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  • 99
    ATCC 293t cells
    Ddx56 interacts with Oct4 and Sox2. a Ddx56-GST and Oct4-SFB plasmids were cotransfected into <t>293T</t> cells, followed by IP and western blotting experiments. GST beads were used for pulldown. Negative controls: Ddx56-GST+GFP-SFB and GFP-GST+Oct4-SFB. Positive control: Sox2-GST+Oct4-SFB. b Ddx56-GST and Sox2-SFB plasmids were cotransfected into 293T cells, followed by IP and western blotting experiments. GST beads were used for pulldown. Negative controls: Ddx56-GST+GFP-SFB and GFP-GST+Sox2-SFB. Positive control: Oct4-GST+Sox2-SFB. c , d GST-tagged Ddx56 truncations and Sox2-HA plasmids were cotransfected into 293T cells, followed by IP and western blotting. e GST-tagged Ddx56 and Ddx56 ΔC-ter plasmids were transfected into 293T cells separately and measured by western blot. His-Sox2 protein was purified by Ni-sepharose purification. Using the same quantity of GST-Ddx56 and Ddx56 ΔC-ter separately, pulldown was performed to detect interaction
    293t Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 12257 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore 293t cells
    Restriction of MoMLV and AKV by rat APOBEC3. (A) Amino acid alignment of rat and mouse APOBEC3 proteins. Yellow boxes indicate major amino acid differences; blue boxes indicate conservative amino acid changes. Residues making up exon 5 and the zinc coordination motif of the first and second protein domains are underlined. (B) Encapsidation of rat APOBEC3 by MoMLV and AKV. FLAG-tagged APOBEC3 proteins were detected in transfected cell lysates or in virions pelleted from culture supernatants by Western blot analysis using a horseradish peroxidase-conjugated anti-FLAG antibody. The lysate immunoblots were stripped and reprobed with an anti-tubulin antibody, whereas the virion blots were reprobed with an anti-p30 (Gag) antibody. hA2, human APOBEC2; hA3G, human APOBEC3G; mA3, mouse APOBEC3; ratA3, rat APOBEC3. (C) The infectivities of MoMLV or AKV particles that were produced by cotransfecting subconfluent <t>293T</t> cells with 1 μg of either pMOV-eGFP or pAKV-NB-eGFP proviral DNA together with 0.2 μg of either the rat, mouse, or human FLAG-APOBEC3-expressing vector (or the human APOBEC2 control) were determined by transferring the virus-containing supernatants to NIH 3T3 cells at 36 h posttransfection and monitoring the percentages of cells displaying eGFP fluorescence after a further 48 h.
    293t Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7711 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher 293t cells
    Direct binding measurements of CD4-Ig and mabs follows neutralization sensitivity of Env+ pseudoviruses. LN8 wt, 375W and 380P Envs were expressed on <t>293T</t> cells before measuring binding of CD4-Ig and mabs using flow cytometry. Boxed values in the right hand, top corner of each flow profile represents the neutralization titer for each reagent and shows that binding closely followed neutralization sensitivity.
    293t Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 37214 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Mirus Bio 293t cells
    Exchangeable apolipoproteins redundantly participate in the formation of infectious HCV particles. (A) BE-KO1 cells infected with HCVcc at an MOI of 1 at 6 h post-transfection with siRNAs targeting ApoA1 (A1), ApoA2 (A2), ApoC1 (C1), ApoC2 (C2), ApoC3 (C3) and ApoH (H) and infectious titers in the culture supernatants were determined by focus-forming assay at 72 h post-infection. (B) ApoA1, ApoA2, ApoC1, ApoC2, ApoC3, ApoE and ApoH were exogenously expressed in BE-KO1 cells by infection with lentiviral vectors, and then infected with HCVcc at an MOI of 1. Expression of the apolipoproteins was determined by immunoblot analysis (upper), and infectious titers in the culture supernatants were determined at 72 h post-infection by focus-forming assay (lower). (C) Extracellular and intracellular HCV RNA in BE-KO1 cells expressing apolipoproteins and infected with HCVcc were determined at 72 h post-infection by qRT-PCR. (D) Specific infectivity was calculated as extracellular infectious titers/extracellular HCV RNA copies in BE-KO1 cells expressing apolipoproteins at 72 h post-infection. (E) <t>293T</t> cells stably expressing CLDN1 and miR-122 (293T-CLDN/miR-122 cells) were infected with the lentiviral vectors, and the expressions of the apolipoproteins were determined by immunoblot analysis (upper). These cells were infected with HCVcc at an MOI of 1, and infectious titers in the supernatants were determined at 72 h post-infection by focus-forming assay (lower). In all cases, asterisks indicate significant differences (*, P
    293t Cells, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 93/100, based on 3248 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC hek293t cells
    Residual enzymatic conversion of 5-formyl-THF to methenyl-THF in lysates (a) Reaction schematic for methenyl-THF, 10-formyl- and 5-formyl-THF interconversion. The proton highlighted in red can exchange with H2O. (b) Labeling kinetics of freshly dissolved unlabeled methenyl-THF standard incubated in D2O phosphate buffer (pH 7) at 4°C. To verify that 5-formyl-THF, unlike 10-formyl-THF, does not interconvert with methenyl-THF under these conditions, an identical experiment was performed with 5-formyl-THF standard (Mean ± SD, N = 3). (c) Folate and metabolite labeling patterns after 24 h incubation of in <t>HEK293T</t> cells with 1 mM [13C,2H]formate in the growth media (Mean ± SD, N ≥ 2). (d) Conversion of 5-formyl-THF to methenyl-THF in cell extracts. HEK293T cell extracts were prepared as described in method, and optionally heated to 60°C for 5 min after addition (when indicated) of 4 pmol 5-formyl-THF standard (Mean ± SD, N = 3).
    Hek293t Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 14151 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Addgene inc 293t cells
    Functional analysis of components of K11/K48-specific quality control A. UBR4 and UBR5 assemble most K11/K48-linked chains during quality control. <t>293T</t> cells were co- depleted of UBR4 and UBR5, treated with MG132, and analyzed for ubiquitin chains of different topologies using Western blotting. B. UBR4 and UBR5 target puromycylated proteins for degradation. 293T cells were co-depleted of UBR4 and UBR5, treated with puromycin, and the stability of puromycylated proteins was determined by cycloheximide chase and Western blotting. C. UBR4 and UBR5 complexes synthesize K11/K48-linked chains in vitro . Endogenous FLAG UBR4 and FLAG UBR5 were affinity-purified from CRISPR/Cas9-edited 293T cells, incubated with E1, the E2 UBE2D3, and ATP (as indicated). Reactions were supplemented with wild-type ubiquitin (WT); a mixture of ubiquitin K11R and ubiquitin K48R (K11R+K48R) or double mutant ubiquitin K11R/K48R (K11R/K48R). Reactions were analyzed for K11/K48-linked chains and the respective E3 enzymes by Western blotting. D. UBR5 assembles branched ubiquitin chains. Affinity-purified FLAG UBR5 were incubated with E1, UBE2D3, and ubiquitin FLAG/TEV . Conjugates were treated with TEV-protease as indicated and analyzed by αFLAG immunoblotting. The presence of two or more FLAG-positive stamps indicates branching. As control, ubiquitylation reactions were performed with UBE2G2/gp78, which only assemble homotypic K48-linked chains. E. UBR5 produces branched ubiquitin trimers. UBR5 and the K11-specific UBE2S were incubated with ubiquitin ∆GG (a mutant that can be modified, but not used as a modifier) and methyl-ubiquitin (which can be transferred to ubiquitin ∆GG , but not further modified), as indicated. Formation of K11- or K48-positive ubiquitin conjugates was analyzed by linkage-specific Western blotting. The presence of both UBE2S and UBR5 leads to formation of branched ubiquitin