glycoproteins showing the number of N -glycoproteins containing the most highly enriched domains and their corresponding P values
    Hek 293t Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 5472 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hek 293 cells
    PKA and AMPK phosphorylate the V-ATPase A subunit in vitro. Recombinant FLAG-tagged A subunit was expressed in <t>HEK-293</t> cells, immunoprecipitated, and incubated with [γ- 32 P]ATP in the presence or absence of PKA catalytic subunit or in the presence or absence of AMPK holoenzyme. A phosphoscreen image ( top ) and immunoblot ( bottom ) of the same membrane are shown (representative of three experiments). In the top , the V-ATPase A subunit becomes phosphorylated in the presence of PKA and also in the presence of AMPK (first and third lanes). Both the PKA catalytic subunit and the AMPK-α subunit become autophosphorylated, as indicated. The immunoblot ( bottom ) reveals similar loading of V-ATPase A subunit in all lanes.
    Hek 293 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12989 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC hek 293 cell
    Conversion of NBCe1 into an anion exchanger. a NBCe1 and AE1 amino-acid alignment showing location of the NBCe1 residues replaced with the corresponding AE1 residues. b Location of the NBCe1 residues replaced with the corresponding AE1 residues located in ion coordination site regions of NBCe1 and AE1 22 (PDB: 4YZF) formed by antiparallel β-strands preceding TMs 3 and 10, and in TM 8 and 10 are shown. c, e-g Loss of Na + -driven base transport and d, h-k gain of Cl − -driven base transport in the mutant constructs. l , m Cell surface expression. Immunoblot analysis of sulfo-NHS-SS-biotin labeled plasma membrane proteins in <t>HEK-293</t> cells-expressing wt-NBCe1 and NBCe1 mutant (A+B+C). l Cell surface expression was normalized to GAPDH (mutant vs. wt, p
    Hek 293 Cell, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    ATCC hek293t cells
    BRD4 mediates HIV D4-A7 RNA splicing. <t>HEK293T</t> cells were co-transfected with the pLTR.gp140/EGFP.Rev∆38/DsRed splicing reporter, 20 ng of pRev and 100 ng of pTat101 (AD8) without (wild-type, WT) or with specific mutations (K28A, K50A, K50/51A, K71A and R53A) then treated with DMSO, 1 μM JQ1 (+) or JQ1 (−). Cells were harvested at 48 h, EGFP and DsRed expression was quantified by flow cytometry. a The flow cytometry gating strategy used to identify % EGFP and % DsRed positive cells in a representative result from n = 3 independent experiments, each conducted in triplicate. b The mean percentage of spliced product DsRed/(DsRed + EGFP) from the 3 independent experiments is shown. Comparisons of each condition to JQ1 (+) were made using Friedman nonparametric test. Only statistically significant comparisons are shown *p
    Hek293t Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 98/100, based on 13906 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Addgene inc hek293t cells
    dLRRK and hLRRK2 promote cap-dependent translation in postsynaptic muscles. ( a ) Quantification of 5′-UTR luciferase reporter activity in response to co-transfection with either wild-type hLRRK2 wt or kinase-dead hLRRK2 3XKD normalized to psiCHECK2 response in <t>HEK293T.</t> n =3 experiments, * P =0.043. See also Supplementary Table 2 . ( b ) Western blot analysis of in vivo Fur1-5′-UTR-eGFP reporter expression when co-expressed with either dLRRK 3KD (+/UAS- Fur1 -5′-UTR- eGFP ; UAS -dLRRK 3KD / MHC-Gal4 ) or wild-type dLRRK wt (UAS -dLRRK wt /UAS- Fur1 -5′-UTR- eGFP ; +/ MHC-Gal4 ). Left: Western blotting probed with (top) anti-GFP and (bottom) anti-Actin as loading control. Right: quantification of Fur1-5′-UTR-eGFP expression normalized to actin levels. n =3 for each. * P
    Hek293t Cells, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 4756 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher hek293t cells
    Association of C4orf14 with the mitochondrial small ribosomal subunit. ( A ) Affinity purified C4orf14.FLAG.StrepII protein was isolated from mitochondria fractions of transgenic <t>HEK293T</t> cells and the concentrated eluted fractions resolved by SDS–PAGE. Proteins identified by MS are indicated on the left and right of the gel. They included 22 polypeptides of the 28S subunit (MRPS; see Supplementary Table S1 ); ( B ) sucrose-gradient purified mitochondria from HEK293T cells were lysed and fractionated on sucrose gradients. Antibodies to MRPS2 and MRPS18, and MRPL3 and MRPL11 were used as markers of the 28S and 39S subunits, respectively. ( C ) 143B cells were transfected with dsRNA (c3 or c6) targeting C4orf14 mRNA and the effects on mitochondrial protein synthesis (i); selected proteins in mitochondria (ii) and mitochondrial ribosomal RNAs (panel D and Supplementary Figure S2 ) were examined 72 h later. GAPDH: glyceraldehyde-3-phosphate dehydrogenase, the outer mitochondrial membrane protein TOM20, a putative mitochondrial RNA helicase DHX30 and components of the 55S ribosome (MRPS2, MRPS29, MRPL3 and MRPL11).
    Hek293t Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 35536 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Mirus Bio hek293t cells
    BORIS isoforms bind and activate the transcription of conserved human and mouse CST testis-specific promoter regions. ( A ) Schematic representation of multiple mouse CST promoters, where exon 1f s has been shown to be testis specific [48] and the BORIS binding site has been mapped to the testis-specific CST promoter [9] . The alignment of human and mouse BORIS binding site in CST promoter is shown at the bottom. The 5 cytosines boxed in the alignment are contact nucleotides for BORIS protein as it was determined by methylation interference assays [9] . Nucleotides that are 100% identical to the human nucleotide sequence are shaded in grey. Dashes indicate insertions or deletions. ( B ) and ( C ) Most BORIS isoforms bind to the human and mouse testis-specific CST promoter in EMSA. The number of ZFs and utilization of alternative N-and C-termini for each BORIS isoform are shown at the bottom of gel. The band shifts specific for particular N- and C-termini are indicated by arrows. Luciferase and CTCF were used as negative and positive controls, respectively. ( D ) The mouse CST promoter is transcriptionally activated by BORIS isoforms in <t>HEK293T</t> cells. HEK293T cells were either co-transfected with mouse Boris, mouse Ctcf, empty pCI vector (EV) or the human BORIS isoform constructs, as well as with pGL-3 containing 359 bp of wild type or mutant CST promoter. Luciferase assays were done 48 h after transfections. All luciferase activities were normalized for transfection efficiency by measuring the Renilla luciferase activity from the co-transfected pRL-TK vector. Error bars are standard deviations.
    Hek293t Cells, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 92/100, based on 1871 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher geneblazer cckbr nfat bla hek293t cells
    BORIS isoforms bind and activate the transcription of conserved human and mouse CST testis-specific promoter regions. ( A ) Schematic representation of multiple mouse CST promoters, where exon 1f s has been shown to be testis specific [48] and the BORIS binding site has been mapped to the testis-specific CST promoter [9] . The alignment of human and mouse BORIS binding site in CST promoter is shown at the bottom. The 5 cytosines boxed in the alignment are contact nucleotides for BORIS protein as it was determined by methylation interference assays [9] . Nucleotides that are 100% identical to the human nucleotide sequence are shaded in grey. Dashes indicate insertions or deletions. ( B ) and ( C ) Most BORIS isoforms bind to the human and mouse testis-specific CST promoter in EMSA. The number of ZFs and utilization of alternative N-and C-termini for each BORIS isoform are shown at the bottom of gel. The band shifts specific for particular N- and C-termini are indicated by arrows. Luciferase and CTCF were used as negative and positive controls, respectively. ( D ) The mouse CST promoter is transcriptionally activated by BORIS isoforms in <t>HEK293T</t> cells. HEK293T cells were either co-transfected with mouse Boris, mouse Ctcf, empty pCI vector (EV) or the human BORIS isoform constructs, as well as with pGL-3 containing 359 bp of wild type or mutant CST promoter. Luciferase assays were done 48 h after transfections. All luciferase activities were normalized for transfection efficiency by measuring the Renilla luciferase activity from the co-transfected pRL-TK vector. Error bars are standard deviations.
    Geneblazer Cckbr Nfat Bla Hek293t Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC hek293t 17 cells
    Localized deactivation of PTP1B PS . a A biosensor for PTP1B activity. Src-mediated phosphorylation of the substrate domain causes it to bind to the SH2 domain, triggering a conformational change that decreases FRET; dephosphorylation by PTP1B increases FRET. b Src increases the donor/acceptor emission ratio in vitro (normalized by the buffer-only condition); EDTA or PTP1B prevent this increase. Error bars denote propagated SE for measurements of n = 3 independent experiments (measurements are normalized to a to a buffer-only condition). c The percent change in donor/acceptor emission ratio over 1 min within 5-μm circular regions located in the cytosol and nucleus of COS-7 cells activated with 457 nm light. Each condition includes the interquartile average, associated data points, and SE for n = 11 biological replicates. The p -values correspond to a two-tailed Student’s t test. d An image of localized illumination (405 nm) of a COS-7 cell expressing both PTP1B PS and biosensor. Circles delineate irradiated (red) and secondary (blue) regions, and colors show the donor/acceptor emission ratio (scale bar, 10 μm). e Time courses of FRET in irradiated and secondary regions. Shading highlights 5-s periods before (gray), during (blue), and after (gray) illumination. f A depiction of a <t>HEK293T/17</t> cell expressing PTP1B PS** . Insulin stimulates phosphorylation of the membrane-bound insulin receptor (IR); PTP1B dephosphorylates it. g ELISA-based measurements of IR phosphorylation in (i) wild-type HEK293T/17 cells and (ii) HEK293T/17 cells stably expressing PTP1B PS** or PTP1B PS** (C450M). Insulin-mediated simulation of IR, BBR-mediated inhibition of PTP1B, and photoinactivation of PTP1B all increase IR phosphorylation. The dark state of PTP1B PS** and the dark and light states of PTP1B PS** (C450M), by contrast, leave IR phosphorylation unaltered from its levels in the wild-type strain (DMSO). The plotted data depict the mean, propagated SE, and associated data points for measurements of n = 3 biological replicates (relative to a buffer-only condition). Source data are provided as a Source Data file.
    Hek293t 17 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 916 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Polyplus Transfection hek293t cells
    Detection of residual PEG. Two gels were loaded with PEG 6000 in decreasing amounts (10, 5, 2.5, 1, 0.5, 0.25, 0.1, 0.01 µg), protein marker, and 6 µg of the final EV samples of <t>HEK293T-CD63-eGF</t> clone B2 and C5 cells. Gels were run under identical conditions. PEG was detectable after barium iodide staining (a); proteins following protein specific imperial stain (b). Signals in (a) were recorded with Image Studio™ Software (LI-COR) and plotted against the PEG concentration of the calibration values (c). The graphic evolution indicates a linear equation of y = 15.692× + 3.4984 which was used to calculate the revealed PEG concentration of 0.02% in the final EV samples.
    Hek293t Cells, supplied by Polyplus Transfection, used in various techniques. Bioz Stars score: 92/100, based on 1097 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Corning Life Sciences hek293t cells
    Effect of FRG1 expression on <t>HEK293T</t> cell proliferation and scratch wound healing ( A ) Shows Western blot to confirm ectopic expression of FRG1 in HEK293T. ( B ) Shows verification of reduced FRG1 levels after RNAi silencing in HEK293T, by Western blot. ( C ) Represents measurement of cell proliferation in HEK293T with ectopic expression of FRG1 compared with empty vector control (pCMV6.XL5), by MTS reagent. ( D ) Represents measurement of cell proliferation in HEK293T with knockdown of FRG1 compared with scrambled vector control (pLKO1.sc), by MTS reagent. ( E ) Shows representative images of scratch wound healing assay of HEK293T cells with ectopic expression of FRG1 and respective vector control (pCMV6.XL5). ( F ) Shows representative images of scratch wound healing assay of HEK293T with FRG1 knockdown and respective scrambled vector control (pLKO1.sc). ( G ) Represents representative graph for scratch wound healing assay of HEK293T cells with ectopic expression of FRG1, compared with empty vector control (pCMV6.XL5). ( H ) Shows representative graph for scratch wound healing assay of HEK293T cells with FRG1 knockdown, compared with empty vector control (pLKO1.sc). # represents P > 0.05 , * represents P
    Hek293t Cells, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 92/100, based on 850 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson hek293t cells
    CEACAM16 interacts homotypically. <t>HEK293T</t> cells either remained untransfected ( A ) or were transiently transfected with expression plasmids encoding full-length murine native ( B ) or GPI-linked CEACAM16 ( C ). The transfected cells were harvested by repeated pipetting, incubated at room temperature, attached to glass slides using the cytospin method, and stained with anti-murine CEACAM16 immune sera by immunocytochemistry ( brown stain ). Note the homotypic sorting of cells expressing CEACAM16 on the cell membrane ( C ) but not of cells with intracellular expression ( B ). This suggests that CEACAM16 can interact homotypically in trans . Aggregation of HEK293T cells was tested as above after transient expression of GPI-anchored human CEACAM16 N1 and N2 domains ( D and E , left panels ). N1- and N2-expressing cells were detected by immunocytochemistry using an anti-myc-tag antibody. Representative regions of the cytospins are shown. The expression levels of myc-tagged fusion proteins and the fraction of positive cells were determined by FACScan analysis using anti-myc and FITC-conjugated secondary antibodies ( D and E , right panels , green curves ). Samples incubated without primary antibody served as a negative control ( blue curves ). Aggregation mediated by N1 and N2 domains was quantitated by determination of the fraction of N1- and N2-positive cells, respectively, in single and double cell populations as well as in aggregates ≥3 using the cytospins shown in D and E ( F ). Note that surface expression of N2 preferentially induces multicellular aggregates. One of two experiments with similar results is shown. Full-length murine native CEACAM16 ( G ) or a deletion constructs lacking N2 ( H ) or N1 and A ( J ) or B and N2 ( K ) or encoding only the N1 or N2 domain either soluble ( I , upper panel ) or GPI-linked ( I , lower panels ) were expressed in HEK293T cells by transient transfection. Cell lysates ( cells ) or cell culture supernatants with or without prior treatment with DTT were separated by gel electrophoresis, and CEACAM16 was detected by Western blot analysis with a monoclonal anti-myc antibody. The domain organization of the encoded proteins is schematically shown to the right . Cysteines possibly involved in intermolecular disulfide bridges are indicated by yellow dots , green tags represent NH 2 -terminally added myc epitopes, and green arrows symbolize GPI anchors. Presumed monomeric, dimeric, and oligomeric forms of CEACAM16 are indicated by arrows . Note that the N2 or the N1 and A domains appear to be dispensable for interaction as dimers are observed in the absence of these domains ( J ). The 60-kDa band. which is observed in the supernatant of transfected and mock-transfected cells ( H, right panel ), is marked by an asterisk ( H , J , and K ).
    Hek293t Cells, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 999 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Thermo Fisher geneblazer vdr uas bla hek 293t cells
    CEACAM16 interacts homotypically. <t>HEK293T</t> cells either remained untransfected ( A ) or were transiently transfected with expression plasmids encoding full-length murine native ( B ) or GPI-linked CEACAM16 ( C ). The transfected cells were harvested by repeated pipetting, incubated at room temperature, attached to glass slides using the cytospin method, and stained with anti-murine CEACAM16 immune sera by immunocytochemistry ( brown stain ). Note the homotypic sorting of cells expressing CEACAM16 on the cell membrane ( C ) but not of cells with intracellular expression ( B ). This suggests that CEACAM16 can interact homotypically in trans . Aggregation of HEK293T cells was tested as above after transient expression of GPI-anchored human CEACAM16 N1 and N2 domains ( D and E , left panels ). N1- and N2-expressing cells were detected by immunocytochemistry using an anti-myc-tag antibody. Representative regions of the cytospins are shown. The expression levels of myc-tagged fusion proteins and the fraction of positive cells were determined by FACScan analysis using anti-myc and FITC-conjugated secondary antibodies ( D and E , right panels , green curves ). Samples incubated without primary antibody served as a negative control ( blue curves ). Aggregation mediated by N1 and N2 domains was quantitated by determination of the fraction of N1- and N2-positive cells, respectively, in single and double cell populations as well as in aggregates ≥3 using the cytospins shown in D and E ( F ). Note that surface expression of N2 preferentially induces multicellular aggregates. One of two experiments with similar results is shown. Full-length murine native CEACAM16 ( G ) or a deletion constructs lacking N2 ( H ) or N1 and A ( J ) or B and N2 ( K ) or encoding only the N1 or N2 domain either soluble ( I , upper panel ) or GPI-linked ( I , lower panels ) were expressed in HEK293T cells by transient transfection. Cell lysates ( cells ) or cell culture supernatants with or without prior treatment with DTT were separated by gel electrophoresis, and CEACAM16 was detected by Western blot analysis with a monoclonal anti-myc antibody. The domain organization of the encoded proteins is schematically shown to the right . Cysteines possibly involved in intermolecular disulfide bridges are indicated by yellow dots , green tags represent NH 2 -terminally added myc epitopes, and green arrows symbolize GPI anchors. Presumed monomeric, dimeric, and oligomeric forms of CEACAM16 are indicated by arrows . Note that the N2 or the N1 and A domains appear to be dispensable for interaction as dimers are observed in the absence of these domains ( J ). The 60-kDa band. which is observed in the supernatant of transfected and mock-transfected cells ( H, right panel ), is marked by an asterisk ( H , J , and K ).
    Geneblazer Vdr Uas Bla Hek 293t Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    SignaGen hek293t cells
    Y247 is the primary residue of EB1 phosphorylated by Src. (A) In vitro kinase assays were performed with purified GST-Src and purified His-EB1 wild-type or mutants. The arrowhead indicates the bands of phosphorylated EB1. (B) <t>HEK293T</t> cells were transfected with the indicated plasmids. Immunoprecipitation and immunoblotting were then performed. (C) In vitro kinase assays were performed with the indicated purified proteins, and immunoblotting were then performed. (D) HUVECs were transfected with GFP-Src and stained with the pY247-EB1 antibody and DAPI. Scale bar, 10 μm. (E) Experiments were performed as in (D), and the relative intensity of pY247-EB1 in the boxed areas was quantified (50 cells were measured for each group). All experiments were replicated three times. **** p
    Hek293t Cells, supplied by SignaGen, used in various techniques. Bioz Stars score: 93/100, based on 613 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC transfections hek293t cells
    Readthrough efficiencies of human UGA_CUAG stop codons. Readthrough efficiencies were determined by dual luciferase assay after <t>transfection</t> of <t>HEK293T</t> cells with dual luciferase reporter constructs consisting of candidate sequences (6 nt 5′ and 12 nt 3′ of stop codon (9 nt 3′ for MS4A5 because of an in-frame stop codon)) shown in Table S1 . AQP4 readthrough has been described previously ( 17 , 21 ) and is included here as an internal readthrough control. A UGA_C control indicated by a dashed line represents background readthrough levels. In each box-whisker plot center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; outliers are represented by dots. n = 12 biological replicates.
    Transfections Hek293t Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Comprehensive analysis of protein N -glycosylation in human cells. a Comparison of unique protein N -glycosylation sites identified in MCF7 cells in duplicate experiments. b Comparison of unique glycosylation sites and glycoproteins identified with the boronic acid derivative magnetic beads (designated as BA, shown in green) and with the dendrimer beads conjugated with the boronic acid derivative (DBA) (blue). c Abundance distributions of N -glycoproteins identified with the BA (green) or DBA (blue) beads. d Overlap of N -glycoproteins in three different types of cells (MCF7, HEK 293T, and Jurkat). e Protein-clustering results for 180 N -glycoproteins identified exclusively in Jurkat cells. f Distribution of membrane proteins (type I, II, III, and IV, and multi-pass transmembrane (TM)) among all identified N -glycoproteins. g N -glycosylation site locations on 301 receptors with X -axis as the TM domain. Each glycoprotein sequence was aligned against the transmembrane domain, and the glycosylation sites are indicated as yellow dots. All sites are located in the extracellular space. h Domain analysis of N -glycoproteins showing the number of N -glycoproteins containing the most highly enriched domains and their corresponding P values

    Journal: Nature Communications

    Article Title: An enrichment method based on synergistic and reversible covalent interactions for large-scale analysis of glycoproteins

    doi: 10.1038/s41467-018-04081-3

    Figure Lengend Snippet: Comprehensive analysis of protein N -glycosylation in human cells. a Comparison of unique protein N -glycosylation sites identified in MCF7 cells in duplicate experiments. b Comparison of unique glycosylation sites and glycoproteins identified with the boronic acid derivative magnetic beads (designated as BA, shown in green) and with the dendrimer beads conjugated with the boronic acid derivative (DBA) (blue). c Abundance distributions of N -glycoproteins identified with the BA (green) or DBA (blue) beads. d Overlap of N -glycoproteins in three different types of cells (MCF7, HEK 293T, and Jurkat). e Protein-clustering results for 180 N -glycoproteins identified exclusively in Jurkat cells. f Distribution of membrane proteins (type I, II, III, and IV, and multi-pass transmembrane (TM)) among all identified N -glycoproteins. g N -glycosylation site locations on 301 receptors with X -axis as the TM domain. Each glycoprotein sequence was aligned against the transmembrane domain, and the glycosylation sites are indicated as yellow dots. All sites are located in the extracellular space. h Domain analysis of N -glycoproteins showing the number of N -glycoproteins containing the most highly enriched domains and their corresponding P values

    Article Snippet: Gang Bao for generously providing the HEK 293T cells (originally from ATCC) and thank Dr. Marshall Bern in Protein Metrics Inc. for his help on protein O -glycosylation analysis using ByonicTM .