trimers. F. UBR5 strongly prefers K48-linkages. Endogenous UBR5 complexes were incubated with indicated single-Lys ubiquitin mutants and analyzed by αUbiquitin immunoblotting. G. K11/K48-branched ubiquitin chains produced by UBR5 show strongly increased affinity to the p97/VCP adaptor NSFL1/p47 than homotypic K48-linked chains. K11/K48-branched or K48-linked chains were assembled by UBR5 using wt-ubiquitin or ubiquitin K48 and incubated with immobilized p97-NSFL1 complexes. Binding reactions were stopped at the indicated times and analyzed by αUbiquitin immunoblotting. H. The proteasome targets puromycylated and K11/K48-labeled proteins for degradation. The stability of puromycylated proteins was analyzed in cells treated with DMSO or MG132, and puromycylated proteins or K11/K48-linked chains were detected by Western blotting using specific antibodies.
    293t Cells, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 4254 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher hek 293t cells
    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 <t>HEK-293T</t> cells transfected with uPARAP
    Hek 293t Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 13802 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa lenti x 293t cells
    Quantitative analysis of TGF-β pathway activation by aqueous humor of patients and controls. Box and whisker plot showing that the aqueous humor of naïve nAMD patients induces less TGF-β pathway activation in <t>Lenti-X</t> <t>293T</t> cells. Cells were co-transfected with the plasmids pNL[NlucP/SBE], expressing NanoLuc luciferase under the control of three SMAD3 binding elements, and pGL4.54[luc2/TK], expressing the Firefly luciferase luc2 (used as transfection normalizer) under the control of the constitutive HSV-TK promoter. Transfected cells were treated with 10 μl of aqueous humor of the 20 nAMD patients, naïve or treated (once or twice, Treat.1 and Treat.2, respectively), or of the 20 control samples (Cntr.) described in Table 1 . Data are presented as the ratio between NanoLuc and luc2 luciferase activity, expressed as Relative Light Units (RLU) (number of replicates for each sample = 3). Asterisks indicate significant differences (**P
    Lenti X 293t Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1770 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson 293t cells
    Targeted transduction and delivery of PSCA antigen gene into dendritic cells (DCs) by DCLV-PSCA. (A) <t>293T</t> cells were transfected transiently with plasmids FUW-Null (mock control, blue line) or FUW-PSCA (red line). Two days later, cells were collected and stained for PSCA expression analyzed by flow cytometry. 293T cells stained with the isotype antibody were included as a control (grey shade area). (B) 293T cells were transfected transiently with plasmids FUW-PSCA, SVGmu, and other necessary lentiviral packaging plasmids to produce DCLV-PSCA vectors. Fresh virus supernatant was used to transduce 293T cells (blue line) or 293T.hDC-SIGN cells (red line) with MOI = 10. PSCA expression was analyzed by flow cytometry 3 days post-transduction. (C) Bone marrow-derived DCs were transduced with a mock vector DC-LV-Null or DC-LV-PSCA vector. Five days later, CD11c and PSCA expression were assessed by flow cytometric analysis. All experiments were repeated three times and the representative data is shown.
    293t Cells, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 1300 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC hek 293 cell lines
    Identification and analysis of global Ca 2+ signals from multiple, individual cells. ( A ) The panel shows a Gaussian blurred image of resting fluorescence of Cal-520 loaded <t>HEK-293</t> WT cells, averaged over ~100 frames prior to stimulation. Imaging was performed by wide-field epifluorescence microscopy at low (10x) magnification. Scale bar = 20 µm ( B ) Binarized image of A, with yellow circles illustrating the identification of individual cells performed by the ‘generate ROIs’ operation in Flika. ( C ) ROIs overlaid on a fluorescence ratio (F/F 0 ) image from the same image sequence as A, at a time when nearly all cells exhibited a large increase in cytosolic Ca 2+ following stimulation by the muscarinic receptor agonist carbachol (CCH). ( D ) Fluorescence traces (ΔF/F 0 ) corresponding to the average change in fluorescence over time from within each ROI shown in C (x axis labels denote frames, acquired at 100 ms intervals).
    Hek 293 Cell Lines, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 146 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Addgene inc hek 293t cells
    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 <t>HEK</t> <t>293T</t> 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.
    Hek 293t Cells, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1675 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Polyplus Transfection hek 293 cells
    Simiate is vital to cells. A) Chariot reagent shuttled rbαSimiate (0.5µg) detects FLAG-Simiate in transfected <t>HEK-293</t> cells. The nuclei are visualized by DAPI staining. The arrows indicate clusters of rbαSimiate and FLAG-Simiate immunofluorescence inside the nucleus. B-D) Apoptosis in rbαSimiate and αrbAlexa568 treated HEK-293 cells. B) 0.25µg rbαSimiate, C) 1.0µg rbαSimiate and D) 1.0µg αrbAlexa568 as negative control. TUNEL staining (in green) served to identify apoptotic cells, while nuclear speckles were outlined with SC35 (in red). E) Quantification of the amount of endogenous Simiate epitopes not targeted by antibodies. F) Quantification of apoptotic cells. The increase in the percentage of TUNEL positive cells after rbαSimiate treatment is extremely significant (Chi 2 : p
    Hek 293 Cells, supplied by Polyplus Transfection, used in various techniques. Bioz Stars score: 93/100, based on 449 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Stratagene 293t cells
    Activation of JNK by v-Crk. ( A ) JNK activities were measured by in vitro kinase assay using GST-c-Jun (1–79) as a substrate with lysates from NIH 3T3 cells and v-Crk NIH 3T3 cells after 24 h serum starvation followed by either serum stimulation for 20 min or not. UV-irradiated NIH 3T3 cells were used as a positive control. The activities of MAPK were also measured in vitro using myelin basic protein (MBP) as a substrate. The levels of endogenous JNK1 and ERKs in total cell lysates were examined by immunoblotting (IB) using anti-JNK1 and mixture of anti-ERK1 and ERK2 antibodies (Santa Cruz Biotechnology). ( B ) JNK activities were measured in NIH 3T3 and v-Crk NIH 3T3 at different growth stages. Cell densities were roughly determined by the percentage of the confluence as 40% (lanes 1 and 6), 60% (lanes 2 and 7), 80% (lanes 3 and 8), 100% (lanes 4 and 9), and 24 h after 100% (lanes 5 and 10). ( C Upper ) JNK activities were assayed in chicken embryo fibroblasts (CEF) uninfected (lane 1) and infected with avian CT10 virus encoding v-Crk (lane 2). Lane 3, CEF with UV irradiation as a control. ( Lower ) The endogenous levels of JNKs were determined by immunoblotting using anti-JNK1 and anti-JNK2 antibodies. Lanes: 1, CEF; 2, CEF infected with CT10 virus; 3, CEF with UV irradiation. HA-JNK1 expressed in mammalian cells (lane 4) and bacterially expressed His-JNK2 (lane 5) were used as positive controls. ( D ) JNK activities in human embryo kidney cells <t>293T</t> that were transiently coexpressing HA-JNK1 with v-Crk, c-Crk-I, or c-Crk-II.
    293t Cells, supplied by Stratagene, used in various techniques. Bioz Stars score: 93/100, based on 1156 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Corning Life Sciences 293t cells