    Techniques: Magnetic Beads, Sequencing

    The synergistic interactions dramatically enhanced the enrichment of O -GlcNAcylated peptides in human cells. a Comparison of glycoproteins with one HexNAc identified with BA and DBA, which clearly shows that the results from DBA are substantially better. b Distribution of O -glycoproteins modified with HexNAc(1) identified in HEK 293T cells based on the cellular compartment. c Proposed mechanism of the interactions between DBA and GlcNAc benefiting from synergistic interactions. d Cellular compartment distributions of glycoproteins containing one HexNAc identified in the three types of human cells (light blue—MCF7, dark blue—Jurkat, and green—HEK 293T)

    Journal: Nature Communications

    Article Title: An enrichment method based on synergistic and reversible covalent interactions for large-scale analysis of glycoproteins

    doi: 10.1038/s41467-018-04081-3

    Figure Lengend Snippet: The synergistic interactions dramatically enhanced the enrichment of O -GlcNAcylated peptides in human cells. a Comparison of glycoproteins with one HexNAc identified with BA and DBA, which clearly shows that the results from DBA are substantially better. b Distribution of O -glycoproteins modified with HexNAc(1) identified in HEK 293T cells based on the cellular compartment. c Proposed mechanism of the interactions between DBA and GlcNAc benefiting from synergistic interactions. d Cellular compartment distributions of glycoproteins containing one HexNAc identified in the three types of human cells (light blue—MCF7, dark blue—Jurkat, and green—HEK 293T)

    Article Snippet: Gang Bao for generously providing the HEK 293T cells (originally from ATCC) and thank Dr. Marshall Bern in Protein Metrics Inc. for his help on protein O -glycosylation analysis using ByonicTM .

    Techniques: Modification

    Mutation of arginine 55 in SLA-DQ beta reduces class II SLA-based xenoantigenicty Cell lines expressed CD74, SLA-DQα1*0101, and mutant or unmodified SLA-DQβ1*0601. Mutant molecules contained a β chain having proline at position 55 (55P). Wild type class II contained arginine at position (55R). (A) White histograms show binding of the CerCLIP.1 antibody to cells expressing either 55R or 55P. Gray histograms show CerCLIP.1 binding to CD74/SLA-deficient HEK 293T cells. (B) 21 human sera were incubated with cells expressing either the 55R or 55P variants of SLA-DQ. These sera were selected from prior experiments suggesting they reacted with SLA-DQα1*0101/SLA-DQβ1*0601. Data from each sample is represented by one red and one black bar (stacked on top of each other). MFI ratios of fluorescent anti-human IgG were calculated to compare binding to 55P versus 55R molecules. The log 10 values of these ratios were plotted for each serum (black bars). The same analysis compared IgG binding to the 55P cells versus SLA-negative parent cells (red bars). The 55P mutation partially reduced IgG binding to the SLA-DQ protein in 17 samples. Three samples had no detectable antibody staining of 55P mutants (#). One sample showed greater binding to the 55P mutant than to 55R (*). The mAB sample indicates the binding of a monoclonal antibody specific for SLA-DQ. This antibody recognized SLA-DQ 55P variants (red bar) almost identically to 55R variants (black bar, Log 10 of 55P/55R = −0.09).

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Examining the Biosynthesis and Xenoantigenicity of Class II SLA Proteins

    doi: 10.4049/jimmunol.1800022

    Figure Lengend Snippet: Mutation of arginine 55 in SLA-DQ beta reduces class II SLA-based xenoantigenicty Cell lines expressed CD74, SLA-DQα1*0101, and mutant or unmodified SLA-DQβ1*0601. Mutant molecules contained a β chain having proline at position 55 (55P). Wild type class II contained arginine at position (55R). (A) White histograms show binding of the CerCLIP.1 antibody to cells expressing either 55R or 55P. Gray histograms show CerCLIP.1 binding to CD74/SLA-deficient HEK 293T cells. (B) 21 human sera were incubated with cells expressing either the 55R or 55P variants of SLA-DQ. These sera were selected from prior experiments suggesting they reacted with SLA-DQα1*0101/SLA-DQβ1*0601. Data from each sample is represented by one red and one black bar (stacked on top of each other). MFI ratios of fluorescent anti-human IgG were calculated to compare binding to 55P versus 55R molecules. The log 10 values of these ratios were plotted for each serum (black bars). The same analysis compared IgG binding to the 55P cells versus SLA-negative parent cells (red bars). The 55P mutation partially reduced IgG binding to the SLA-DQ protein in 17 samples. Three samples had no detectable antibody staining of 55P mutants (#). One sample showed greater binding to the 55P mutant than to 55R (*). The mAB sample indicates the binding of a monoclonal antibody specific for SLA-DQ. This antibody recognized SLA-DQ 55P variants (red bar) almost identically to 55R variants (black bar, Log 10 of 55P/55R = −0.09).

    Article Snippet: Human embryonic kidney (HEK) 293T cells (ATCC CRL-3216) were cultured in minimum essential media (MEM- α) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan UT) in collagen-I-coated plates (Becton Dickinson, Bedford, MA) at 37°C and 5% CO2 .

    Techniques: Mutagenesis, Binding Assay, Expressing, Incubation, Staining

    Effect of A3 proteins levels in virus producer cells on HIV-1 infection. VSV-G pseudotyped NL4-3-ΔEnv/ΔVif/eGFP was produced by co-transfection in 293T cells along with 30ng ( A and B ), 100ng (C and D) or 250ng ( E and F ) of A3 expression plasmids. Gel exposure controls, Comparative 1 and 2, represent co-transfection with 100ng of A3 W94A and A3 W127A plasmids, respectively. Control lane represents transfection with pcDNA3 only; virus lane is the co-transfection of pseudotyped virus along with the indicated amount (30ng, 100ng or 250ng) of pcDNA3 plasmid. CEM-SS cells were infected with pre-determined levels of capsid (p24) protein (20 ng, 100 ng or 500 ng) as measured by ELISA. The percentage of infected cells was determined by flow cytometry. Productively infected cells express the eGFP reporter gene encoded in the viral genome ( A, C and E ). Production of intracellular viral proteins was analyzed by Western blotting of transfected virus producer cells using anti-FLAG, anti-p24CA or anti-β tubulin antibodies ( B, D and F ).

    Journal: bioRxiv

    Article Title: Antiretroviral APOBEC3 Cytidine Deaminases Alter HIV-1 Provirus Integration Site Profiles

    doi: 10.1101/833475

    Figure Lengend Snippet: Effect of A3 proteins levels in virus producer cells on HIV-1 infection. VSV-G pseudotyped NL4-3-ΔEnv/ΔVif/eGFP was produced by co-transfection in 293T cells along with 30ng ( A and B ), 100ng (C and D) or 250ng ( E and F ) of A3 expression plasmids. Gel exposure controls, Comparative 1 and 2, represent co-transfection with 100ng of A3 W94A and A3 W127A plasmids, respectively. Control lane represents transfection with pcDNA3 only; virus lane is the co-transfection of pseudotyped virus along with the indicated amount (30ng, 100ng or 250ng) of pcDNA3 plasmid. CEM-SS cells were infected with pre-determined levels of capsid (p24) protein (20 ng, 100 ng or 500 ng) as measured by ELISA. The percentage of infected cells was determined by flow cytometry. Productively infected cells express the eGFP reporter gene encoded in the viral genome ( A, C and E ). Production of intracellular viral proteins was analyzed by Western blotting of transfected virus producer cells using anti-FLAG, anti-p24CA or anti-β tubulin antibodies ( B, D and F ).

    Article Snippet: HEK 293T cells (ATCC CRL-3216™) were maintained in complete DMEM with high glucose.

    Techniques: Infection, Produced, Cotransfection, Expressing, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Western Blot

    HOTAIRM1 regulates autophagy-associated gene expression by competing with the binding sites of miRNAs. ( a ) HOTAIRM1 transcripts bind to AGO2 directly. AGO2 was assayed by western blotting after acquiring the possible protein complex binding to HOTAIRM1. Proteins bound to the antisense of HOTAIRM1 served as loading controls. ( b ) RNA immunoprecipitation was performed to acquire the RNA that interacted with AGO2 protein. The qRT-PCR product of HOTAIRM1 was tested by agarose gel electrophoresis. ( c ) Luciferase reporter assays analyzing the binding of HOTAIRM1 to miRNAs. NC and miRNAs duplexes were co-transfected with psiCHECH-2 plasmids containing the 59 nt of HOTAIRM1-WT or HOTAIRM1-MUT. The firefly luciferase activity of each sample was normalized to the Renilla luciferase activity ( n =3 independent experiments performed in triplicate). ( d ) Luciferase reporter assays analyzing the genes potentially influenced by HOTAIRM1. NC and two HOTAIRM1-siRNAs were co-transfected with psiCHECH-2 plasmids with the 59-nt of miRNAs targeting the wild-type or mutant gene position in HEK-293 T cells. The firefly luciferase activity of each sample was normalized to the Renilla luciferase activity. Values are derived from  n =3 independent experiments, and data are reported as the mean±S.D. ( e ) Western blotting detecting the proteins expression levels of genes regulated by HOTAIRM1. ULK1, E2F1 and DRAM2 were all downregulated when HOTAIRM1 was knocked down in NB4 cells with or without 1  μ M ATRA treatment; GAPDH was used as a loading control. The densitometric ratio normalized to GAPDH was recorded by quantity one. Values are derived from  n =3 independent experiments, and data are reported as the mean±S.D. (down). ( f–h ) The qRT-PCR testing of DRMA1, LC3B and ULK1, three direct targets of E2F1. Experiments were performed in triplicate and are reported as the mean±S.D. ( i ) The qRT-PCR testing of miRNAs in NB4-Lv-NC and NB4-Lv-shHOTAIRM1 cells after treatment with ATRA (1  μ M, 48 h). Experiments were performed in triplicate and normalized to GAPDH. ( j ) The detection of PML-RARA by western bloting after transient overexpression of miR-20a and miR-106b mimics in NB4 cells. Experiments were performed in triplicate. PML-RARA/GAPDH densitometric ratios were recorded. Differences in  c - i  were considered significant at * P

    Journal: Cell Death and Differentiation

    Article Title: The lncRNA HOTAIRM1 regulates the degradation of PML-RARA oncoprotein and myeloid cell differentiation by enhancing the autophagy pathway

    doi: 10.1038/cdd.2016.111

    Figure Lengend Snippet: HOTAIRM1 regulates autophagy-associated gene expression by competing with the binding sites of miRNAs. ( a ) HOTAIRM1 transcripts bind to AGO2 directly. AGO2 was assayed by western blotting after acquiring the possible protein complex binding to HOTAIRM1. Proteins bound to the antisense of HOTAIRM1 served as loading controls. ( b ) RNA immunoprecipitation was performed to acquire the RNA that interacted with AGO2 protein. The qRT-PCR product of HOTAIRM1 was tested by agarose gel electrophoresis. ( c ) Luciferase reporter assays analyzing the binding of HOTAIRM1 to miRNAs. NC and miRNAs duplexes were co-transfected with psiCHECH-2 plasmids containing the 59 nt of HOTAIRM1-WT or HOTAIRM1-MUT. The firefly luciferase activity of each sample was normalized to the Renilla luciferase activity ( n =3 independent experiments performed in triplicate). ( d ) Luciferase reporter assays analyzing the genes potentially influenced by HOTAIRM1. NC and two HOTAIRM1-siRNAs were co-transfected with psiCHECH-2 plasmids with the 59-nt of miRNAs targeting the wild-type or mutant gene position in HEK-293 T cells. The firefly luciferase activity of each sample was normalized to the Renilla luciferase activity. Values are derived from n =3 independent experiments, and data are reported as the mean±S.D. ( e ) Western blotting detecting the proteins expression levels of genes regulated by HOTAIRM1. ULK1, E2F1 and DRAM2 were all downregulated when HOTAIRM1 was knocked down in NB4 cells with or without 1  μ M ATRA treatment; GAPDH was used as a loading control. The densitometric ratio normalized to GAPDH was recorded by quantity one. Values are derived from n =3 independent experiments, and data are reported as the mean±S.D. (down). ( f–h ) The qRT-PCR testing of DRMA1, LC3B and ULK1, three direct targets of E2F1. Experiments were performed in triplicate and are reported as the mean±S.D. ( i ) The qRT-PCR testing of miRNAs in NB4-Lv-NC and NB4-Lv-shHOTAIRM1 cells after treatment with ATRA (1  μ M, 48 h). Experiments were performed in triplicate and normalized to GAPDH. ( j ) The detection of PML-RARA by western bloting after transient overexpression of miR-20a and miR-106b mimics in NB4 cells. Experiments were performed in triplicate. PML-RARA/GAPDH densitometric ratios were recorded. Differences in c - i were considered significant at * P

    Article Snippet: Cell lines and cultures Leukemia cells (NB4 and U937-PR9cells) and HEK-293 T cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA.) and were cultured in RPMI Medium Modified (HyClone, South Logan, UT, USA) and DMEM (HyClone), respectively, containing 10% fetal bovine serum (Gibco, ThermoFisher Scientific, Waltham, MA, USA).

    Techniques: Expressing, Binding Assay, Western Blot, Immunoprecipitation, Quantitative RT-PCR, Agarose Gel Electrophoresis, Luciferase, Transfection, Activity Assay, Mutagenesis, Derivative Assay, Over Expression

    Tid1 interacts with Galectin-7 through N-linked glycosylation to mediate Galectin-7-induced malignancy in HNSCC cells. (A) 293T cells were transfected with Myc-tagged Galectin-7 alone or together with either Tid1-L-wt-HA or Tid1-L-mut-HA plasmids. Total protein lysates of the transfected cells were precipitated (IP) with either anti-HA (Tid1) or anti-Myc (Gal-7) antibody followed by immunoblotting (IB) with the indicated antibodies. (B) Immunofluorescence staining showing the subcellular localization of Gal-7 (Red) in 293T cells expressing Tid1-L-wt, or Tid1-L-mut, respectively. Co-localization of Tid1 and Gal-7 (yellow) was detected in the cytosol, as indicated by white arrows. (C) Subcellular localization of Myc (Gal-7), actin and Lamin B proteins in the nuclear and cytosolic fractions, prepared from 293T cells transfected with indicated plasmids, was determined. Actin was detectable mainly in the cytoplasmic extract, whereas Lamin B was mainly visible in the nuclear extract. (D) 293T cells were transfected with Tid1-L-wt-HA plasmids. Protein lysates of the transfected cells were treated without or with PNGase F, followed by immunoprecipitation with an anti-HA antibody, then, immunoblotted with antibody against HA or Myc, respectively. (E) 293T cells were co-transfected with Tid1-L-wt-HA and Gal-7-myc. Protein lysates of the transfected cells were incubated with lactose or sucrose, respectively, followed by immunoprecipitation with anti-HA, then, immunoblotted with antibody against HA and Myc. (F) Various plasmids of Tid1-L N-link glycosylation site mutants (Tid1N102A and Tid1N372A) were co-transfected with Gal-7-Myc into 293T cells. Protein lysates of the transfected cells were immunoprecipitated with anti-HA, and immunoblotted with antibody against HA and Myc antibodies, respectively. (G) Confocal fluorescence microscopy showed the intracellular localization of Tid1N102A or Tid1N372A plus Gal-7-Myc detected by double staining with antibodies against HA and Myc tag. Co-localization of Tid1 and Gal-7 is indicated with white arrows. (H) 293T cells were transfected with the HA-tagged WT Tid1 (Tid1-L-wt-HA) or Tid1-L mutants (Tid1N102A and Tid1N372A) plasmid and then pulled down by PNA agarose beads. The pulled-down proteins were separated by SDS-PAGE and analyzed by IB with anti-HA antibody. SAS cells were transfected with various combinations of the indicated plasmids. The transfected cells were examined for Transwell® migration ability (I) , and anchorage-independent growth (J) . The histograms shown are the mean ± SD from three independent experiments and analyzed by Student's t-test (∗p

    Journal: Theranostics

    Article Title: HSP40 co-chaperone protein Tid1 suppresses metastasis of head and neck cancer by inhibiting Galectin-7-TCF3-MMP9 axis signaling

    doi: 10.7150/thno.25784

    Figure Lengend Snippet: Tid1 interacts with Galectin-7 through N-linked glycosylation to mediate Galectin-7-induced malignancy in HNSCC cells. (A) 293T cells were transfected with Myc-tagged Galectin-7 alone or together with either Tid1-L-wt-HA or Tid1-L-mut-HA plasmids. Total protein lysates of the transfected cells were precipitated (IP) with either anti-HA (Tid1) or anti-Myc (Gal-7) antibody followed by immunoblotting (IB) with the indicated antibodies. (B) Immunofluorescence staining showing the subcellular localization of Gal-7 (Red) in 293T cells expressing Tid1-L-wt, or Tid1-L-mut, respectively. Co-localization of Tid1 and Gal-7 (yellow) was detected in the cytosol, as indicated by white arrows. (C) Subcellular localization of Myc (Gal-7), actin and Lamin B proteins in the nuclear and cytosolic fractions, prepared from 293T cells transfected with indicated plasmids, was determined. Actin was detectable mainly in the cytoplasmic extract, whereas Lamin B was mainly visible in the nuclear extract. (D) 293T cells were transfected with Tid1-L-wt-HA plasmids. Protein lysates of the transfected cells were treated without or with PNGase F, followed by immunoprecipitation with an anti-HA antibody, then, immunoblotted with antibody against HA or Myc, respectively. (E) 293T cells were co-transfected with Tid1-L-wt-HA and Gal-7-myc. Protein lysates of the transfected cells were incubated with lactose or sucrose, respectively, followed by immunoprecipitation with anti-HA, then, immunoblotted with antibody against HA and Myc. (F) Various plasmids of Tid1-L N-link glycosylation site mutants (Tid1N102A and Tid1N372A) were co-transfected with Gal-7-Myc into 293T cells. Protein lysates of the transfected cells were immunoprecipitated with anti-HA, and immunoblotted with antibody against HA and Myc antibodies, respectively. (G) Confocal fluorescence microscopy showed the intracellular localization of Tid1N102A or Tid1N372A plus Gal-7-Myc detected by double staining with antibodies against HA and Myc tag. Co-localization of Tid1 and Gal-7 is indicated with white arrows. (H) 293T cells were transfected with the HA-tagged WT Tid1 (Tid1-L-wt-HA) or Tid1-L mutants (Tid1N102A and Tid1N372A) plasmid and then pulled down by PNA agarose beads. The pulled-down proteins were separated by SDS-PAGE and analyzed by IB with anti-HA antibody. SAS cells were transfected with various combinations of the indicated plasmids. The transfected cells were examined for Transwell® migration ability (I) , and anchorage-independent growth (J) . The histograms shown are the mean ± SD from three independent experiments and analyzed by Student's t-test (∗p

    Article Snippet: The human embryonic kidney cell line 293T was originally from ATCC and maintained under recommended culture conditions.