    Analysis of proteolytic activity of viral VP4 protein. (A) <t>293T</t> cells were transfected with the recombinant wild-type A-segment plasmid or the Ala or Asp substituted A-segment plasmids at sites pSer538, pTyr611 and pThr674 within VP4. At 24 h post-transfection, cell samples were harvested and electrophoresed on 12% SDS-PAGE gels for Western blot analysis with mAbs specific to VP3 and VP4 proteins. GAPDH was used as a loading control. (B) Recombinant plasmids used in (A) were translated with the TNT T7 Quick Coupled Transcription/Translation System, and expressed proteins were detected with mAbs specific for VP3 and VP4 proteins. (C) Analysis of co-localization between IBDV-encoding proteins within segment A. 293T cells were transfected with the IBDV A-segment mutant with the single dephospho- and phospho-mimicking VP4 gene. Wild-type IBDV A-segment transfected cells were used as a positive control. At 24 h post-transfection, the cells were fixed and probed with chicken anti-VP2 pAb, mouse anti-VP3 mAb and rabbit anti-VP4 pAb followed by FITC-conjugated goat anti-chicken IgG (green), Alexa Fluor 647 donkey anti-mouse IgG (blue) and Alexa Fluor 546 donkey ant-rabbit IgG (red). Nuclei were counterstained with DAPI (grey). The cells were observed with a laser Zeiss LSM510 laser confocal microscope. Cells transfected with the A segment with the Tyr611Asp and Thr674 Ala/Asp substitutions revealed co-localization between the IBDV-encoded proteins.
    293t Cells, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 93/100, based on 590 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
    Analysis of proteolytic activity of viral VP4 protein. (A) <t>293T</t> cells were transfected with the recombinant wild-type A-segment plasmid or the Ala or Asp substituted A-segment plasmids at sites pSer538, pTyr611 and pThr674 within VP4. At 24 h post-transfection, cell samples were harvested and electrophoresed on 12% SDS-PAGE gels for Western blot analysis with mAbs specific to VP3 and VP4 proteins. GAPDH was used as a loading control. (B) Recombinant plasmids used in (A) were translated with the TNT T7 Quick Coupled Transcription/Translation System, and expressed proteins were detected with mAbs specific for VP3 and VP4 proteins. (C) Analysis of co-localization between IBDV-encoding proteins within segment A. 293T cells were transfected with the IBDV A-segment mutant with the single dephospho- and phospho-mimicking VP4 gene. Wild-type IBDV A-segment transfected cells were used as a positive control. At 24 h post-transfection, the cells were fixed and probed with chicken anti-VP2 pAb, mouse anti-VP3 mAb and rabbit anti-VP4 pAb followed by FITC-conjugated goat anti-chicken IgG (green), Alexa Fluor 647 donkey anti-mouse IgG (blue) and Alexa Fluor 546 donkey ant-rabbit IgG (red). Nuclei were counterstained with DAPI (grey). The cells were observed with a laser Zeiss LSM510 laser confocal microscope. Cells transfected with the A segment with the Tyr611Asp and Thr674 Ala/Asp substitutions revealed co-localization between the IBDV-encoded proteins.
    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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    Ddx56 interacts with Oct4 and Sox2. a Ddx56-GST and Oct4-SFB plasmids were cotransfected into 293T cells, followed by IP and western blotting experiments. GST beads were used for pulldown. Negative controls: Ddx56-GST+GFP-SFB and GFP-GST+Oct4-SFB. Positive control: Sox2-GST+Oct4-SFB. b Ddx56-GST and Sox2-SFB plasmids were cotransfected into 293T cells, followed by IP and western blotting experiments. GST beads were used for pulldown. Negative controls: Ddx56-GST+GFP-SFB and GFP-GST+Sox2-SFB. Positive control: Oct4-GST+Sox2-SFB. c , d GST-tagged Ddx56 truncations and Sox2-HA plasmids were cotransfected into 293T cells, followed by IP and western blotting. e GST-tagged Ddx56 and Ddx56 ΔC-ter plasmids were transfected into 293T cells separately and measured by western blot. His-Sox2 protein was purified by Ni-sepharose purification. Using the same quantity of GST-Ddx56 and Ddx56 ΔC-ter separately, pulldown was performed to detect interaction