    Techniques: Transfection, Immunofluorescence, Staining, Expressing, Immunoprecipitation, Incubation, Fluorescence, Microscopy, Double Staining, Plasmid Preparation, SDS Page, Migration

    Tid1 reduces the protein level of Galectin-7 by promoting ubiquitination, and the ubiquitination of Galectin-7 is required for inhibiting the malignancy of HNSCC cells. (A-B) 293T cells were transfected with various combinations of indicated plasmids. 24 h after transfection, the cells were treated with MG132 for 6 h. Protein lysates from the transfected cells were immunoprecipitated with anti-Myc (Gal-7), then, immunoblotted with antibody against Flag (Ubiquitin, Ub). (C) Various plasmids of Gal-7 ubiquitination site mutants (K7A-HA, K65A-HA, and K99A-HA) were transiently introduced into 293T cells. The expression of wild-type and mutant Gal-7 protein was determined by immunoblotting assays. (D) Various plasmids of Gal-7 mutants were co-transfected with Tid1-L-wt and Ub-Flag plasmids into 293T cells. Protein lysates from the transfected cells were immunoprecipitated with anti-HA (Gal-7) and immunoblotted with antibody against Flag (Ubiquitin). SAS cells were transfected with various combinations of indicated plasmids. Transwell® migration ability (E) and anchorage-independent growth (F) of the transfected cells were examined. The histograms shown are the mean ± SD from three independent experiments and analyzed by Student's t-test (∗p

    Journal: Theranostics

    Article Title: HSP40 co-chaperone protein Tid1 suppresses metastasis of head and neck cancer by inhibiting Galectin-7-TCF3-MMP9 axis signaling

    doi: 10.7150/thno.25784

    Figure Lengend Snippet: Tid1 reduces the protein level of Galectin-7 by promoting ubiquitination, and the ubiquitination of Galectin-7 is required for inhibiting the malignancy of HNSCC cells. (A-B) 293T cells were transfected with various combinations of indicated plasmids. 24 h after transfection, the cells were treated with MG132 for 6 h. Protein lysates from the transfected cells were immunoprecipitated with anti-Myc (Gal-7), then, immunoblotted with antibody against Flag (Ubiquitin, Ub). (C) Various plasmids of Gal-7 ubiquitination site mutants (K7A-HA, K65A-HA, and K99A-HA) were transiently introduced into 293T cells. The expression of wild-type and mutant Gal-7 protein was determined by immunoblotting assays. (D) Various plasmids of Gal-7 mutants were co-transfected with Tid1-L-wt and Ub-Flag plasmids into 293T cells. Protein lysates from the transfected cells were immunoprecipitated with anti-HA (Gal-7) and immunoblotted with antibody against Flag (Ubiquitin). SAS cells were transfected with various combinations of indicated plasmids. Transwell® migration ability (E) and anchorage-independent growth (F) of the transfected cells were examined. The histograms shown are the mean ± SD from three independent experiments and analyzed by Student's t-test (∗p

    Article Snippet: The human embryonic kidney cell line 293T was originally from ATCC and maintained under recommended culture conditions.

    Techniques: Transfection, Immunoprecipitation, Expressing, Mutagenesis, Migration

    S5 localizes to detergent-resistant membranes (DRMs) (A) HEK293T cells expressing S5-FLAG or S2-FLAG were lysed and fractionated using a flotation gradient as described in Methods. Cells were homogenized in a Triton X-100 detergent solution and fractionated by discontinuous sucrose gradient centrifugation (5–40%). Gradient fractions 1–12 were analyzed by Western blotting using anti-FLAG antibodies. As positive controls, similar analysis was performed using HEK293T cells separately transfected with Caveolin-1 fused to GFP (Caveolin-1-GFP), which localizes to DRMs. As a negative control, we studied the membrane localization of the receptor TVB S3 fused to GFP (TVB S3 -GFP), which does not localize to DRMs. Caveolin-1-GFP and TVB S3 -GFP expression was detected by Western blotting using anti-GFP antibodies. Fractions for S2 and S5 from three independent experiments were quantified using a Li-Cor instrument, and they are shown as % of total FLAG protein with standard deviations (lower panel). (B) Similar fractionation experiments were performed in human HEK293T cells producing HIV-1 SF162 viruses in the presence of S5-FLAG or S2-FLAG. The DNA mix for each transfection included 8 μg NL4–3ΔNef, 0.5 μg SF162, plus either empty vector, S2-FLAG, S5-FLAG, caveolin-1-GFP, or TVB-1. Forty-eight hours post-transfection cells were lyzed and fractionated as described in Methods. Fractions were analyzed by Western blotting using anti-FLAG, anti-GFP, or anti-Env antibodies. Fractions for S2 and S5 from three independent experiments were quantified using a Li-Cor instrument, and they are shown as % of total FLAG protein with standard deviations (lower panel).

    Journal: Virology

    Article Title: Localization to Detergent-Resistant Membranes and HIV-1 Core Entry Inhibition Correlate with HIV-1 Restriction by SERINC5

    doi: 10.1016/j.virol.2017.12.005

    Figure Lengend Snippet: S5 localizes to detergent-resistant membranes (DRMs) (A) HEK293T cells expressing S5-FLAG or S2-FLAG were lysed and fractionated using a flotation gradient as described in Methods. Cells were homogenized in a Triton X-100 detergent solution and fractionated by discontinuous sucrose gradient centrifugation (5–40%). Gradient fractions 1–12 were analyzed by Western blotting using anti-FLAG antibodies. As positive controls, similar analysis was performed using HEK293T cells separately transfected with Caveolin-1 fused to GFP (Caveolin-1-GFP), which localizes to DRMs. As a negative control, we studied the membrane localization of the receptor TVB S3 fused to GFP (TVB S3 -GFP), which does not localize to DRMs. Caveolin-1-GFP and TVB S3 -GFP expression was detected by Western blotting using anti-GFP antibodies. Fractions for S2 and S5 from three independent experiments were quantified using a Li-Cor instrument, and they are shown as % of total FLAG protein with standard deviations (lower panel). (B) Similar fractionation experiments were performed in human HEK293T cells producing HIV-1 SF162 viruses in the presence of S5-FLAG or S2-FLAG. The DNA mix for each transfection included 8 μg NL4–3ΔNef, 0.5 μg SF162, plus either empty vector, S2-FLAG, S5-FLAG, caveolin-1-GFP, or TVB-1. Forty-eight hours post-transfection cells were lyzed and fractionated as described in Methods. Fractions were analyzed by Western blotting using anti-FLAG, anti-GFP, or anti-Env antibodies. Fractions for S2 and S5 from three independent experiments were quantified using a Li-Cor instrument, and they are shown as % of total FLAG protein with standard deviations (lower panel).

    Article Snippet: Human embryonic kidney HEK293T cells were obtained from ATCC (CRL-3216).

    Techniques: Expressing, Gradient Centrifugation, Western Blot, Transfection, Negative Control, Fractionation, Plasmid Preparation

    The S5 region L5-TM6-L6 is required for incorporation into HIV-1 virions (A) Ability of S2 proteins containing S5 regions to restrict HIV-1 infection and be incorporated into virions. S2 proteins containing individual elements (L5, TM6 or L6), or the entire L5-TM6-L6 region of S5, were analyzed for the ability to restrict HIV-1 (fold-restriction) and be incorporated into virions (viral particle incorporation ratio). Fold-restriction is defined as the ratio of %infection by viruses produced in the presence of empty vector to %infection by viruses produced in the presence of the indicated S5-S2 chimera. The HIV-1 fold-restriction where expression of the chimera did not affect viral production is shown with a standard deviation from six independent experiments. Incorporation of the chimera into viral particles is defined as the ratio of protein incorporated into virions to the protein expressed in producer cells. Viral particle incorporation ratio where expression of the chimera did not affect viral production is shown with a standard deviation from three independent experiments. Schematic representations of S2 proteins containing S5 regions are shown with protein regions of S2 and S5 colored in black and orange, respectively. (B) The ability of chimera#7 and chimera#9 to restrict HIV-1 is shown. Black arrows point to the experiments where the levels of chimera expression did not affect virus production as measured by p24. Experiments were repeated at least three times and the Western blot from a representative example is shown. (C) The ability of the indicated HIV-1 neutralizing antibodies to block HIV-1 SF162 viruses produced in the presence of chimera#7 was measured. Viruses produced in the presence of the indicated SERINC proteins were incubated with increasing amounts of the indicated neutralizing antibody for 1 hr at 37 °C. The virus-antibody mixture was then used to infect TZM-bl GFP-reporter cells. Infectivity was determined by measuring the percentage of GFP-positive cells at 24 h post-infection. Infection values were normalized to the infection of virions that were not incubated with neutralizing antibodies. Infections were performed in triplicates and standard deviations are shown. (D) Human 293T cells expressing the indicated proteins were lysed in Triton X-100 and fractionated on a discontinuous sucrose gradient (5–40%) as described in Methods. As controls, human HEK293T cells expressing caveoilin-1 fused to GFP (Caveolin-1-GFP) and tumor virus B receptor (TVB S3 -GFP) were analyzed as probes for proteins that did or did not localize to DRMs, respectively. Twelve fractions (1–12) were analyzed per sample using anti-FLAG or anti-GFP antibodies. Fractions for the indicated proteins from three independent experiments were quantified using a Li-Cor instrument, and they are shown as % of total FLAG protein with standard deviations (lower panel). (E) The ability of chimera#11 to block HIV-1 infection was measured, as described above.

    Journal: Virology

    Article Title: Localization to Detergent-Resistant Membranes and HIV-1 Core Entry Inhibition Correlate with HIV-1 Restriction by SERINC5

    doi: 10.1016/j.virol.2017.12.005

    Figure Lengend Snippet: The S5 region L5-TM6-L6 is required for incorporation into HIV-1 virions (A) Ability of S2 proteins containing S5 regions to restrict HIV-1 infection and be incorporated into virions. S2 proteins containing individual elements (L5, TM6 or L6), or the entire L5-TM6-L6 region of S5, were analyzed for the ability to restrict HIV-1 (fold-restriction) and be incorporated into virions (viral particle incorporation ratio). Fold-restriction is defined as the ratio of %infection by viruses produced in the presence of empty vector to %infection by viruses produced in the presence of the indicated S5-S2 chimera. The HIV-1 fold-restriction where expression of the chimera did not affect viral production is shown with a standard deviation from six independent experiments. Incorporation of the chimera into viral particles is defined as the ratio of protein incorporated into virions to the protein expressed in producer cells. Viral particle incorporation ratio where expression of the chimera did not affect viral production is shown with a standard deviation from three independent experiments. Schematic representations of S2 proteins containing S5 regions are shown with protein regions of S2 and S5 colored in black and orange, respectively. (B) The ability of chimera#7 and chimera#9 to restrict HIV-1 is shown. Black arrows point to the experiments where the levels of chimera expression did not affect virus production as measured by p24. Experiments were repeated at least three times and the Western blot from a representative example is shown. (C) The ability of the indicated HIV-1 neutralizing antibodies to block HIV-1 SF162 viruses produced in the presence of chimera#7 was measured. Viruses produced in the presence of the indicated SERINC proteins were incubated with increasing amounts of the indicated neutralizing antibody for 1 hr at 37 °C. The virus-antibody mixture was then used to infect TZM-bl GFP-reporter cells. Infectivity was determined by measuring the percentage of GFP-positive cells at 24 h post-infection. Infection values were normalized to the infection of virions that were not incubated with neutralizing antibodies. Infections were performed in triplicates and standard deviations are shown. (D) Human 293T cells expressing the indicated proteins were lysed in Triton X-100 and fractionated on a discontinuous sucrose gradient (5–40%) as described in Methods. As controls, human HEK293T cells expressing caveoilin-1 fused to GFP (Caveolin-1-GFP) and tumor virus B receptor (TVB S3 -GFP) were analyzed as probes for proteins that did or did not localize to DRMs, respectively. Twelve fractions (1–12) were analyzed per sample using anti-FLAG or anti-GFP antibodies. Fractions for the indicated proteins from three independent experiments were quantified using a Li-Cor instrument, and they are shown as % of total FLAG protein with standard deviations (lower panel). (E) The ability of chimera#11 to block HIV-1 infection was measured, as described above.

    Article Snippet: Human embryonic kidney HEK293T cells were obtained from ATCC (CRL-3216).

    Techniques: Infection, Produced, Plasmid Preparation, Expressing, Standard Deviation, Western Blot, Blocking Assay, Incubation

    S5 affects the conformation of the HIV-1 envelope (A) The ability of different HIV-1 neutralizing antibodies to block HIV-1 SF162 viruses produced in the presence S5 was measured. HIV-1 SF162 viruses produced in human HEK293T cells in the presence of S5, S2, or empty vector were incubated with increasing amounts of the indicated neutralizing antibody for 1 h at 37 °C. The virus- antibody mixture was then used to infect TZM-bl GFP-reporter cells. Infectivity was determined by measuring the percentage of GFP-positive cells twenty-four hours post-infection. Infection values were normalized to the infection of viruses that were not incubated with neutralizing antibodies. (B) Similar neutralization experiments were performed using HIV-1 SF162 viruses with anti-gp41 and anti-gp120 antibodies, which either don´t neutralize SF162 viruses or with anti-gp120 antibodies, which strongly neutralize SF162 viruses. (C) Neutralization experiments were also performed using HIV-1 SF162 viruses produced in human Jurkat TAg cells that do or do not express S3 and S5 (S3/S5 KO). (D) HIV-1 SF162 viruses produced in HEK293T (top) or Jurkat TAg S3/S5 KO cells (bottom) in the presence of S5, S2, or empty vector were used to challenge TZM-bl GFP-reporter cells in the presence of increasing concentrations of the small molecule inhibitor 484. Infectivity was determined by measuring the % of GFP-positive cells 24 h post-infection. Infection values were normalized to the infection of viruses that were not incubated with the drug. Infections were performed in triplicates and standard deviations are shown.

    Journal: Virology

    Article Title: Localization to Detergent-Resistant Membranes and HIV-1 Core Entry Inhibition Correlate with HIV-1 Restriction by SERINC5

    doi: 10.1016/j.virol.2017.12.005

    Figure Lengend Snippet: S5 affects the conformation of the HIV-1 envelope (A) The ability of different HIV-1 neutralizing antibodies to block HIV-1 SF162 viruses produced in the presence S5 was measured. HIV-1 SF162 viruses produced in human HEK293T cells in the presence of S5, S2, or empty vector were incubated with increasing amounts of the indicated neutralizing antibody for 1 h at 37 °C. The virus- antibody mixture was then used to infect TZM-bl GFP-reporter cells. Infectivity was determined by measuring the percentage of GFP-positive cells twenty-four hours post-infection. Infection values were normalized to the infection of viruses that were not incubated with neutralizing antibodies. (B) Similar neutralization experiments were performed using HIV-1 SF162 viruses with anti-gp41 and anti-gp120 antibodies, which either don´t neutralize SF162 viruses or with anti-gp120 antibodies, which strongly neutralize SF162 viruses. (C) Neutralization experiments were also performed using HIV-1 SF162 viruses produced in human Jurkat TAg cells that do or do not express S3 and S5 (S3/S5 KO). (D) HIV-1 SF162 viruses produced in HEK293T (top) or Jurkat TAg S3/S5 KO cells (bottom) in the presence of S5, S2, or empty vector were used to challenge TZM-bl GFP-reporter cells in the presence of increasing concentrations of the small molecule inhibitor 484. Infectivity was determined by measuring the % of GFP-positive cells 24 h post-infection. Infection values were normalized to the infection of viruses that were not incubated with the drug. Infections were performed in triplicates and standard deviations are shown.

    Article Snippet: Human embryonic kidney HEK293T cells were obtained from ATCC (CRL-3216).

    Techniques: Blocking Assay, Produced, Plasmid Preparation, Incubation, Infection, Neutralization

    Infection of MDM by HIV-1 PV. Monocytes were isolated from the peripheral blood of healthy donors and culture for 7 days in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/ml), streptomycin (100 µg/ml), and M-CSF (250 ng/ml) to allow the cell differentiation. The MDM cells were infected with HIV-1 PV at a multiplicity of infection (MOI) of ∼3 in the presence of 10 ng/ml of polybrene for 4 hr and cultured for 7 more days. MDM from the same donor treated with polybrene at 10 ng/ml and mock infected with the supernatant of HEK 293T/17 cells transfected with SG3ΔEnv only were used as control. The cells were analyzed with immunofluorescence staining ( A ) and Western blotting ( C ) for the viral p24 protein expression, and by HIV DNA PCR ( B ) for the presence of integrated proviral DNA.

    Journal: PLoS ONE

    Article Title: HIV Infection Enhances TRAIL-Induced Cell Death in Macrophage by Down-Regulating Decoy Receptor Expression and Generation of Reactive Oxygen Species

    doi: 10.1371/journal.pone.0018291

    Figure Lengend Snippet: Infection of MDM by HIV-1 PV. Monocytes were isolated from the peripheral blood of healthy donors and culture for 7 days in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/ml), streptomycin (100 µg/ml), and M-CSF (250 ng/ml) to allow the cell differentiation. The MDM cells were infected with HIV-1 PV at a multiplicity of infection (MOI) of ∼3 in the presence of 10 ng/ml of polybrene for 4 hr and cultured for 7 more days. MDM from the same donor treated with polybrene at 10 ng/ml and mock infected with the supernatant of HEK 293T/17 cells transfected with SG3ΔEnv only were used as control. The cells were analyzed with immunofluorescence staining ( A ) and Western blotting ( C ) for the viral p24 protein expression, and by HIV DNA PCR ( B ) for the presence of integrated proviral DNA.

    Article Snippet: The medium from HEK 293T/17 cells transfected with pSG3△Env only was used as mock infection control.