    Journal: Stem Cell Research & Therapy

    Article Title: Ddx56 maintains proliferation of mouse embryonic stem cells via ribosome assembly and interaction with the Oct4/Sox2 complex

    doi: 10.1186/s13287-020-01800-w

    Figure Lengend Snippet: Ddx56 interacts with Oct4 and Sox2. a Ddx56-GST and Oct4-SFB plasmids were cotransfected into 293T cells, followed by IP and western blotting experiments. GST beads were used for pulldown. Negative controls: Ddx56-GST+GFP-SFB and GFP-GST+Oct4-SFB. Positive control: Sox2-GST+Oct4-SFB. b Ddx56-GST and Sox2-SFB plasmids were cotransfected into 293T cells, followed by IP and western blotting experiments. GST beads were used for pulldown. Negative controls: Ddx56-GST+GFP-SFB and GFP-GST+Sox2-SFB. Positive control: Oct4-GST+Sox2-SFB. c , d GST-tagged Ddx56 truncations and Sox2-HA plasmids were cotransfected into 293T cells, followed by IP and western blotting. e GST-tagged Ddx56 and Ddx56 ΔC-ter plasmids were transfected into 293T cells separately and measured by western blot. His-Sox2 protein was purified by Ni-sepharose purification. Using the same quantity of GST-Ddx56 and Ddx56 ΔC-ter separately, pulldown was performed to detect interaction

    Article Snippet: 293T cells were collected in 48 h after transfected with Ddx56 full length and ΔC-ter plasmids, and grayscale in western blot experiment was used to balance the quantity of protein.

    Techniques: Western Blot, Positive Control, Transfection, Purification

    PRRSV nsp11 inhibits PCSK9 expression through its endoribonuclease activity. ( A ) HEK-293T cells were cotransfected with different combinations of vectors as indicated. Cell lysates were harvested at 24 hpt, and immunoprecipitation was performed using an antibody against HA, followed by WB analysis. ( B and C ) 855 bp PCSK9 promoter sequence was cloned into the pGL3-Basic vector. pGL3-PCSK9 was cotransfected with pCAGGS-HA-Nsp11(1 μg) or pCAGGS empty vector (1 μg) as a negative control into HEK-293T cells. Error bar: mean ± SEM; ***, p ≤ 0.001.; ns: no significant. ( D ) HEK-293T cells were transfected with pCAGGS-PCSK9-Flag (2 μg) and/or pCAGGS-HA-Nsp11 (2 μg) as indicated. At 18 hpt, the cells were further treated with/without the proteasome inhibitor MG132 or lysosomal inhibitor chloroquine (CQ) or vehicle, DMSO, for 6 h. Then, cell lysates were collected and analyzed by WB to assess PCSK9 and nsp11 expression. ( E ) A set of nsp11 constructs containing mutations that could inactivate endoribonuclease activity (nsp11-H129A, nsp11-H144A, nsp11-K173A, nsp11-H129H144A, and nsp11-C112K173A) or deubiquitinating activity (nsp11-C112A) (2 μg) were generated. pCAGGS-PCSK9-Flag (2 μg) was cotransfected with nsp11 mutants and wild type nsp11 into HEK-293T cells. WB was performed to analyze PCSK9 and nsp11 expression.

    Journal: Viruses

    Article Title: Porcine Reproductive and Respiratory Syndrome Virus Antagonizes PCSK9’s Antiviral Effect via Nsp11 Endoribonuclease Activity

    doi: 10.3390/v12060655

    Figure Lengend Snippet: PRRSV nsp11 inhibits PCSK9 expression through its endoribonuclease activity. ( A ) HEK-293T cells were cotransfected with different combinations of vectors as indicated. Cell lysates were harvested at 24 hpt, and immunoprecipitation was performed using an antibody against HA, followed by WB analysis. ( B and C ) 855 bp PCSK9 promoter sequence was cloned into the pGL3-Basic vector. pGL3-PCSK9 was cotransfected with pCAGGS-HA-Nsp11(1 μg) or pCAGGS empty vector (1 μg) as a negative control into HEK-293T cells. Error bar: mean ± SEM; ***, p ≤ 0.001.; ns: no significant. ( D ) HEK-293T cells were transfected with pCAGGS-PCSK9-Flag (2 μg) and/or pCAGGS-HA-Nsp11 (2 μg) as indicated. At 18 hpt, the cells were further treated with/without the proteasome inhibitor MG132 or lysosomal inhibitor chloroquine (CQ) or vehicle, DMSO, for 6 h. Then, cell lysates were collected and analyzed by WB to assess PCSK9 and nsp11 expression. ( E ) A set of nsp11 constructs containing mutations that could inactivate endoribonuclease activity (nsp11-H129A, nsp11-H144A, nsp11-K173A, nsp11-H129H144A, and nsp11-C112K173A) or deubiquitinating activity (nsp11-C112A) (2 μg) were generated. pCAGGS-PCSK9-Flag (2 μg) was cotransfected with nsp11 mutants and wild type nsp11 into HEK-293T cells. WB was performed to analyze PCSK9 and nsp11 expression.

    Article Snippet: To explore which nsps of PRRSV were involved in the down-regulation of PCSK9 expression, several nsps including nsp1α, nsp1β, nsp2, nsp4, nsp9, nsp10, nsp11, and nsp12 of PRRSV were cloned and cotransfected with pCAGGS-PCSK9-Flag into HEK-293T cells.

    Techniques: Expressing, Activity Assay, Immunoprecipitation, Western Blot, Sequencing, Clone Assay, Plasmid Preparation, Negative Control, Transfection, Construct, Generated

    PCSK9 promotes interferon production in a dose-dependent manner. ( A ) MARC-145 cells were transfected with either pCAGGS-PCSK9-flag or the empty vector pCAGGS. At different time points as indicated after transfection, the cells were harvested and total RNAs were purified. The expression of IFN-β was assessed by RT-qPCR with specific primers for IFN-β. Error bar: mean ± SEM; *, p ≤ 0.05; ***, p ≤ 0.001. ( B ) HEK-293T cells were transfected with different amounts of pCAGGS-PCSK9-flag or the empty vector pCAGGS then treated with or without Poly (I:C). The activity of luciferase was monitored to evaluate the activity of the IFN-β promoter.

    Journal: Viruses

    Article Title: Porcine Reproductive and Respiratory Syndrome Virus Antagonizes PCSK9’s Antiviral Effect via Nsp11 Endoribonuclease Activity

    doi: 10.3390/v12060655

    Figure Lengend Snippet: PCSK9 promotes interferon production in a dose-dependent manner. ( A ) MARC-145 cells were transfected with either pCAGGS-PCSK9-flag or the empty vector pCAGGS. At different time points as indicated after transfection, the cells were harvested and total RNAs were purified. The expression of IFN-β was assessed by RT-qPCR with specific primers for IFN-β. Error bar: mean ± SEM; *, p ≤ 0.05; ***, p ≤ 0.001. ( B ) HEK-293T cells were transfected with different amounts of pCAGGS-PCSK9-flag or the empty vector pCAGGS then treated with or without Poly (I:C). The activity of luciferase was monitored to evaluate the activity of the IFN-β promoter.

    Article Snippet: To explore which nsps of PRRSV were involved in the down-regulation of PCSK9 expression, several nsps including nsp1α, nsp1β, nsp2, nsp4, nsp9, nsp10, nsp11, and nsp12 of PRRSV were cloned and cotransfected with pCAGGS-PCSK9-Flag into HEK-293T cells.