    Techniques: Infection, Isolation, Cell Differentiation, Cell Culture, Transfection, Immunofluorescence, Staining, Western Blot, Expressing, Polymerase Chain Reaction

    PKA and AMPK phosphorylate the V-ATPase A subunit in vitro. Recombinant FLAG-tagged A subunit was expressed in HEK-293 cells, immunoprecipitated, and incubated with [γ- 32 P]ATP in the presence or absence of PKA catalytic subunit or in the presence or absence of AMPK holoenzyme. A phosphoscreen image ( top ) and immunoblot ( bottom ) of the same membrane are shown (representative of three experiments). In the top , the V-ATPase A subunit becomes phosphorylated in the presence of PKA and also in the presence of AMPK (first and third lanes). Both the PKA catalytic subunit and the AMPK-α subunit become autophosphorylated, as indicated. The immunoblot ( bottom ) reveals similar loading of V-ATPase A subunit in all lanes.

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H+-ATPase accumulation in epididymal clear cells

    doi: 10.1152/ajpcell.00004.2009

    Figure Lengend Snippet: PKA and AMPK phosphorylate the V-ATPase A subunit in vitro. Recombinant FLAG-tagged A subunit was expressed in HEK-293 cells, immunoprecipitated, and incubated with [γ- 32 P]ATP in the presence or absence of PKA catalytic subunit or in the presence or absence of AMPK holoenzyme. A phosphoscreen image ( top ) and immunoblot ( bottom ) of the same membrane are shown (representative of three experiments). In the top , the V-ATPase A subunit becomes phosphorylated in the presence of PKA and also in the presence of AMPK (first and third lanes). Both the PKA catalytic subunit and the AMPK-α subunit become autophosphorylated, as indicated. The immunoblot ( bottom ) reveals similar loading of V-ATPase A subunit in all lanes.

    Article Snippet: Briefly, HEK-293 cells were transiently transfected to express FLAG-V-ATPase A subunit using Lipofectamine 2000 (Invitrogen), and cells were lysed 2 days after transfection.

    Techniques: In Vitro, Recombinant, Immunoprecipitation, Incubation

    Tetracycline-inducible knockdown of AMPK-α1 in HEK-293 cells. Immunoblots for AMPK-α1 (top band) and β-actin (bottom band) of lysates derived from HEK-293 cells that were stably transfected with either a control short hairpin RNA (shRNA) that does not silence any known mammalian gene (CON) or an shRNA designed to specifically knock down AMPK-α (KD). Upon exposure to doxycycline for 3 days a ∼50% decrease in AMPK-α1 protein expression normalized to β-actin was observed.

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H+-ATPase accumulation in epididymal clear cells

    doi: 10.1152/ajpcell.00004.2009

    Figure Lengend Snippet: Tetracycline-inducible knockdown of AMPK-α1 in HEK-293 cells. Immunoblots for AMPK-α1 (top band) and β-actin (bottom band) of lysates derived from HEK-293 cells that were stably transfected with either a control short hairpin RNA (shRNA) that does not silence any known mammalian gene (CON) or an shRNA designed to specifically knock down AMPK-α (KD). Upon exposure to doxycycline for 3 days a ∼50% decrease in AMPK-α1 protein expression normalized to β-actin was observed.

    Article Snippet: Briefly, HEK-293 cells were transiently transfected to express FLAG-V-ATPase A subunit using Lipofectamine 2000 (Invitrogen), and cells were lysed 2 days after transfection.

    Techniques: Western Blot, Derivative Assay, Stable Transfection, Transfection, shRNA, Expressing

    Conversion of NBCe1 into an anion exchanger. a NBCe1 and AE1 amino-acid alignment showing location of the NBCe1 residues replaced with the corresponding AE1 residues. b Location of the NBCe1 residues replaced with the corresponding AE1 residues located in ion coordination site regions of NBCe1 and AE1 22 (PDB: 4YZF) formed by antiparallel β-strands preceding TMs 3 and 10, and in TM 8 and 10 are shown. c, e-g Loss of Na + -driven base transport and d, h-k gain of Cl − -driven base transport in the mutant constructs. l , m Cell surface expression. Immunoblot analysis of sulfo-NHS-SS-biotin labeled plasma membrane proteins in HEK-293 cells-expressing wt-NBCe1 and NBCe1 mutant (A+B+C). l Cell surface expression was normalized to GAPDH (mutant vs. wt, p

    Journal: Nature Communications

    Article Title: CryoEM structure of the human SLC4A4 sodium-coupled acid-base transporter NBCe1

    doi: 10.1038/s41467-018-03271-3

    Figure Lengend Snippet: Conversion of NBCe1 into an anion exchanger. a NBCe1 and AE1 amino-acid alignment showing location of the NBCe1 residues replaced with the corresponding AE1 residues. b Location of the NBCe1 residues replaced with the corresponding AE1 residues located in ion coordination site regions of NBCe1 and AE1 22 (PDB: 4YZF) formed by antiparallel β-strands preceding TMs 3 and 10, and in TM 8 and 10 are shown. c, e-g Loss of Na + -driven base transport and d, h-k gain of Cl − -driven base transport in the mutant constructs. l , m Cell surface expression. Immunoblot analysis of sulfo-NHS-SS-biotin labeled plasma membrane proteins in HEK-293 cells-expressing wt-NBCe1 and NBCe1 mutant (A+B+C). l Cell surface expression was normalized to GAPDH (mutant vs. wt, p

    Article Snippet: Expression and purification of NBCe1 Human N-terminally Strep(II)-tagged wild-type (wt) NBCe1-A in the pTT vector (Addgene) was transfected into HEK-293 cell (ATCC) monolayers using the calcium phosphate method.

    Techniques: Mutagenesis, Construct, Expressing, Labeling

    BRD4 mediates HIV D4-A7 RNA splicing. HEK293T cells were co-transfected with the pLTR.gp140/EGFP.Rev∆38/DsRed splicing reporter, 20 ng of pRev and 100 ng of pTat101 (AD8) without (wild-type, WT) or with specific mutations (K28A, K50A, K50/51A, K71A and R53A) then treated with DMSO, 1 μM JQ1 (+) or JQ1 (−). Cells were harvested at 48 h, EGFP and DsRed expression was quantified by flow cytometry. a The flow cytometry gating strategy used to identify % EGFP and % DsRed positive cells in a representative result from n = 3 independent experiments, each conducted in triplicate. b The mean percentage of spliced product DsRed/(DsRed + EGFP) from the 3 independent experiments is shown. Comparisons of each condition to JQ1 (+) were made using Friedman nonparametric test. Only statistically significant comparisons are shown *p

    Journal: Retrovirology

    Article Title: HIV latency reversing agents act through Tat post translational modifications

    doi: 10.1186/s12977-018-0421-6

    Figure Lengend Snippet: BRD4 mediates HIV D4-A7 RNA splicing. HEK293T cells were co-transfected with the pLTR.gp140/EGFP.Rev∆38/DsRed splicing reporter, 20 ng of pRev and 100 ng of pTat101 (AD8) without (wild-type, WT) or with specific mutations (K28A, K50A, K50/51A, K71A and R53A) then treated with DMSO, 1 μM JQ1 (+) or JQ1 (−). Cells were harvested at 48 h, EGFP and DsRed expression was quantified by flow cytometry. a The flow cytometry gating strategy used to identify % EGFP and % DsRed positive cells in a representative result from n = 3 independent experiments, each conducted in triplicate. b The mean percentage of spliced product DsRed/(DsRed + EGFP) from the 3 independent experiments is shown. Comparisons of each condition to JQ1 (+) were made using Friedman nonparametric test. Only statistically significant comparisons are shown *p

    Article Snippet: Briefly, 2 × 104 HEK293T cells (human embryonic kidney cells that stably express the SV40 large T antigen; American Tissue Culture Collection) were seeded per well into 96-well plates with DMEM (Gibco) + 10% FBS with Penicillin (100U/ml)/Streptomycin (100 μg/ml) and cultured overnight.

    Techniques: Transfection, Expressing, Flow Cytometry, Cytometry

    Mutations in Tat at lysine and arginine residues reduce the efficiency of HIV transcription. a Schematic diagram showing Tat protein sub-domains and residues that are post-translationally modified. Specific basic lysine (K) and arginine (R) residues were mutated to alanine (A) in pTat101 (AD8)-Flag using site-directed mutagenesis. b Western Blot analysis of wild-type (WT) or mutant Tat expression in HEK293T cells 48 h post-transfection using anti-Flag antibody. GAPDH was used as a loading control. c Luciferase expression of TZMbl cells 48 h following transfection of Tat mutants compared to WT Tat101, represented as the % of WT activity. Only statistically significant comparisons are shown ****p

    Journal: Retrovirology

    Article Title: HIV latency reversing agents act through Tat post translational modifications

    doi: 10.1186/s12977-018-0421-6

    Figure Lengend Snippet: Mutations in Tat at lysine and arginine residues reduce the efficiency of HIV transcription. a Schematic diagram showing Tat protein sub-domains and residues that are post-translationally modified. Specific basic lysine (K) and arginine (R) residues were mutated to alanine (A) in pTat101 (AD8)-Flag using site-directed mutagenesis. b Western Blot analysis of wild-type (WT) or mutant Tat expression in HEK293T cells 48 h post-transfection using anti-Flag antibody. GAPDH was used as a loading control. c Luciferase expression of TZMbl cells 48 h following transfection of Tat mutants compared to WT Tat101, represented as the % of WT activity. Only statistically significant comparisons are shown ****p

    Article Snippet: Briefly, 2 × 104 HEK293T cells (human embryonic kidney cells that stably express the SV40 large T antigen; American Tissue Culture Collection) were seeded per well into 96-well plates with DMEM (Gibco) + 10% FBS with Penicillin (100U/ml)/Streptomycin (100 μg/ml) and cultured overnight.

    Techniques: Modification, Mutagenesis, Western Blot, Expressing, Transfection, Luciferase, Activity Assay

    JQ1 can rescue the splicing activity lost by Tat mutants. HEK293T cells were transfected with the pLTR.gp140/EGFP.Rev∆38/DsRed splicing reporter and 100 ng of pTat101 (AD8) WT or with specific mutations (K28A, K50A, K50/51A, K71A and R53A). Cells were treated with DMSO (D, 1:5000) or JQ1 (J, 1 μM), harvested at 48 h and DsRed and EGFP expression quantified using flow cytometry. a Data were represented as the proportion of spliced product [DsRed/(DsRed + EGFP) × 100] relative to WT Tat+ DMSO. Comparisons between DMSO and JQ1 for either no Tat, WT Tat or each mutant Tat were made using multiple paired T tests. b The difference between JQ1 and DMSO in the proportion of spliced product [DsRed/(DsRed + EGFP) × 100] was calculated for each Tat mutant, no Tat or WT Tat, and then compared to the difference with WT Tat101. Only statistically significant comparisons are shown *p

    Journal: Retrovirology

    Article Title: HIV latency reversing agents act through Tat post translational modifications

    doi: 10.1186/s12977-018-0421-6

    Figure Lengend Snippet: JQ1 can rescue the splicing activity lost by Tat mutants. HEK293T cells were transfected with the pLTR.gp140/EGFP.Rev∆38/DsRed splicing reporter and 100 ng of pTat101 (AD8) WT or with specific mutations (K28A, K50A, K50/51A, K71A and R53A). Cells were treated with DMSO (D, 1:5000) or JQ1 (J, 1 μM), harvested at 48 h and DsRed and EGFP expression quantified using flow cytometry. a Data were represented as the proportion of spliced product [DsRed/(DsRed + EGFP) × 100] relative to WT Tat+ DMSO. Comparisons between DMSO and JQ1 for either no Tat, WT Tat or each mutant Tat were made using multiple paired T tests. b The difference between JQ1 and DMSO in the proportion of spliced product [DsRed/(DsRed + EGFP) × 100] was calculated for each Tat mutant, no Tat or WT Tat, and then compared to the difference with WT Tat101. Only statistically significant comparisons are shown *p

    Article Snippet: Briefly, 2 × 104 HEK293T cells (human embryonic kidney cells that stably express the SV40 large T antigen; American Tissue Culture Collection) were seeded per well into 96-well plates with DMEM (Gibco) + 10% FBS with Penicillin (100U/ml)/Streptomycin (100 μg/ml) and cultured overnight.

    Techniques: Activity Assay, Transfection, Expressing, Flow Cytometry, Cytometry, Mutagenesis

    Specific mutations within Tat reduce HIV D4-A7 splicing. HEK293T cells were co-transfected with the pLTR.gp140/EGFP.Rev∆38/DsRed splicing reporter, 20 ng of pRev and 100 ng of pTat101 without (WT) or with specific mutations (K28A, K50A, K50/51A, K71A and R53A). Cells were harvested at 48 h and DsRed expression was quantified by flow cytometry. a A representative example of the gating strategy used to identify % DsRed positive cells that represent spliced product. b The proportion of spliced product [DsRed/(DsRed + EGFP) × 100] relative to WT Tat is shown from n = 5 independent experiments, each conducted in triplicate. Comparisons of each condition to DMSO were made using a Paired T test. Only statistically significant comparisons are shown **p

    Journal: Retrovirology

    Article Title: HIV latency reversing agents act through Tat post translational modifications

    doi: 10.1186/s12977-018-0421-6

    Figure Lengend Snippet: Specific mutations within Tat reduce HIV D4-A7 splicing. HEK293T cells were co-transfected with the pLTR.gp140/EGFP.Rev∆38/DsRed splicing reporter, 20 ng of pRev and 100 ng of pTat101 without (WT) or with specific mutations (K28A, K50A, K50/51A, K71A and R53A). Cells were harvested at 48 h and DsRed expression was quantified by flow cytometry. a A representative example of the gating strategy used to identify % DsRed positive cells that represent spliced product. b The proportion of spliced product [DsRed/(DsRed + EGFP) × 100] relative to WT Tat is shown from n = 5 independent experiments, each conducted in triplicate. Comparisons of each condition to DMSO were made using a Paired T test. Only statistically significant comparisons are shown **p

    Article Snippet: Briefly, 2 × 104 HEK293T cells (human embryonic kidney cells that stably express the SV40 large T antigen; American Tissue Culture Collection) were seeded per well into 96-well plates with DMEM (Gibco) + 10% FBS with Penicillin (100U/ml)/Streptomycin (100 μg/ml) and cultured overnight.

    Techniques: Transfection, Expressing, Flow Cytometry, Cytometry

    JQ1 consistently rescued HIV D4A7 splicing. HEK293T cells were transfected with the pLTR.gp140/EGFP.Rev∆38/DsRed splicing reporter without ( a ) or with 100 ng of pTat101 (AD8)-Flag that was either wild-type (shown in Fig. 2 ) or had the specific mutations: b K28A, c K50A, d K50/51A, e K71A or f R53A. Transfected cells were then treated with a panel of LRAs for 48 h, harvested and DsRed expression was quantified using flow cytometry. The fold change in the proportion of spliced product [DsRed/(DsRed + EGFP) × 100] in the no Tat or Tat mutants relative to WT Tat+ DMSO is represented. Comparisons of each condition to DMSO were made using the 2-way ANOVA test. Only statistically significant comparisons are shown *p

    Journal: Retrovirology

    Article Title: HIV latency reversing agents act through Tat post translational modifications

    doi: 10.1186/s12977-018-0421-6

    Figure Lengend Snippet: JQ1 consistently rescued HIV D4A7 splicing. HEK293T cells were transfected with the pLTR.gp140/EGFP.Rev∆38/DsRed splicing reporter without ( a ) or with 100 ng of pTat101 (AD8)-Flag that was either wild-type (shown in Fig. 2 ) or had the specific mutations: b K28A, c K50A, d K50/51A, e K71A or f R53A. Transfected cells were then treated with a panel of LRAs for 48 h, harvested and DsRed expression was quantified using flow cytometry. The fold change in the proportion of spliced product [DsRed/(DsRed + EGFP) × 100] in the no Tat or Tat mutants relative to WT Tat+ DMSO is represented. Comparisons of each condition to DMSO were made using the 2-way ANOVA test. Only statistically significant comparisons are shown *p

    Article Snippet: Briefly, 2 × 104 HEK293T cells (human embryonic kidney cells that stably express the SV40 large T antigen; American Tissue Culture Collection) were seeded per well into 96-well plates with DMEM (Gibco) + 10% FBS with Penicillin (100U/ml)/Streptomycin (100 μg/ml) and cultured overnight.

    Techniques: Transfection, Expressing, Flow Cytometry, Cytometry

    Model used to determine the effects of LRAs on LTR-driven transcription and splicing in the presence and absence of Tat. a Schematic of the in vitro model used in this study to determine the effects of LRAs on LTR-driven transcription and splicing. HEK293T cells were co-transfected with the LTR reporter construct together with a plasmid expressing Rev, with or without a plasmid expressing Tat for 48 h, followed by analysis using flow cytometry. The LTR construct expresses either unspliced protein fused to enhanced green fluorescent protein (EGFP) or spliced ∆38rev protein fused with DsRed. b Gating strategy of a representative sample used to identify the percentage of cells expressing EGFP or DsRed. c – e The combined results of 5 independent experiments showing the percentage of cells expressing EGFP ( c ), DsRed ( d ) or the percentage of spliced products (DsRed/DsRed+ EGFP) ( e ). The mean ± SEM of the 5 separate experiments, each run in triplicate, is shown. Comparisons were made to cells only (−) using a paired T test. Only statistically significant comparisons are shown **p

    Journal: Retrovirology

    Article Title: HIV latency reversing agents act through Tat post translational modifications

    doi: 10.1186/s12977-018-0421-6

    Figure Lengend Snippet: Model used to determine the effects of LRAs on LTR-driven transcription and splicing in the presence and absence of Tat. a Schematic of the in vitro model used in this study to determine the effects of LRAs on LTR-driven transcription and splicing. HEK293T cells were co-transfected with the LTR reporter construct together with a plasmid expressing Rev, with or without a plasmid expressing Tat for 48 h, followed by analysis using flow cytometry. The LTR construct expresses either unspliced protein fused to enhanced green fluorescent protein (EGFP) or spliced ∆38rev protein fused with DsRed. b Gating strategy of a representative sample used to identify the percentage of cells expressing EGFP or DsRed. c – e The combined results of 5 independent experiments showing the percentage of cells expressing EGFP ( c ), DsRed ( d ) or the percentage of spliced products (DsRed/DsRed+ EGFP) ( e ). The mean ± SEM of the 5 separate experiments, each run in triplicate, is shown. Comparisons were made to cells only (−) using a paired T test. Only statistically significant comparisons are shown **p

    Article Snippet: Briefly, 2 × 104 HEK293T cells (human embryonic kidney cells that stably express the SV40 large T antigen; American Tissue Culture Collection) were seeded per well into 96-well plates with DMEM (Gibco) + 10% FBS with Penicillin (100U/ml)/Streptomycin (100 μg/ml) and cultured overnight.

    Techniques: In Vitro, Transfection, Construct, Plasmid Preparation, Expressing, Flow Cytometry, Cytometry

    JQ1 but not HDACi can increase splicing in the absence and presence of Tat. HEK293T cells were co-transfected with the pLTR.gp140/EGFP.Rev∆38/DsRed splicing reporter together with a plasmid expressing Rev, in the absence ( a , b ) or presence ( c , d ) of 100 ng of pTat101 (AD8)-Flag expression plasmid and then treated with a panel of LRAs or DMSO diluent control (n = 5). EGFP (unspliced) and DsRed (spliced) expression were measured using flow cytometry. Comparisons of each condition to DMSO were made using 2-way ANOVA test. Only statistically significant comparisons are shown *p

    Journal: Retrovirology

    Article Title: HIV latency reversing agents act through Tat post translational modifications

    doi: 10.1186/s12977-018-0421-6

    Figure Lengend Snippet: JQ1 but not HDACi can increase splicing in the absence and presence of Tat. HEK293T cells were co-transfected with the pLTR.gp140/EGFP.Rev∆38/DsRed splicing reporter together with a plasmid expressing Rev, in the absence ( a , b ) or presence ( c , d ) of 100 ng of pTat101 (AD8)-Flag expression plasmid and then treated with a panel of LRAs or DMSO diluent control (n = 5). EGFP (unspliced) and DsRed (spliced) expression were measured using flow cytometry. Comparisons of each condition to DMSO were made using 2-way ANOVA test. Only statistically significant comparisons are shown *p

    Article Snippet: Briefly, 2 × 104 HEK293T cells (human embryonic kidney cells that stably express the SV40 large T antigen; American Tissue Culture Collection) were seeded per well into 96-well plates with DMEM (Gibco) + 10% FBS with Penicillin (100U/ml)/Streptomycin (100 μg/ml) and cultured overnight.

    Techniques: Transfection, Plasmid Preparation, Expressing, Flow Cytometry, Cytometry

    JQ1 affects alternative splicing by modulating hnRNP protein levels. Alternative splicing pattern of CD46 ( a ), ATF2 ( b ) and ABI1 ( c ) in HEK293T cells following 24 h of JQ1 (1 μM) or DMSO treatment. Inclusion and exclusion of exons are indicated on the right of the gel with white boxes, while grey boxes represent constitutively included exons. NTC = No Template Control, -RT corresponds to minus reverse transcriptase control. d Western Blot analysis of splicing factors following treatment with JQ1 or DMSO diluent control

    Journal: Retrovirology

    Article Title: HIV latency reversing agents act through Tat post translational modifications

    doi: 10.1186/s12977-018-0421-6

    Figure Lengend Snippet: JQ1 affects alternative splicing by modulating hnRNP protein levels. Alternative splicing pattern of CD46 ( a ), ATF2 ( b ) and ABI1 ( c ) in HEK293T cells following 24 h of JQ1 (1 μM) or DMSO treatment. Inclusion and exclusion of exons are indicated on the right of the gel with white boxes, while grey boxes represent constitutively included exons. NTC = No Template Control, -RT corresponds to minus reverse transcriptase control. d Western Blot analysis of splicing factors following treatment with JQ1 or DMSO diluent control

    Article Snippet: Briefly, 2 × 104 HEK293T cells (human embryonic kidney cells that stably express the SV40 large T antigen; American Tissue Culture Collection) were seeded per well into 96-well plates with DMEM (Gibco) + 10% FBS with Penicillin (100U/ml)/Streptomycin (100 μg/ml) and cultured overnight.

    Techniques: Western Blot

    JQ1 induces HIV spliced RNA accumulation in the absence and presence of Tat. a Schematic of unspliced and spliced mRNAs produced following HEK293T cell transfection with the pLTR.gp140/EGFP.Rev∆38/DsRed splicing reporter. The different sets of primers-probe used for HIV RNA quantification are displayed as coloured arrows for primers, and linked coloured spots for the probe. The env set detects unspliced mRNA (green), rev set detects spliced mRNA (red) and the viral set detects all viral mRNAs including the co-transfected pRev NL4.3 mRNA (brown). b – d HEK293T cells transfected in the absence or presence of 100 ng of pTat101 (AD8)-Flag expression plasmid were treated for 24 h with JQ1 (1 μM) or DMSO diluent control (n = 4). Cells were then harvested and EGFP (unspliced) and DsRed (spliced) protein expression measured using flow cytometry (Fig. S2), while HIV unspliced (US), spliced (D4-A7) and all viral RNA expression levels (copies/μl) were quantified by droplet digital PCR (ddPCR) ( b – d ). The ratio of US ( c ) and spliced ( d ) over all viral RNAs is displayed as fold-change (FC) over DMSO. Comparisons of each condition to DMSO were made using a paired T test. Only statistically significant comparisons are shown *p

    Journal: Retrovirology

    Article Title: HIV latency reversing agents act through Tat post translational modifications

    doi: 10.1186/s12977-018-0421-6

    Figure Lengend Snippet: JQ1 induces HIV spliced RNA accumulation in the absence and presence of Tat. a Schematic of unspliced and spliced mRNAs produced following HEK293T cell transfection with the pLTR.gp140/EGFP.Rev∆38/DsRed splicing reporter. The different sets of primers-probe used for HIV RNA quantification are displayed as coloured arrows for primers, and linked coloured spots for the probe. The env set detects unspliced mRNA (green), rev set detects spliced mRNA (red) and the viral set detects all viral mRNAs including the co-transfected pRev NL4.3 mRNA (brown). b – d HEK293T cells transfected in the absence or presence of 100 ng of pTat101 (AD8)-Flag expression plasmid were treated for 24 h with JQ1 (1 μM) or DMSO diluent control (n = 4). Cells were then harvested and EGFP (unspliced) and DsRed (spliced) protein expression measured using flow cytometry (Fig. S2), while HIV unspliced (US), spliced (D4-A7) and all viral RNA expression levels (copies/μl) were quantified by droplet digital PCR (ddPCR) ( b – d ). The ratio of US ( c ) and spliced ( d ) over all viral RNAs is displayed as fold-change (FC) over DMSO. Comparisons of each condition to DMSO were made using a paired T test. Only statistically significant comparisons are shown *p

    Article Snippet: Briefly, 2 × 104 HEK293T cells (human embryonic kidney cells that stably express the SV40 large T antigen; American Tissue Culture Collection) were seeded per well into 96-well plates with DMEM (Gibco) + 10% FBS with Penicillin (100U/ml)/Streptomycin (100 μg/ml) and cultured overnight.

    Techniques: Produced, Transfection, Expressing, Plasmid Preparation, Flow Cytometry, Cytometry, RNA Expression, Digital PCR

    dLRRK and hLRRK2 promote cap-dependent translation in postsynaptic muscles. ( a ) Quantification of 5′-UTR luciferase reporter activity in response to co-transfection with either wild-type hLRRK2 wt or kinase-dead hLRRK2 3XKD normalized to psiCHECK2 response in HEK293T. n =3 experiments, * P =0.043. See also Supplementary Table 2 . ( b ) Western blot analysis of in vivo Fur1-5′-UTR-eGFP reporter expression when co-expressed with either dLRRK 3KD (+/UAS- Fur1 -5′-UTR- eGFP ; UAS -dLRRK 3KD / MHC-Gal4 ) or wild-type dLRRK wt (UAS -dLRRK wt /UAS- Fur1 -5′-UTR- eGFP ; +/ MHC-Gal4 ). Left: Western blotting probed with (top) anti-GFP and (bottom) anti-Actin as loading control. Right: quantification of Fur1-5′-UTR-eGFP expression normalized to actin levels. n =3 for each. * P

    Journal: Nature Communications

    Article Title: LRRK2 regulates retrograde synaptic compensation at the Drosophila neuromuscular junction

    doi: 10.1038/ncomms12188

    Figure Lengend Snippet: dLRRK and hLRRK2 promote cap-dependent translation in postsynaptic muscles. ( a ) Quantification of 5′-UTR luciferase reporter activity in response to co-transfection with either wild-type hLRRK2 wt or kinase-dead hLRRK2 3XKD normalized to psiCHECK2 response in HEK293T. n =3 experiments, * P =0.043. See also Supplementary Table 2 . ( b ) Western blot analysis of in vivo Fur1-5′-UTR-eGFP reporter expression when co-expressed with either dLRRK 3KD (+/UAS- Fur1 -5′-UTR- eGFP ; UAS -dLRRK 3KD / MHC-Gal4 ) or wild-type dLRRK wt (UAS -dLRRK wt /UAS- Fur1 -5′-UTR- eGFP ; +/ MHC-Gal4 ). Left: Western blotting probed with (top) anti-GFP and (bottom) anti-Actin as loading control. Right: quantification of Fur1-5′-UTR-eGFP expression normalized to actin levels. n =3 for each. * P

    Article Snippet: HEK293T cells were transfected with control or 5′-UTR Luciferase sensors with or without co-transfection with hLRRK2 plasmid (hLRRK-WT (Addgene#17609) was a gift from Ted Dawson and hLRRK2-3xKD (Addgene#25366) was a gift from Mark Cookson) using Lipofectamine 2,000 (Invitrogen).

    Techniques: Luciferase, Activity Assay, Cotransfection, Western Blot, In Vivo, Expressing

    Rab37 is a novel MetAP-2 substrate ( A and B ) LC-MS analysis of N-terminal peptides from MPE cells differentially labeled with [ 13 C 6 ]Arg before treatment with vehicle or TNP-470 (50 nM, 16 hr). Unprocessed N-terminal tryptic peptides are highlighted in the ovals. Note the peak height difference in the light, vehicle-treated and heavy (i.e., [ 13 C 6 ]Arg labeled) TNP-470 treated peptides for the known MetAP-2 substrate GAPDH. No light, vehicle-treated N-terminal trypic peptide was detected for the novel MetAP-2 substrate Rab37, suggesting that this protein is destabilized by NME. ( C ) Diagram of Rab37 constructs used in this study. ( D ) Whole cell lysates from HEK 293T cells transiently transfected with WT- or T2E-Rab37 (50 ng) or empty vector followed by incubation with vehicle or TNP-470 (50 nM, 16 hr) were immunoblotted with specific antibodies as indicated.

    Journal: Chemistry & biology

    Article Title: Disruption of Wnt Planar Cell Polarity Signaling by Aberrant Accumulation of the MetAP-2 Substrate Rab37

    doi: 10.1016/j.chembiol.2011.07.020

    Figure Lengend Snippet: Rab37 is a novel MetAP-2 substrate ( A and B ) LC-MS analysis of N-terminal peptides from MPE cells differentially labeled with [ 13 C 6 ]Arg before treatment with vehicle or TNP-470 (50 nM, 16 hr). Unprocessed N-terminal tryptic peptides are highlighted in the ovals. Note the peak height difference in the light, vehicle-treated and heavy (i.e., [ 13 C 6 ]Arg labeled) TNP-470 treated peptides for the known MetAP-2 substrate GAPDH. No light, vehicle-treated N-terminal trypic peptide was detected for the novel MetAP-2 substrate Rab37, suggesting that this protein is destabilized by NME. ( C ) Diagram of Rab37 constructs used in this study. ( D ) Whole cell lysates from HEK 293T cells transiently transfected with WT- or T2E-Rab37 (50 ng) or empty vector followed by incubation with vehicle or TNP-470 (50 nM, 16 hr) were immunoblotted with specific antibodies as indicated.

    Article Snippet: For stable knockdown of Rab37, HEK 293T cells were co-transfected in a 1:3:3 ratio with pMD2.G and psPAX2 (cat# 12260, Addgene) along with pLKO shRNA vectors targeting Rab37 (cat# RHS4552- ; Open Biosystems, Huntsville, AL) or a non-targeting scramble shRNA (cat# 1864, Addgene).

    Techniques: Liquid Chromatography with Mass Spectroscopy, Labeling, Construct, Transfection, Plasmid Preparation, Incubation

    Localization of Rab37 GTPase cycle mutants suggests a role in plasma membrane to Golgi trafficking ( A ) HUVE cells electroporated with WT- or T2E-Rab37 were cultured on gelatin-coated slides followed by staining with antibodies for FLAG and the Golgi marker Giantin. ( B ) HEK 293T cells transiently transfected with WT-Rab37, Rab37-Q89L or Rab37-T43N were cultured on poly-L-lysine coated slides followed by staining with antibodies specific for FLAG and the Golgi marker Giantin. Scale bar .

    Journal: Chemistry & biology

    Article Title: Disruption of Wnt Planar Cell Polarity Signaling by Aberrant Accumulation of the MetAP-2 Substrate Rab37

    doi: 10.1016/j.chembiol.2011.07.020

    Figure Lengend Snippet: Localization of Rab37 GTPase cycle mutants suggests a role in plasma membrane to Golgi trafficking ( A ) HUVE cells electroporated with WT- or T2E-Rab37 were cultured on gelatin-coated slides followed by staining with antibodies for FLAG and the Golgi marker Giantin. ( B ) HEK 293T cells transiently transfected with WT-Rab37, Rab37-Q89L or Rab37-T43N were cultured on poly-L-lysine coated slides followed by staining with antibodies specific for FLAG and the Golgi marker Giantin. Scale bar .

    Article Snippet: For stable knockdown of Rab37, HEK 293T cells were co-transfected in a 1:3:3 ratio with pMD2.G and psPAX2 (cat# 12260, Addgene) along with pLKO shRNA vectors targeting Rab37 (cat# RHS4552- ; Open Biosystems, Huntsville, AL) or a non-targeting scramble shRNA (cat# 1864, Addgene).

    Techniques: Cell Culture, Staining, Marker, Transfection

    Aberrant Rab37 accumulation disrupts endothelial cell function ( A ) Whole cell lysates from HUVE or HEK 293T cells stably expressing GFP or WT- or T2E-Rab37 were immunoblotted with specific antibodies as indicated. ( B ) Effect of stably expressing GFP or WT- or T2E-Rab37 on [ 3 H]-thymidine incorporation in HEK 293T cells ( black bars ) or HUVE cells ( white bars ). ( C ) HUVE cells stably expressing GFP (●, solid line ), WT-Rab37 (■, dashed line ) or T2E-Rab37 (▲, dotted line ) were treated with the indicated concentrations of TNP-470 for 20 hr followed by labeling with [ 3 H]-thymidine (20 μCi/mL; 4 hr) before harvesting of nuclei. ( D ) Stable retroviral infection with WT- or T2E-Rab37 suppresses network formation in HUVE cells plated on Matrigel ™ relative to cells stably infected with GFP or mock infected cells. Scale bar , 100 μm. ( E ) Whole cell lysates from HUVE cells stably expressing HA-ΔDIX-Dvl2 along with GFP or T2E-Rab37 were immunoblotted with specific antibodies as indicated. ( F ) Effect of stably expressing GFP ( black bars ) T2E-Rab37 ( white bars ) on [ 3 .

    Journal: Chemistry & biology

    Article Title: Disruption of Wnt Planar Cell Polarity Signaling by Aberrant Accumulation of the MetAP-2 Substrate Rab37

    doi: 10.1016/j.chembiol.2011.07.020

    Figure Lengend Snippet: Aberrant Rab37 accumulation disrupts endothelial cell function ( A ) Whole cell lysates from HUVE or HEK 293T cells stably expressing GFP or WT- or T2E-Rab37 were immunoblotted with specific antibodies as indicated. ( B ) Effect of stably expressing GFP or WT- or T2E-Rab37 on [ 3 H]-thymidine incorporation in HEK 293T cells ( black bars ) or HUVE cells ( white bars ). ( C ) HUVE cells stably expressing GFP (●, solid line ), WT-Rab37 (■, dashed line ) or T2E-Rab37 (▲, dotted line ) were treated with the indicated concentrations of TNP-470 for 20 hr followed by labeling with [ 3 H]-thymidine (20 μCi/mL; 4 hr) before harvesting of nuclei. ( D ) Stable retroviral infection with WT- or T2E-Rab37 suppresses network formation in HUVE cells plated on Matrigel ™ relative to cells stably infected with GFP or mock infected cells. Scale bar , 100 μm. ( E ) Whole cell lysates from HUVE cells stably expressing HA-ΔDIX-Dvl2 along with GFP or T2E-Rab37 were immunoblotted with specific antibodies as indicated. ( F ) Effect of stably expressing GFP ( black bars ) T2E-Rab37 ( white bars ) on [ 3 .

    Article Snippet: For stable knockdown of Rab37, HEK 293T cells were co-transfected in a 1:3:3 ratio with pMD2.G and psPAX2 (cat# 12260, Addgene) along with pLKO shRNA vectors targeting Rab37 (cat# RHS4552- ; Open Biosystems, Huntsville, AL) or a non-targeting scramble shRNA (cat# 1864, Addgene).

    Techniques: Cell Function Assay, Stable Transfection, Expressing, Labeling, Infection

    Association of C4orf14 with the mitochondrial small ribosomal subunit. ( A ) Affinity purified C4orf14.FLAG.StrepII protein was isolated from mitochondria fractions of transgenic HEK293T cells and the concentrated eluted fractions resolved by SDS–PAGE. Proteins identified by MS are indicated on the left and right of the gel. They included 22 polypeptides of the 28S subunit (MRPS; see Supplementary Table S1 ); ( B ) sucrose-gradient purified mitochondria from HEK293T cells were lysed and fractionated on sucrose gradients. Antibodies to MRPS2 and MRPS18, and MRPL3 and MRPL11 were used as markers of the 28S and 39S subunits, respectively. ( C ) 143B cells were transfected with dsRNA (c3 or c6) targeting C4orf14 mRNA and the effects on mitochondrial protein synthesis (i); selected proteins in mitochondria (ii) and mitochondrial ribosomal RNAs (panel D and Supplementary Figure S2 ) were examined 72 h later. GAPDH: glyceraldehyde-3-phosphate dehydrogenase, the outer mitochondrial membrane protein TOM20, a putative mitochondrial RNA helicase DHX30 and components of the 55S ribosome (MRPS2, MRPS29, MRPL3 and MRPL11).

    Journal: Nucleic Acids Research

    Article Title: Human C4orf14 interacts with the mitochondrial nucleoid and is involved in the biogenesis of the small mitochondrial ribosomal subunit

    doi: 10.1093/nar/gks257

    Figure Lengend Snippet: Association of C4orf14 with the mitochondrial small ribosomal subunit. ( A ) Affinity purified C4orf14.FLAG.StrepII protein was isolated from mitochondria fractions of transgenic HEK293T cells and the concentrated eluted fractions resolved by SDS–PAGE. Proteins identified by MS are indicated on the left and right of the gel. They included 22 polypeptides of the 28S subunit (MRPS; see Supplementary Table S1 ); ( B ) sucrose-gradient purified mitochondria from HEK293T cells were lysed and fractionated on sucrose gradients. Antibodies to MRPS2 and MRPS18, and MRPL3 and MRPL11 were used as markers of the 28S and 39S subunits, respectively. ( C ) 143B cells were transfected with dsRNA (c3 or c6) targeting C4orf14 mRNA and the effects on mitochondrial protein synthesis (i); selected proteins in mitochondria (ii) and mitochondrial ribosomal RNAs (panel D and Supplementary Figure S2 ) were examined 72 h later. GAPDH: glyceraldehyde-3-phosphate dehydrogenase, the outer mitochondrial membrane protein TOM20, a putative mitochondrial RNA helicase DHX30 and components of the 55S ribosome (MRPS2, MRPS29, MRPL3 and MRPL11).

    Article Snippet: Affinity purification of C4orf14 and TFAM from HEK293T cells Human complementary DNAs (cDNAs) of TFAM or C4orf14, with a carboxy-terminal Strep II followed by a FLAG tag, were introduced into HEK293T cells (Invitrogen), to establish inducible, transgenic cell lines.

    Techniques: Affinity Purification, Isolation, Transgenic Assay, SDS Page, Mass Spectrometry, Purification, Transfection

    C4orf14 co-purifies with FLAG-StrepII-tagged TFAM and C4orf14.FLAG.StrepII is targeted to mitochondria in HEK293T cells. ( A ) Affinity purified TFAM.FLAG.StrepII protein was isolated from mitochondria fractions of HEK293T cells. Proteins from various stages of the purification procedure were analysed by immunoblotting, after separation via 4–12% SDS–PAGE. S, supernatant; F, flow-through; w, washes; e, eluted fractions. Pvu II digested mtDNA was detected by Southern hybridization. ( B ) Immunocytochemistry of C4orf14. FLAG.StrepII expressing HEK293T cells with an anti-FLAG antibody (green) 24 h after transgene induction. Additionally, TFAM was labelled with an antibody (false-colour violet), mitochondria were stained with MitoTracker (false-colour red) and the nucleus was stained with DAPI (blue). Bottom right: merged image of C4orf14 and TFAM. The 4 × merged image (bottom left) is C4orf14, TFAM, DAPI and MitoTracker. White arrows in the enlarged images (bottom centre) indicate foci where TFAM and C4orf14 coincide within the mitochondrial network. The ellipses each enclose two TFAM labeled foci, one of which coincides with C4orf14.

    Journal: Nucleic Acids Research

    Article Title: Human C4orf14 interacts with the mitochondrial nucleoid and is involved in the biogenesis of the small mitochondrial ribosomal subunit

    doi: 10.1093/nar/gks257

    Figure Lengend Snippet: C4orf14 co-purifies with FLAG-StrepII-tagged TFAM and C4orf14.FLAG.StrepII is targeted to mitochondria in HEK293T cells. ( A ) Affinity purified TFAM.FLAG.StrepII protein was isolated from mitochondria fractions of HEK293T cells. Proteins from various stages of the purification procedure were analysed by immunoblotting, after separation via 4–12% SDS–PAGE. S, supernatant; F, flow-through; w, washes; e, eluted fractions. Pvu II digested mtDNA was detected by Southern hybridization. ( B ) Immunocytochemistry of C4orf14. FLAG.StrepII expressing HEK293T cells with an anti-FLAG antibody (green) 24 h after transgene induction. Additionally, TFAM was labelled with an antibody (false-colour violet), mitochondria were stained with MitoTracker (false-colour red) and the nucleus was stained with DAPI (blue). Bottom right: merged image of C4orf14 and TFAM. The 4 × merged image (bottom left) is C4orf14, TFAM, DAPI and MitoTracker. White arrows in the enlarged images (bottom centre) indicate foci where TFAM and C4orf14 coincide within the mitochondrial network. The ellipses each enclose two TFAM labeled foci, one of which coincides with C4orf14.

    Article Snippet: Affinity purification of C4orf14 and TFAM from HEK293T cells Human complementary DNAs (cDNAs) of TFAM or C4orf14, with a carboxy-terminal Strep II followed by a FLAG tag, were introduced into HEK293T cells (Invitrogen), to establish inducible, transgenic cell lines.

    Techniques: Affinity Purification, Isolation, Purification, SDS Page, Flow Cytometry, Hybridization, Immunocytochemistry, Expressing, Staining, Labeling

    A critical FN-II domain loop from uPARAP reconstitutes collagen internalization function in uPARAP chimeras with PLA 2 R and DEC-205 FN-II domains. A , internalization of radiolabeled collagen type I (100 ng/ml) by HEK-293T cells transfected with uPARAP

    Journal: The Journal of Biological Chemistry

    Article Title: Complex Determinants in Specific Members of the Mannose Receptor Family Govern Collagen Endocytosis *

    doi: 10.1074/jbc.M113.512780

    Figure Lengend Snippet: A critical FN-II domain loop from uPARAP reconstitutes collagen internalization function in uPARAP chimeras with PLA 2 R and DEC-205 FN-II domains. A , internalization of radiolabeled collagen type I (100 ng/ml) by HEK-293T cells transfected with uPARAP

    Article Snippet: In this system, HEK-293T cells were transiently transfected with cDNA encoding full-length uPARAP, MR, PLA2 R, or DEC-205, respectively.

    Techniques: Proximity Ligation Assay, Transfection

    Ligands internalized by each receptor are degraded lysosomally. The intracellular accumulation of radiolabeled collagen type I (100 ng/ml) in HEK-293T cells transfected with uPARAP ( A ), MR ( B ), PLA 2 R ( C , left panel ), and DEC-205 ( D , left panel ) is shown

    Journal: The Journal of Biological Chemistry

    Article Title: Complex Determinants in Specific Members of the Mannose Receptor Family Govern Collagen Endocytosis *

    doi: 10.1074/jbc.M113.512780

    Figure Lengend Snippet: Ligands internalized by each receptor are degraded lysosomally. The intracellular accumulation of radiolabeled collagen type I (100 ng/ml) in HEK-293T cells transfected with uPARAP ( A ), MR ( B ), PLA 2 R ( C , left panel ), and DEC-205 ( D , left panel ) is shown

    Article Snippet: In this system, HEK-293T cells were transiently transfected with cDNA encoding full-length uPARAP, MR, PLA2 R, or DEC-205, respectively.

    Techniques: Transfection, Proximity Ligation Assay

    Endocytosis of radiolabeled ligands mediated by uPARAP, MR, PLA 2 R, and DEC-205. HEK-293T cells were transfected with uPARAP ( A ), MR ( B ), PLA 2 R ( C ), and DEC-205 ( D ). In each panel, black columns show the internalization of a radiolabeled positive control

    Journal: The Journal of Biological Chemistry

    Article Title: Complex Determinants in Specific Members of the Mannose Receptor Family Govern Collagen Endocytosis *

    doi: 10.1074/jbc.M113.512780

    Figure Lengend Snippet: Endocytosis of radiolabeled ligands mediated by uPARAP, MR, PLA 2 R, and DEC-205. HEK-293T cells were transfected with uPARAP ( A ), MR ( B ), PLA 2 R ( C ), and DEC-205 ( D ). In each panel, black columns show the internalization of a radiolabeled positive control

    Article Snippet: In this system, HEK-293T cells were transiently transfected with cDNA encoding full-length uPARAP, MR, PLA2 R, or DEC-205, respectively.

    Techniques: Proximity Ligation Assay, Transfection, Positive Control

    Transfected HEK-293T cells express members of the MR protein family. Western blot analysis of uPARAP ( A ), MR ( B ), PLA 2 R ( C ), and DEC-205 ( D ) expression in whole cell lysates from HEK-293T cells 24 h post transfection. In each panel, duplicate samples

    Journal: The Journal of Biological Chemistry

    Article Title: Complex Determinants in Specific Members of the Mannose Receptor Family Govern Collagen Endocytosis *

    doi: 10.1074/jbc.M113.512780

    Figure Lengend Snippet: Transfected HEK-293T cells express members of the MR protein family. Western blot analysis of uPARAP ( A ), MR ( B ), PLA 2 R ( C ), and DEC-205 ( D ) expression in whole cell lysates from HEK-293T cells 24 h post transfection. In each panel, duplicate samples

    Article Snippet: In this system, HEK-293T cells were transiently transfected with cDNA encoding full-length uPARAP, MR, PLA2 R, or DEC-205, respectively.

    Techniques: Transfection, Western Blot, Proximity Ligation Assay, Expressing

    BORIS isoforms bind and activate the transcription of conserved human and mouse CST testis-specific promoter regions. ( A ) Schematic representation of multiple mouse CST promoters, where exon 1f s has been shown to be testis specific [48] and the BORIS binding site has been mapped to the testis-specific CST promoter [9] . The alignment of human and mouse BORIS binding site in CST promoter is shown at the bottom. The 5 cytosines boxed in the alignment are contact nucleotides for BORIS protein as it was determined by methylation interference assays [9] . Nucleotides that are 100% identical to the human nucleotide sequence are shaded in grey. Dashes indicate insertions or deletions. ( B ) and ( C ) Most BORIS isoforms bind to the human and mouse testis-specific CST promoter in EMSA. The number of ZFs and utilization of alternative N-and C-termini for each BORIS isoform are shown at the bottom of gel. The band shifts specific for particular N- and C-termini are indicated by arrows. Luciferase and CTCF were used as negative and positive controls, respectively. ( D ) The mouse CST promoter is transcriptionally activated by BORIS isoforms in HEK293T cells. HEK293T cells were either co-transfected with mouse Boris, mouse Ctcf, empty pCI vector (EV) or the human BORIS isoform constructs, as well as with pGL-3 containing 359 bp of wild type or mutant CST promoter. Luciferase assays were done 48 h after transfections. All luciferase activities were normalized for transfection efficiency by measuring the Renilla luciferase activity from the co-transfected pRL-TK vector. Error bars are standard deviations.

    Journal: PLoS ONE

    Article Title: The Structural Complexity of the Human BORIS Gene in Gametogenesis and Cancer

    doi: 10.1371/journal.pone.0013872

    Figure Lengend Snippet: BORIS isoforms bind and activate the transcription of conserved human and mouse CST testis-specific promoter regions. ( A ) Schematic representation of multiple mouse CST promoters, where exon 1f s has been shown to be testis specific [48] and the BORIS binding site has been mapped to the testis-specific CST promoter [9] . The alignment of human and mouse BORIS binding site in CST promoter is shown at the bottom. The 5 cytosines boxed in the alignment are contact nucleotides for BORIS protein as it was determined by methylation interference assays [9] . Nucleotides that are 100% identical to the human nucleotide sequence are shaded in grey. Dashes indicate insertions or deletions. ( B ) and ( C ) Most BORIS isoforms bind to the human and mouse testis-specific CST promoter in EMSA. The number of ZFs and utilization of alternative N-and C-termini for each BORIS isoform are shown at the bottom of gel. The band shifts specific for particular N- and C-termini are indicated by arrows. Luciferase and CTCF were used as negative and positive controls, respectively. ( D ) The mouse CST promoter is transcriptionally activated by BORIS isoforms in HEK293T cells. HEK293T cells were either co-transfected with mouse Boris, mouse Ctcf, empty pCI vector (EV) or the human BORIS isoform constructs, as well as with pGL-3 containing 359 bp of wild type or mutant CST promoter. Luciferase assays were done 48 h after transfections. All luciferase activities were normalized for transfection efficiency by measuring the Renilla luciferase activity from the co-transfected pRL-TK vector. Error bars are standard deviations.

    Article Snippet: All transfections were done with the jetPEI Cationic Polymer Transfection reagent (PolyPlus-transfection, Illkirch, France) according to the manufacturer's instructions, except HEK293T cells, which were transfected using TransIt transfection agent according to manufacturer's protocol (Mirus Bio, Madison, WI).

    Techniques: Binding Assay, Methylation, Sequencing, Luciferase, Transfection, Plasmid Preparation, Construct, Mutagenesis, Activity Assay

    BORIS isoforms are translated into proteins with nuclear localization in vivo . ( A ) Immunoblotting detection of ectopically expressed BORIS isoform proteins. Expression vectors with HA-Tag fused BORIS isoforms were transiently transfected into HEK293T cell line. The transfection efficiency, monitored by co-transfection with a GFP-expressing plasmid, was similar for all transfected plasmids. Total protein was extracted, separated on a 12% SDS-PAGE gel, and probed with rat monoclonal anti-HA antibody (Roche). Molecular mass standards are on the left. The sizes (in kDa) of BORIS isoform proteins correspond to thier predicted molecular masses, as shown in Table S3 . ( B ) Nuclear localization of CTCF, B0, and C5 isoproteins. The localization of the rest BORIS isoproteins is shown in Fig. S3 . HEK293T cells transiently transfected with either BORIS or CTCF fused at the N-terminus to dsRFP or GFP , respectively, were analyzed for RFP and GFP fluorescence by microscopy. dsRED protein (Empty Vector (EV)-dsRED) served as a marker for cytoplasmic location. Cells were also stained with DAPI to visualize nuclear DNA. Both BORIS and CTCF were detected in the nuclei.

    Journal: PLoS ONE

    Article Title: The Structural Complexity of the Human BORIS Gene in Gametogenesis and Cancer

    doi: 10.1371/journal.pone.0013872

    Figure Lengend Snippet: BORIS isoforms are translated into proteins with nuclear localization in vivo . ( A ) Immunoblotting detection of ectopically expressed BORIS isoform proteins. Expression vectors with HA-Tag fused BORIS isoforms were transiently transfected into HEK293T cell line. The transfection efficiency, monitored by co-transfection with a GFP-expressing plasmid, was similar for all transfected plasmids. Total protein was extracted, separated on a 12% SDS-PAGE gel, and probed with rat monoclonal anti-HA antibody (Roche). Molecular mass standards are on the left. The sizes (in kDa) of BORIS isoform proteins correspond to thier predicted molecular masses, as shown in Table S3 . ( B ) Nuclear localization of CTCF, B0, and C5 isoproteins. The localization of the rest BORIS isoproteins is shown in Fig. S3 . HEK293T cells transiently transfected with either BORIS or CTCF fused at the N-terminus to dsRFP or GFP , respectively, were analyzed for RFP and GFP fluorescence by microscopy. dsRED protein (Empty Vector (EV)-dsRED) served as a marker for cytoplasmic location. Cells were also stained with DAPI to visualize nuclear DNA. Both BORIS and CTCF were detected in the nuclei.

    Article Snippet: All transfections were done with the jetPEI Cationic Polymer Transfection reagent (PolyPlus-transfection, Illkirch, France) according to the manufacturer's instructions, except HEK293T cells, which were transfected using TransIt transfection agent according to manufacturer's protocol (Mirus Bio, Madison, WI).

    Techniques: In Vivo, Expressing, Transfection, Cotransfection, Plasmid Preparation, SDS Page, Fluorescence, Microscopy, Marker, Staining

    Localized deactivation of PTP1B PS . a A biosensor for PTP1B activity. Src-mediated phosphorylation of the substrate domain causes it to bind to the SH2 domain, triggering a conformational change that decreases FRET; dephosphorylation by PTP1B increases FRET. b Src increases the donor/acceptor emission ratio in vitro (normalized by the buffer-only condition); EDTA or PTP1B prevent this increase. Error bars denote propagated SE for measurements of n = 3 independent experiments (measurements are normalized to a to a buffer-only condition). c The percent change in donor/acceptor emission ratio over 1 min within 5-μm circular regions located in the cytosol and nucleus of COS-7 cells activated with 457 nm light. Each condition includes the interquartile average, associated data points, and SE for n = 11 biological replicates. The p -values correspond to a two-tailed Student’s t test. d An image of localized illumination (405 nm) of a COS-7 cell expressing both PTP1B PS and biosensor. Circles delineate irradiated (red) and secondary (blue) regions, and colors show the donor/acceptor emission ratio (scale bar, 10 μm). e Time courses of FRET in irradiated and secondary regions. Shading highlights 5-s periods before (gray), during (blue), and after (gray) illumination. f A depiction of a HEK293T/17 cell expressing PTP1B PS** . Insulin stimulates phosphorylation of the membrane-bound insulin receptor (IR); PTP1B dephosphorylates it. g ELISA-based measurements of IR phosphorylation in (i) wild-type HEK293T/17 cells and (ii) HEK293T/17 cells stably expressing PTP1B PS** or PTP1B PS** (C450M). Insulin-mediated simulation of IR, BBR-mediated inhibition of PTP1B, and photoinactivation of PTP1B all increase IR phosphorylation. The dark state of PTP1B PS** and the dark and light states of PTP1B PS** (C450M), by contrast, leave IR phosphorylation unaltered from its levels in the wild-type strain (DMSO). The plotted data depict the mean, propagated SE, and associated data points for measurements of n = 3 biological replicates (relative to a buffer-only condition). Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Minimally disruptive optical control of protein tyrosine phosphatase 1B

    doi: 10.1038/s41467-020-14567-8

    Figure Lengend Snippet: Localized deactivation of PTP1B PS . a A biosensor for PTP1B activity. Src-mediated phosphorylation of the substrate domain causes it to bind to the SH2 domain, triggering a conformational change that decreases FRET; dephosphorylation by PTP1B increases FRET. b Src increases the donor/acceptor emission ratio in vitro (normalized by the buffer-only condition); EDTA or PTP1B prevent this increase. Error bars denote propagated SE for measurements of n = 3 independent experiments (measurements are normalized to a to a buffer-only condition). c The percent change in donor/acceptor emission ratio over 1 min within 5-μm circular regions located in the cytosol and nucleus of COS-7 cells activated with 457 nm light. Each condition includes the interquartile average, associated data points, and SE for n = 11 biological replicates. The p -values correspond to a two-tailed Student’s t test. d An image of localized illumination (405 nm) of a COS-7 cell expressing both PTP1B PS and biosensor. Circles delineate irradiated (red) and secondary (blue) regions, and colors show the donor/acceptor emission ratio (scale bar, 10 μm). e Time courses of FRET in irradiated and secondary regions. Shading highlights 5-s periods before (gray), during (blue), and after (gray) illumination. f A depiction of a HEK293T/17 cell expressing PTP1B PS** . Insulin stimulates phosphorylation of the membrane-bound insulin receptor (IR); PTP1B dephosphorylates it. g ELISA-based measurements of IR phosphorylation in (i) wild-type HEK293T/17 cells and (ii) HEK293T/17 cells stably expressing PTP1B PS** or PTP1B PS** (C450M). Insulin-mediated simulation of IR, BBR-mediated inhibition of PTP1B, and photoinactivation of PTP1B all increase IR phosphorylation. The dark state of PTP1B PS** and the dark and light states of PTP1B PS** (C450M), by contrast, leave IR phosphorylation unaltered from its levels in the wild-type strain (DMSO). The plotted data depict the mean, propagated SE, and associated data points for measurements of n = 3 biological replicates (relative to a buffer-only condition). Source data are provided as a Source Data file.

    Article Snippet: ELISA of insulin receptor phosphorylation We examined IR phosphorylation in HEK293T/17 cells exposed to various conditions by using an enzyme-linked immunosorbent assay (ELISA).

    Techniques: Activity Assay, De-Phosphorylation Assay, In Vitro, Two Tailed Test, Expressing, Irradiation, Enzyme-linked Immunosorbent Assay, Stable Transfection, Inhibition

    Detection of residual PEG. Two gels were loaded with PEG 6000 in decreasing amounts (10, 5, 2.5, 1, 0.5, 0.25, 0.1, 0.01 µg), protein marker, and 6 µg of the final EV samples of HEK293T-CD63-eGF clone B2 and C5 cells. Gels were run under identical conditions. PEG was detectable after barium iodide staining (a); proteins following protein specific imperial stain (b). Signals in (a) were recorded with Image Studio™ Software (LI-COR) and plotted against the PEG concentration of the calibration values (c). The graphic evolution indicates a linear equation of y = 15.692× + 3.4984 which was used to calculate the revealed PEG concentration of 0.02% in the final EV samples.

    Journal: Journal of Extracellular Vesicles

    Article Title: Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales

    doi: 10.1080/20013078.2018.1528109

    Figure Lengend Snippet: Detection of residual PEG. Two gels were loaded with PEG 6000 in decreasing amounts (10, 5, 2.5, 1, 0.5, 0.25, 0.1, 0.01 µg), protein marker, and 6 µg of the final EV samples of HEK293T-CD63-eGF clone B2 and C5 cells. Gels were run under identical conditions. PEG was detectable after barium iodide staining (a); proteins following protein specific imperial stain (b). Signals in (a) were recorded with Image Studio™ Software (LI-COR) and plotted against the PEG concentration of the calibration values (c). The graphic evolution indicates a linear equation of y = 15.692× + 3.4984 which was used to calculate the revealed PEG concentration of 0.02% in the final EV samples.

    Article Snippet: To test for the appropriate subcellular distribution of the encoded CD63-eGFP-CD63 fusion protein, the obtained pEGFP-N1-CD63 plasmid and the original pEGFP1 plasmid were transfected into HEK293T cells using Jetpei (Polyplus, Illkirch Cedex, France) transfection reagent according to the manufacturer’s recommendations.

    Techniques: Marker, Staining, Software, Concentration Assay

    PEG-precipitation versus differential centrifugation (DC). EVs from 10 mL HEK293T-CD63-eGFP cell conditioned media were either prepared by PEG precipitation or differential centrifugation. The particle concentration per fraction ( n = 3; SD, (a) and their average size distribution ( n = 3; SD, (b)) were assessed by NTA (ZetaView). The purity of the obtained samples was determined as particle numbers – measured by NTA – per mg protein content – measured by the BCA assay (c). The presence of the EV marker proteins HSP70, CD63, CD81 and CD9, and within the individual PEG samples was analysed by western blot; 5 µg proteins were loaded per lane (d). The content of CD81, CD9 and BSA was compared in final EV samples harvested from of HEK293T-CD63-eGFP clones C5, F5, E7, either harvested by PEG 6000 precipitation (PUP) or differential centrifugation (DCP). Proteins obtained from supernatants of the final PEG pellets (PUS) or the ultracentrifugation pellet (DCS) obtained in the differential centrifugation method served as controls. 2.5 µg proteins were loaded per lane (e). Of note, following analyses of CD81 blots were stripped and re-analysed for the presence of BSA.

    Journal: Journal of Extracellular Vesicles

    Article Title: Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales

    doi: 10.1080/20013078.2018.1528109

    Figure Lengend Snippet: PEG-precipitation versus differential centrifugation (DC). EVs from 10 mL HEK293T-CD63-eGFP cell conditioned media were either prepared by PEG precipitation or differential centrifugation. The particle concentration per fraction ( n = 3; SD, (a) and their average size distribution ( n = 3; SD, (b)) were assessed by NTA (ZetaView). The purity of the obtained samples was determined as particle numbers – measured by NTA – per mg protein content – measured by the BCA assay (c). The presence of the EV marker proteins HSP70, CD63, CD81 and CD9, and within the individual PEG samples was analysed by western blot; 5 µg proteins were loaded per lane (d). The content of CD81, CD9 and BSA was compared in final EV samples harvested from of HEK293T-CD63-eGFP clones C5, F5, E7, either harvested by PEG 6000 precipitation (PUP) or differential centrifugation (DCP). Proteins obtained from supernatants of the final PEG pellets (PUS) or the ultracentrifugation pellet (DCS) obtained in the differential centrifugation method served as controls. 2.5 µg proteins were loaded per lane (e). Of note, following analyses of CD81 blots were stripped and re-analysed for the presence of BSA.

    Article Snippet: To test for the appropriate subcellular distribution of the encoded CD63-eGFP-CD63 fusion protein, the obtained pEGFP-N1-CD63 plasmid and the original pEGFP1 plasmid were transfected into HEK293T cells using Jetpei (Polyplus, Illkirch Cedex, France) transfection reagent according to the manufacturer’s recommendations.

    Techniques: Centrifugation, Concentration Assay, BIA-KA, Marker, Western Blot, Clone Assay

    CD63-eGFP but not eGFP expressing HEK293T cells release high quantities of eGFP-labelled particles into their extracellular environment. After processing of supernatants of HEK293T cells either transfected with eGFP-encoding plasmids (left side) or CD63-eGFP-encoding plasmids (right side) with ultracentrifugation, 1 µL of each resolved pellet was transferred onto a microscopic slide and allowed to dry (results in crystal formation). At the original border of the HEK293T CD63-eGFP drop, high amounts of GFP + -particles were concentrated. Upper row: fluorescent image, lower row: overlay fluorescent and bright field images; scale bar: 50 µm for all images.

    Journal: Journal of Extracellular Vesicles

    Article Title: Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales

    doi: 10.1080/20013078.2018.1528109

    Figure Lengend Snippet: CD63-eGFP but not eGFP expressing HEK293T cells release high quantities of eGFP-labelled particles into their extracellular environment. After processing of supernatants of HEK293T cells either transfected with eGFP-encoding plasmids (left side) or CD63-eGFP-encoding plasmids (right side) with ultracentrifugation, 1 µL of each resolved pellet was transferred onto a microscopic slide and allowed to dry (results in crystal formation). At the original border of the HEK293T CD63-eGFP drop, high amounts of GFP + -particles were concentrated. Upper row: fluorescent image, lower row: overlay fluorescent and bright field images; scale bar: 50 µm for all images.

    Article Snippet: To test for the appropriate subcellular distribution of the encoded CD63-eGFP-CD63 fusion protein, the obtained pEGFP-N1-CD63 plasmid and the original pEGFP1 plasmid were transfected into HEK293T cells using Jetpei (Polyplus, Illkirch Cedex, France) transfection reagent according to the manufacturer’s recommendations.

    Techniques: Expressing, Transfection

    Comparison of different methods to enrich nano-sized EVs. EVs from 10 mL HEK293T-CD63-eGFP (single-cell clones C5, F5, E7 and B2) cell conditioned media were either prepared by direct ultracentrifugation (UC), differential centrifugation (DC), PEG 6000 or PEG 8000 precipitation, density gradient centrifugation (DG), or size-exclusion chromatography (SE). The particle concentration ( n = 3; SD, (a) and their average size distribution ( n = 3; SD, (b) were assessed by NTA (NanoSight LM10). The purity of the obtained samples was determined as particle numbers – measured by NTA – per µg protein content – measured by the BCA assay ( n = 3; SD, (c). The presence of the exosomal marker protein Tsg101 was analysed by western blot; 10 µg of each fraction were loaded per lane (d). Electron micrographs of samples from UC, DC, DG and PEG enriched EVs were acquired; scale bars 200 nm (e). To test for potential impacts on the physiology of the obtained EVs, uptake experiments were performed: 1 × 10 8 particles as estimated by NTA were supplemented to the media of N-KM cells. After 14–16 h, pictures were taken (f) before the amount of the eGFP labelling was quantified as the mean fluorescence intensity (MFI) by flow cytometry ( n = 3; SD, t-test,* p > 0.05, (g)). Scale bars 10 µm.

    Journal: Journal of Extracellular Vesicles

    Article Title: Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales

    doi: 10.1080/20013078.2018.1528109

    Figure Lengend Snippet: Comparison of different methods to enrich nano-sized EVs. EVs from 10 mL HEK293T-CD63-eGFP (single-cell clones C5, F5, E7 and B2) cell conditioned media were either prepared by direct ultracentrifugation (UC), differential centrifugation (DC), PEG 6000 or PEG 8000 precipitation, density gradient centrifugation (DG), or size-exclusion chromatography (SE). The particle concentration ( n = 3; SD, (a) and their average size distribution ( n = 3; SD, (b) were assessed by NTA (NanoSight LM10). The purity of the obtained samples was determined as particle numbers – measured by NTA – per µg protein content – measured by the BCA assay ( n = 3; SD, (c). The presence of the exosomal marker protein Tsg101 was analysed by western blot; 10 µg of each fraction were loaded per lane (d). Electron micrographs of samples from UC, DC, DG and PEG enriched EVs were acquired; scale bars 200 nm (e). To test for potential impacts on the physiology of the obtained EVs, uptake experiments were performed: 1 × 10 8 particles as estimated by NTA were supplemented to the media of N-KM cells. After 14–16 h, pictures were taken (f) before the amount of the eGFP labelling was quantified as the mean fluorescence intensity (MFI) by flow cytometry ( n = 3; SD, t-test,* p > 0.05, (g)). Scale bars 10 µm.

    Article Snippet: To test for the appropriate subcellular distribution of the encoded CD63-eGFP-CD63 fusion protein, the obtained pEGFP-N1-CD63 plasmid and the original pEGFP1 plasmid were transfected into HEK293T cells using Jetpei (Polyplus, Illkirch Cedex, France) transfection reagent according to the manufacturer’s recommendations.

    Techniques: Clone Assay, Centrifugation, Gradient Centrifugation, Size-exclusion Chromatography, Concentration Assay, BIA-KA, Marker, Western Blot, Fluorescence, Flow Cytometry, Cytometry

    Up-scaling of PEG-precipitation. EVs from 360 mL HEK293T-CD63-eGFP cell conditioned media were prepared by PEG precipitation. The particle concentration per fraction ( n = 3; SD, (a) and their average size distribution ( n = 3; SD, (b) were assessed by NTA (ZetaView). The purity of the obtained samples was determined as particle numbers – measured by NTA – per mg protein content – measured by the BCA assay (c). The presence of the EV marker proteins HSP70, Tsg101, CD63, CD81 and CD9 within the individual PEG samples was analysed by western blot. Additionally, the content of BSA, Syntenin and Prohibitin within the final samples was compared; 5 µg proteins were loaded per lane (d). To test for potential impacts on the physiology of the obtained EVs, uptake experiments were performed: 30–50 µg of the obtained samples were supplemented to the cell culture media of N-KM cells. After 14–16 h pictures were taken, then, the amount of the eGFP labelling was quantified as the mean fluorescence intensity (MFI) by flow cytometry (e). Scale bars 20 µm.

    Journal: Journal of Extracellular Vesicles

    Article Title: Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales

    doi: 10.1080/20013078.2018.1528109

    Figure Lengend Snippet: Up-scaling of PEG-precipitation. EVs from 360 mL HEK293T-CD63-eGFP cell conditioned media were prepared by PEG precipitation. The particle concentration per fraction ( n = 3; SD, (a) and their average size distribution ( n = 3; SD, (b) were assessed by NTA (ZetaView). The purity of the obtained samples was determined as particle numbers – measured by NTA – per mg protein content – measured by the BCA assay (c). The presence of the EV marker proteins HSP70, Tsg101, CD63, CD81 and CD9 within the individual PEG samples was analysed by western blot. Additionally, the content of BSA, Syntenin and Prohibitin within the final samples was compared; 5 µg proteins were loaded per lane (d). To test for potential impacts on the physiology of the obtained EVs, uptake experiments were performed: 30–50 µg of the obtained samples were supplemented to the cell culture media of N-KM cells. After 14–16 h pictures were taken, then, the amount of the eGFP labelling was quantified as the mean fluorescence intensity (MFI) by flow cytometry (e). Scale bars 20 µm.

    Article Snippet: To test for the appropriate subcellular distribution of the encoded CD63-eGFP-CD63 fusion protein, the obtained pEGFP-N1-CD63 plasmid and the original pEGFP1 plasmid were transfected into HEK293T cells using Jetpei (Polyplus, Illkirch Cedex, France) transfection reagent according to the manufacturer’s recommendations.

    Techniques: Concentration Assay, BIA-KA, Marker, Western Blot, Cell Culture, Fluorescence, Flow Cytometry, Cytometry

    Effect of FRG1 expression on HEK293T cell proliferation and scratch wound healing ( A ) Shows Western blot to confirm ectopic expression of FRG1 in HEK293T. ( B ) Shows verification of reduced FRG1 levels after RNAi silencing in HEK293T, by Western blot. ( C ) Represents measurement of cell proliferation in HEK293T with ectopic expression of FRG1 compared with empty vector control (pCMV6.XL5), by MTS reagent. ( D ) Represents measurement of cell proliferation in HEK293T with knockdown of FRG1 compared with scrambled vector control (pLKO1.sc), by MTS reagent. ( E ) Shows representative images of scratch wound healing assay of HEK293T cells with ectopic expression of FRG1 and respective vector control (pCMV6.XL5). ( F ) Shows representative images of scratch wound healing assay of HEK293T with FRG1 knockdown and respective scrambled vector control (pLKO1.sc). ( G ) Represents representative graph for scratch wound healing assay of HEK293T cells with ectopic expression of FRG1, compared with empty vector control (pCMV6.XL5). ( H ) Shows representative graph for scratch wound healing assay of HEK293T cells with FRG1 knockdown, compared with empty vector control (pLKO1.sc). # represents P > 0.05 , * represents P

    Journal: Bioscience Reports

    Article Title: Increased FSHD region gene1 expression reduces in vitro cell migration, invasion, and angiogenesis, ex vivo supported by reduced expression in tumors

    doi: 10.1042/BSR20171062

    Figure Lengend Snippet: Effect of FRG1 expression on HEK293T cell proliferation and scratch wound healing ( A ) Shows Western blot to confirm ectopic expression of FRG1 in HEK293T. ( B ) Shows verification of reduced FRG1 levels after RNAi silencing in HEK293T, by Western blot. ( C ) Represents measurement of cell proliferation in HEK293T with ectopic expression of FRG1 compared with empty vector control (pCMV6.XL5), by MTS reagent. ( D ) Represents measurement of cell proliferation in HEK293T with knockdown of FRG1 compared with scrambled vector control (pLKO1.sc), by MTS reagent. ( E ) Shows representative images of scratch wound healing assay of HEK293T cells with ectopic expression of FRG1 and respective vector control (pCMV6.XL5). ( F ) Shows representative images of scratch wound healing assay of HEK293T with FRG1 knockdown and respective scrambled vector control (pLKO1.sc). ( G ) Represents representative graph for scratch wound healing assay of HEK293T cells with ectopic expression of FRG1, compared with empty vector control (pCMV6.XL5). ( H ) Shows representative graph for scratch wound healing assay of HEK293T cells with FRG1 knockdown, compared with empty vector control (pLKO1.sc). # represents P > 0.05 , * represents P

    Article Snippet: Transwell invasion and migration assay Matrigel invasion assay was performed using Millipore Transwell chambers (8 μm: pore size); 2 × 104 HEK293T cells were seeded in the upper chamber of a 12-well plate, coated with growth factor reduced Matrigel (Corning), in 500 μl serum-free medium.

    Techniques: Expressing, Western Blot, Plasmid Preparation, Wound Healing Assay

    Effect of FRG1 expression on transwell migration and invasion ( A ) Shows representative images of transwell migration assay of HEK293T cells with ectopic expression of FRG1 and respective vector control (pCMV6.XL5). ( B ) Shows representative images of transwell migration assay of HEK293T with FRG1 knockdown and respective scrambled vector control (pLKO1.sc). ( C ) Represents representative graph for transwell migration assay of HEK293T cells with ectopic expression of FRG1, compared with empty vector control (pCMV6.XL5). ( D ) Shows representative graph for transwell migration assay of HEK293T cells with FRG1 knockdown, compared with empty vector control (pLKO1.sc). ( E ) Shows representative images of Matrigel invasion assay of HEK293T cells with ectopic expression of FRG1 and respective vector control (pCMV6.XL5). ( F ) Shows representative images of Matrigel invasion assay of HEK293T with FRG1 knockdown and respective scrambled vector control (pLKO1.sc). ( G ) Represents representative graph for Matrigel invasion assay of HEK293T cells with ectopic expression of FRG1, compared with empty vector control (pCMV6.XL5). ( H ) Shows representative graph for Matrigel invasion assay of HEK293T cells with FRG1 knockdown, compared with empty vector control (pLKO1.sc). * represents P

    Journal: Bioscience Reports

    Article Title: Increased FSHD region gene1 expression reduces in vitro cell migration, invasion, and angiogenesis, ex vivo supported by reduced expression in tumors

    doi: 10.1042/BSR20171062

    Figure Lengend Snippet: Effect of FRG1 expression on transwell migration and invasion ( A ) Shows representative images of transwell migration assay of HEK293T cells with ectopic expression of FRG1 and respective vector control (pCMV6.XL5). ( B ) Shows representative images of transwell migration assay of HEK293T with FRG1 knockdown and respective scrambled vector control (pLKO1.sc). ( C ) Represents representative graph for transwell migration assay of HEK293T cells with ectopic expression of FRG1, compared with empty vector control (pCMV6.XL5). ( D ) Shows representative graph for transwell migration assay of HEK293T cells with FRG1 knockdown, compared with empty vector control (pLKO1.sc). ( E ) Shows representative images of Matrigel invasion assay of HEK293T cells with ectopic expression of FRG1 and respective vector control (pCMV6.XL5). ( F ) Shows representative images of Matrigel invasion assay of HEK293T with FRG1 knockdown and respective scrambled vector control (pLKO1.sc). ( G ) Represents representative graph for Matrigel invasion assay of HEK293T cells with ectopic expression of FRG1, compared with empty vector control (pCMV6.XL5). ( H ) Shows representative graph for Matrigel invasion assay of HEK293T cells with FRG1 knockdown, compared with empty vector control (pLKO1.sc). * represents P

    Article Snippet: Transwell invasion and migration assay Matrigel invasion assay was performed using Millipore Transwell chambers (8 μm: pore size); 2 × 104 HEK293T cells were seeded in the upper chamber of a 12-well plate, coated with growth factor reduced Matrigel (Corning), in 500 μl serum-free medium.

    Techniques: Expressing, Migration, Transwell Migration Assay, Plasmid Preparation, Invasion Assay

    Effect of FRG1 expression on cell signaling molecules ( A ) qRT-PCR expression analysis shows effect of FRG1 expression on levels of various cytokines and MMPs, in HEK293T cells, transfected with FRG1 expression vector, compared with empty vector control. ( B ) Expression analysis of various cytokines and MMPs, in HEK293T cells with FRG1 knockdown, compared with scrambled vector control; X-axis shows the name of various signaling molecules and Y-axis shows the fold-change in expression of these molecules compared with their controls. ** represents P

    Journal: Bioscience Reports

    Article Title: Increased FSHD region gene1 expression reduces in vitro cell migration, invasion, and angiogenesis, ex vivo supported by reduced expression in tumors

    doi: 10.1042/BSR20171062

    Figure Lengend Snippet: Effect of FRG1 expression on cell signaling molecules ( A ) qRT-PCR expression analysis shows effect of FRG1 expression on levels of various cytokines and MMPs, in HEK293T cells, transfected with FRG1 expression vector, compared with empty vector control. ( B ) Expression analysis of various cytokines and MMPs, in HEK293T cells with FRG1 knockdown, compared with scrambled vector control; X-axis shows the name of various signaling molecules and Y-axis shows the fold-change in expression of these molecules compared with their controls. ** represents P

    Article Snippet: Transwell invasion and migration assay Matrigel invasion assay was performed using Millipore Transwell chambers (8 μm: pore size); 2 × 104 HEK293T cells were seeded in the upper chamber of a 12-well plate, coated with growth factor reduced Matrigel (Corning), in 500 μl serum-free medium.

    Techniques: Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation

    Effect of FRG1 expression on endothelial cell function ( A ) Representative image showing Matrigel tubule formation assay in HUVECs treated with conditioned medium, obtained from FRG1 expressing HEK293T cells and respective vector control; image shows reduced tubule formation in FRG1 overexpression set. ( B ) Graphical representation of cell proliferation assay data showing no significant change in proliferation of HUVECs when treated with conditioned medium obtained from HEK293T transfected with respective sets. ( C ) Representative images of HUVEC transwell migration assay in co-culture with HEK293T cells, transfected with FRG1 expression vector and empty vector control. ( D ) Graphical representation of HUVEC transwell migration assay showing significant reduction in HUVEC migration co-cultured with HEK293T expressing FRG1 (pCMV6.XL5.FRG1), compared with empty vector (pCMV6.XL5). # represents P > 0.05 , ** represents P

    Journal: Bioscience Reports

    Article Title: Increased FSHD region gene1 expression reduces in vitro cell migration, invasion, and angiogenesis, ex vivo supported by reduced expression in tumors

    doi: 10.1042/BSR20171062

    Figure Lengend Snippet: Effect of FRG1 expression on endothelial cell function ( A ) Representative image showing Matrigel tubule formation assay in HUVECs treated with conditioned medium, obtained from FRG1 expressing HEK293T cells and respective vector control; image shows reduced tubule formation in FRG1 overexpression set. ( B ) Graphical representation of cell proliferation assay data showing no significant change in proliferation of HUVECs when treated with conditioned medium obtained from HEK293T transfected with respective sets. ( C ) Representative images of HUVEC transwell migration assay in co-culture with HEK293T cells, transfected with FRG1 expression vector and empty vector control. ( D ) Graphical representation of HUVEC transwell migration assay showing significant reduction in HUVEC migration co-cultured with HEK293T expressing FRG1 (pCMV6.XL5.FRG1), compared with empty vector (pCMV6.XL5). # represents P > 0.05 , ** represents P

    Article Snippet: Transwell invasion and migration assay Matrigel invasion assay was performed using Millipore Transwell chambers (8 μm: pore size); 2 × 104 HEK293T cells were seeded in the upper chamber of a 12-well plate, coated with growth factor reduced Matrigel (Corning), in 500 μl serum-free medium.

    Techniques: Expressing, Cell Function Assay, Tube Formation Assay, Plasmid Preparation, Over Expression, Proliferation Assay, Transfection, Transwell Migration Assay, Co-Culture Assay, Migration, Cell Culture

    CEACAM16 interacts homotypically. HEK293T cells either remained untransfected ( A ) or were transiently transfected with expression plasmids encoding full-length murine native ( B ) or GPI-linked CEACAM16 ( C ). The transfected cells were harvested by repeated pipetting, incubated at room temperature, attached to glass slides using the cytospin method, and stained with anti-murine CEACAM16 immune sera by immunocytochemistry ( brown stain ). Note the homotypic sorting of cells expressing CEACAM16 on the cell membrane ( C ) but not of cells with intracellular expression ( B ). This suggests that CEACAM16 can interact homotypically in trans . Aggregation of HEK293T cells was tested as above after transient expression of GPI-anchored human CEACAM16 N1 and N2 domains ( D and E , left panels ). N1- and N2-expressing cells were detected by immunocytochemistry using an anti-myc-tag antibody. Representative regions of the cytospins are shown. The expression levels of myc-tagged fusion proteins and the fraction of positive cells were determined by FACScan analysis using anti-myc and FITC-conjugated secondary antibodies ( D and E , right panels , green curves ). Samples incubated without primary antibody served as a negative control ( blue curves ). Aggregation mediated by N1 and N2 domains was quantitated by determination of the fraction of N1- and N2-positive cells, respectively, in single and double cell populations as well as in aggregates ≥3 using the cytospins shown in D and E ( F ). Note that surface expression of N2 preferentially induces multicellular aggregates. One of two experiments with similar results is shown. Full-length murine native CEACAM16 ( G ) or a deletion constructs lacking N2 ( H ) or N1 and A ( J ) or B and N2 ( K ) or encoding only the N1 or N2 domain either soluble ( I , upper panel ) or GPI-linked ( I , lower panels ) were expressed in HEK293T cells by transient transfection. Cell lysates ( cells ) or cell culture supernatants with or without prior treatment with DTT were separated by gel electrophoresis, and CEACAM16 was detected by Western blot analysis with a monoclonal anti-myc antibody. The domain organization of the encoded proteins is schematically shown to the right . Cysteines possibly involved in intermolecular disulfide bridges are indicated by yellow dots , green tags represent NH 2 -terminally added myc epitopes, and green arrows symbolize GPI anchors. Presumed monomeric, dimeric, and oligomeric forms of CEACAM16 are indicated by arrows . Note that the N2 or the N1 and A domains appear to be dispensable for interaction as dimers are observed in the absence of these domains ( J ). The 60-kDa band. which is observed in the supernatant of transfected and mock-transfected cells ( H, right panel ), is marked by an asterisk ( H , J , and K ).

    Journal: The Journal of Biological Chemistry

    Article Title: Loss of Mammal-specific Tectorial Membrane Component Carcinoembryonic Antigen Cell Adhesion Molecule 16 (CEACAM16) Leads to Hearing Impairment at Low and High Frequencies *

    doi: 10.1074/jbc.M111.320481

    Figure Lengend Snippet: CEACAM16 interacts homotypically. HEK293T cells either remained untransfected ( A ) or were transiently transfected with expression plasmids encoding full-length murine native ( B ) or GPI-linked CEACAM16 ( C ). The transfected cells were harvested by repeated pipetting, incubated at room temperature, attached to glass slides using the cytospin method, and stained with anti-murine CEACAM16 immune sera by immunocytochemistry ( brown stain ). Note the homotypic sorting of cells expressing CEACAM16 on the cell membrane ( C ) but not of cells with intracellular expression ( B ). This suggests that CEACAM16 can interact homotypically in trans . Aggregation of HEK293T cells was tested as above after transient expression of GPI-anchored human CEACAM16 N1 and N2 domains ( D and E , left panels ). N1- and N2-expressing cells were detected by immunocytochemistry using an anti-myc-tag antibody. Representative regions of the cytospins are shown. The expression levels of myc-tagged fusion proteins and the fraction of positive cells were determined by FACScan analysis using anti-myc and FITC-conjugated secondary antibodies ( D and E , right panels , green curves ). Samples incubated without primary antibody served as a negative control ( blue curves ). Aggregation mediated by N1 and N2 domains was quantitated by determination of the fraction of N1- and N2-positive cells, respectively, in single and double cell populations as well as in aggregates ≥3 using the cytospins shown in D and E ( F ). Note that surface expression of N2 preferentially induces multicellular aggregates. One of two experiments with similar results is shown. Full-length murine native CEACAM16 ( G ) or a deletion constructs lacking N2 ( H ) or N1 and A ( J ) or B and N2 ( K ) or encoding only the N1 or N2 domain either soluble ( I , upper panel ) or GPI-linked ( I , lower panels ) were expressed in HEK293T cells by transient transfection. Cell lysates ( cells ) or cell culture supernatants with or without prior treatment with DTT were separated by gel electrophoresis, and CEACAM16 was detected by Western blot analysis with a monoclonal anti-myc antibody. The domain organization of the encoded proteins is schematically shown to the right . Cysteines possibly involved in intermolecular disulfide bridges are indicated by yellow dots , green tags represent NH 2 -terminally added myc epitopes, and green arrows symbolize GPI anchors. Presumed monomeric, dimeric, and oligomeric forms of CEACAM16 are indicated by arrows . Note that the N2 or the N1 and A domains appear to be dispensable for interaction as dimers are observed in the absence of these domains ( J ). The 60-kDa band. which is observed in the supernatant of transfected and mock-transfected cells ( H, right panel ), is marked by an asterisk ( H , J , and K ).

    Article Snippet: Expression levels of CEACAM16 domains on HEK293T cells were quantified after incubation with anti-c-myc antibody (100 μg/ml) followed by FITC-coupled goat anti-mouse Ig antibody (75 μg/ml) using FACScan analysis (FACSCalibur, BD Biosciences).

    Techniques: Transfection, Expressing, Incubation, Staining, Immunocytochemistry, Negative Control, Construct, Cell Culture, Nucleic Acid Electrophoresis, Western Blot

    Y247 is the primary residue of EB1 phosphorylated by Src. (A) In vitro kinase assays were performed with purified GST-Src and purified His-EB1 wild-type or mutants. The arrowhead indicates the bands of phosphorylated EB1. (B) HEK293T cells were transfected with the indicated plasmids. Immunoprecipitation and immunoblotting were then performed. (C) In vitro kinase assays were performed with the indicated purified proteins, and immunoblotting were then performed. (D) HUVECs were transfected with GFP-Src and stained with the pY247-EB1 antibody and DAPI. Scale bar, 10 μm. (E) Experiments were performed as in (D), and the relative intensity of pY247-EB1 in the boxed areas was quantified (50 cells were measured for each group). All experiments were replicated three times. **** p

    Journal: Theranostics

    Article Title: Proto-Oncogenic Src Phosphorylates EB1 to Regulate the Microtubule-Focal Adhesion Crosstalk and Stimulate Cell Migration

    doi: 10.7150/thno.16356

    Figure Lengend Snippet: Y247 is the primary residue of EB1 phosphorylated by Src. (A) In vitro kinase assays were performed with purified GST-Src and purified His-EB1 wild-type or mutants. The arrowhead indicates the bands of phosphorylated EB1. (B) HEK293T cells were transfected with the indicated plasmids. Immunoprecipitation and immunoblotting were then performed. (C) In vitro kinase assays were performed with the indicated purified proteins, and immunoblotting were then performed. (D) HUVECs were transfected with GFP-Src and stained with the pY247-EB1 antibody and DAPI. Scale bar, 10 μm. (E) Experiments were performed as in (D), and the relative intensity of pY247-EB1 in the boxed areas was quantified (50 cells were measured for each group). All experiments were replicated three times. **** p

    Article Snippet: We then investigated whether Src interacts with EB1 in cells, by transfecting HEK293T cells with GFP-Src and HA-EB1 and performing immunoprecipitation assays.

    Techniques: In Vitro, Purification, Transfection, Immunoprecipitation, Staining

    Src-mediated EB1 phosphorylation diminishes its interactions with APC-C and MCAK. (A) HEK293T cells were transfected with GFP-EB1 and HA-EB1 wild-type or Y247D. Immunoprecipitation and immunoblotting were then performed with the indicated antibodies. (B) HEK293T cells were transfected with GFP-APC-C and HA-EB1 wild-type, Y247F, or Y247D, or the HA vector. Immunoprecipitation and immunoblotting were then performed as indicated. (C) Experiments were performed as in (B) except that GFP-MCAK was used instead of GFP-APC-C. (D) Structural analysis of the interaction between the SxIP motif (violet) and EB1 wild-type, Y247F, or Y247D. (E) HEK293T cells were transfected with GFP-APC-C and treated with SU6656 (10 μM), PP2 (10 μM), or equal amount of DMSO for 12 h or co-transfected with HA-Src or the HA vector. Immunoprecipitation and immunoblotting were then performed. The arrowhead indicates the bands of GFP-APC-C. (F) Experiments were performed as in (D) except that GFP-MCAK was used instead of GFP-APC-C. The arrowhead indicates the bands of GFP-MCAK. All experiments were replicated three times.

    Journal: Theranostics

    Article Title: Proto-Oncogenic Src Phosphorylates EB1 to Regulate the Microtubule-Focal Adhesion Crosstalk and Stimulate Cell Migration

    doi: 10.7150/thno.16356

    Figure Lengend Snippet: Src-mediated EB1 phosphorylation diminishes its interactions with APC-C and MCAK. (A) HEK293T cells were transfected with GFP-EB1 and HA-EB1 wild-type or Y247D. Immunoprecipitation and immunoblotting were then performed with the indicated antibodies. (B) HEK293T cells were transfected with GFP-APC-C and HA-EB1 wild-type, Y247F, or Y247D, or the HA vector. Immunoprecipitation and immunoblotting were then performed as indicated. (C) Experiments were performed as in (B) except that GFP-MCAK was used instead of GFP-APC-C. (D) Structural analysis of the interaction between the SxIP motif (violet) and EB1 wild-type, Y247F, or Y247D. (E) HEK293T cells were transfected with GFP-APC-C and treated with SU6656 (10 μM), PP2 (10 μM), or equal amount of DMSO for 12 h or co-transfected with HA-Src or the HA vector. Immunoprecipitation and immunoblotting were then performed. The arrowhead indicates the bands of GFP-APC-C. (F) Experiments were performed as in (D) except that GFP-MCAK was used instead of GFP-APC-C. The arrowhead indicates the bands of GFP-MCAK. All experiments were replicated three times.

    Article Snippet: We then investigated whether Src interacts with EB1 in cells, by transfecting HEK293T cells with GFP-Src and HA-EB1 and performing immunoprecipitation assays.

    Techniques: Transfection, Immunoprecipitation, Plasmid Preparation

    Src interacts with and phosphorylates EB1 both in cells and in vitro . (A) HEK293T cells were transfected with HA-EB1 and GFP-Src or the GFP vector. Immunoprecipitation (IP) and immunoblotting (IB) were then performed with the indicated antibodies. (B) HEK293T cells were treated with SU6656 (10 μM), PP2 (10 μM), or equal amount of the vehicle DMSO for 12 h. Immunoprecipitation and immunoblotting were then performed as indicated. (C) In vitro kinase assays were performed with purified His-EB1 and the GFP or GFP-Src immunoprecipitate from HEK293T cells, and examined by immunoblotting. (D) In vitro kinase assays were performed with purified His-EB1 and the GFP-Src immunoprecipitate, in the absence (Mock) or presence of SU6656 (50 μM), PP2 (50 μM), or equal amount of DMSO, and analyzed by immunoblotting. (E) In vitro kinase assays were performed with purified His-EB1 and the GFP-Src immunoprecipitate, in the absence (Mock) or presence of λPPase, and analyzed by immunoblotting. (F) In vitro kinase assays were performed with purified His-EB1 and purified GST or GST-Src and analyzed by immunoblotting. (G) HEK293T cells were transfected with GFP-Src or the GFP vector. Immunoprecipitation and immunoblotting were then performed with the indicated antibodies. (H) Purified His-EB1 was incubated with purified GST or GST-Src. GST pulldown and immunoblotting were then performed as indicated. (I) The lysate of HUVECs was immunoprecipitated with the EB1 antibody or IgG control. The interaction between endogenous Src and EB1 was then examined by immunoblotting of the precipitates. All experiments were replicated three times.

    Journal: Theranostics

    Article Title: Proto-Oncogenic Src Phosphorylates EB1 to Regulate the Microtubule-Focal Adhesion Crosstalk and Stimulate Cell Migration

    doi: 10.7150/thno.16356

    Figure Lengend Snippet: Src interacts with and phosphorylates EB1 both in cells and in vitro . (A) HEK293T cells were transfected with HA-EB1 and GFP-Src or the GFP vector. Immunoprecipitation (IP) and immunoblotting (IB) were then performed with the indicated antibodies. (B) HEK293T cells were treated with SU6656 (10 μM), PP2 (10 μM), or equal amount of the vehicle DMSO for 12 h. Immunoprecipitation and immunoblotting were then performed as indicated. (C) In vitro kinase assays were performed with purified His-EB1 and the GFP or GFP-Src immunoprecipitate from HEK293T cells, and examined by immunoblotting. (D) In vitro kinase assays were performed with purified His-EB1 and the GFP-Src immunoprecipitate, in the absence (Mock) or presence of SU6656 (50 μM), PP2 (50 μM), or equal amount of DMSO, and analyzed by immunoblotting. (E) In vitro kinase assays were performed with purified His-EB1 and the GFP-Src immunoprecipitate, in the absence (Mock) or presence of λPPase, and analyzed by immunoblotting. (F) In vitro kinase assays were performed with purified His-EB1 and purified GST or GST-Src and analyzed by immunoblotting. (G) HEK293T cells were transfected with GFP-Src or the GFP vector. Immunoprecipitation and immunoblotting were then performed with the indicated antibodies. (H) Purified His-EB1 was incubated with purified GST or GST-Src. GST pulldown and immunoblotting were then performed as indicated. (I) The lysate of HUVECs was immunoprecipitated with the EB1 antibody or IgG control. The interaction between endogenous Src and EB1 was then examined by immunoblotting of the precipitates. All experiments were replicated three times.

    Article Snippet: We then investigated whether Src interacts with EB1 in cells, by transfecting HEK293T cells with GFP-Src and HA-EB1 and performing immunoprecipitation assays.

    Techniques: In Vitro, Transfection, Plasmid Preparation, Immunoprecipitation, Purification, Incubation

    Readthrough efficiencies of human UGA_CUAG stop codons. Readthrough efficiencies were determined by dual luciferase assay after transfection of HEK293T cells with dual luciferase reporter constructs consisting of candidate sequences (6 nt 5′ and 12 nt 3′ of stop codon (9 nt 3′ for MS4A5 because of an in-frame stop codon)) shown in Table S1 . AQP4 readthrough has been described previously ( 17 , 21 ) and is included here as an internal readthrough control. A UGA_C control indicated by a dashed line represents background readthrough levels. In each box-whisker plot center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; outliers are represented by dots. n = 12 biological replicates.

    Journal: The Journal of Biological Chemistry

    Article Title: Stop codon readthrough generates a C-terminally extended variant of the human vitamin D receptor with reduced calcitriol response

    doi: 10.1074/jbc.M117.818526

    Figure Lengend Snippet: Readthrough efficiencies of human UGA_CUAG stop codons. Readthrough efficiencies were determined by dual luciferase assay after transfection of HEK293T cells with dual luciferase reporter constructs consisting of candidate sequences (6 nt 5′ and 12 nt 3′ of stop codon (9 nt 3′ for MS4A5 because of an in-frame stop codon)) shown in Table S1 . AQP4 readthrough has been described previously ( 17 , 21 ) and is included here as an internal readthrough control. A UGA_C control indicated by a dashed line represents background readthrough levels. In each box-whisker plot center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; outliers are represented by dots. n = 12 biological replicates.

    Article Snippet: Cell culture and transfections HEK293T cells (ATCC) and HeLa cells (ATCC) were maintained in DMEM supplemented with 10% FBS, 1 mm l -glutamine, and antibiotics.

    Techniques: Luciferase, Transfection, Construct, Whisker Assay, Software

    VDRx is less responsive than VDR to calcitriol. A , relative luciferase activities determined by dual luciferase assay after co-transfection of HEK293T cells with plasmids expressing HA-tagged VDR proteins (or mock transfected) as indicated, together with firefly luciferase reporter constructs driven by a minimal promoter with either tandem VDR elements (VDRE) from the rat osteocalcin gene ( WT ) or control firefly luciferase reporters harboring mutated VDREs ( Mu ). A Renilla luciferase plasmid was also co-transfected to allow firefly luciferase activity normalization. Transfectants that were treated with vehicle (ethanol) control are indicated by the minus , and the plus indicates activities from calcitriol-stimulated (1 n m ) cells. Representative anti-HA Western blot of the same lysates are shown under the histogram. In each box-whisker plot center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; and outliers are represented by dots. n = 4 biological replicates. B , relative luciferase activities determined by dual luciferase assay of HEK293T cells transfected as in ( A ) then treated with varying concentrations of calcitriol as indicated for 24 h. Dashed lines indicate relative luciferase activities when HA-RXRα was co-transfected along with VDR variants. Error bars , mean ± S.D. are shown from n = 8 biological replicates.

    Journal: The Journal of Biological Chemistry

    Article Title: Stop codon readthrough generates a C-terminally extended variant of the human vitamin D receptor with reduced calcitriol response

    doi: 10.1074/jbc.M117.818526

    Figure Lengend Snippet: VDRx is less responsive than VDR to calcitriol. A , relative luciferase activities determined by dual luciferase assay after co-transfection of HEK293T cells with plasmids expressing HA-tagged VDR proteins (or mock transfected) as indicated, together with firefly luciferase reporter constructs driven by a minimal promoter with either tandem VDR elements (VDRE) from the rat osteocalcin gene ( WT ) or control firefly luciferase reporters harboring mutated VDREs ( Mu ). A Renilla luciferase plasmid was also co-transfected to allow firefly luciferase activity normalization. Transfectants that were treated with vehicle (ethanol) control are indicated by the minus , and the plus indicates activities from calcitriol-stimulated (1 n m ) cells. Representative anti-HA Western blot of the same lysates are shown under the histogram. In each box-whisker plot center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; and outliers are represented by dots. n = 4 biological replicates. B , relative luciferase activities determined by dual luciferase assay of HEK293T cells transfected as in ( A ) then treated with varying concentrations of calcitriol as indicated for 24 h. Dashed lines indicate relative luciferase activities when HA-RXRα was co-transfected along with VDR variants. Error bars , mean ± S.D. are shown from n = 8 biological replicates.

    Article Snippet: Cell culture and transfections HEK293T cells (ATCC) and HeLa cells (ATCC) were maintained in DMEM supplemented with 10% FBS, 1 mm l -glutamine, and antibiotics.

    Techniques: Luciferase, Cotransfection, Expressing, Transfection, Construct, Plasmid Preparation, Activity Assay, Western Blot, Whisker Assay, Software