    Techniques: Transfection, Plasmid Preparation, Purification, Expressing, Quantitative RT-PCR, Activity Assay, Luciferase

    PCSK9 degrades the PRRSV receptor CD163 through the lysosome pathway ( A ) MARC-145 cells were transfected with either pCAGGS-PCSK9-flag or the empty vector pCAGGS. At 24 hpt, total RNAs were isolated and RT-qPCR was carried out to evaluate the relative expression of CD163, CD151, and vimentin. The mRNA expression levels were determined relative to GAPDH. Error bar: mean ± SEM; ns: no significant. ( B ) HEK-293T cells were transfected with pCAGGS-CD163-HA and pCAGGS-PCSK9-Flag or pCAGGS vector. The cell lysates were analyzed by WB for the PCSK9 protein and CD163 protein. ( C and D ) HEK-293T cells were cotransfected with different combinations of vectors as indicated. Cell lysates were harvested at 24 hpt, and immunoprecipitation was performed using antibodies against HA ( C ) or Flag ( D ), followed by WB analysis. Samples of input were included as controls. ( E ) HeLa cells were transfected with pCAGGS-PCSK9-flag and/or pCAGGS-CD163-HA. The cells were then fixed and permeabilized with 0.5% Triton X-100 for immunofluorescent staining with a mouse anti-HA antibody (red) and a rabbit anti-Flag antibody (green). Representative images are shown. ( F ). Top panel: HEK-293T cells were transfected with pCAGGS-PCSK9-Flag and/or pCAGGS-CD163-HA as indicated. At 18 hpt, the cells were further treated with/without the proteasome inhibitor MG132 or the lysosomal inhibitor chloroquine (CQ) or the vehicle, DMSO, for 6 h. Then, cell lysates were collected and analyzed by WB for PCSK9 and CD163 expression. Bottom panel: HEK-293T cells were transfected with pCAGGS-CD163-HA only, and at 18 hpt, the cells were further treated with CQ or the vehicle, DMSO, for 6 h. Then, cell lysates were collected and analyzed by WB for CD163 expression.

    Journal: Viruses

    Article Title: Porcine Reproductive and Respiratory Syndrome Virus Antagonizes PCSK9’s Antiviral Effect via Nsp11 Endoribonuclease Activity

    doi: 10.3390/v12060655

    Figure Lengend Snippet: PCSK9 degrades the PRRSV receptor CD163 through the lysosome pathway ( A ) MARC-145 cells were transfected with either pCAGGS-PCSK9-flag or the empty vector pCAGGS. At 24 hpt, total RNAs were isolated and RT-qPCR was carried out to evaluate the relative expression of CD163, CD151, and vimentin. The mRNA expression levels were determined relative to GAPDH. Error bar: mean ± SEM; ns: no significant. ( B ) HEK-293T cells were transfected with pCAGGS-CD163-HA and pCAGGS-PCSK9-Flag or pCAGGS vector. The cell lysates were analyzed by WB for the PCSK9 protein and CD163 protein. ( C and D ) HEK-293T cells were cotransfected with different combinations of vectors as indicated. Cell lysates were harvested at 24 hpt, and immunoprecipitation was performed using antibodies against HA ( C ) or Flag ( D ), followed by WB analysis. Samples of input were included as controls. ( E ) HeLa cells were transfected with pCAGGS-PCSK9-flag and/or pCAGGS-CD163-HA. The cells were then fixed and permeabilized with 0.5% Triton X-100 for immunofluorescent staining with a mouse anti-HA antibody (red) and a rabbit anti-Flag antibody (green). Representative images are shown. ( F ). Top panel: HEK-293T cells were transfected with pCAGGS-PCSK9-Flag and/or pCAGGS-CD163-HA as indicated. At 18 hpt, the cells were further treated with/without the proteasome inhibitor MG132 or the lysosomal inhibitor chloroquine (CQ) or the vehicle, DMSO, for 6 h. Then, cell lysates were collected and analyzed by WB for PCSK9 and CD163 expression. Bottom panel: HEK-293T cells were transfected with pCAGGS-CD163-HA only, and at 18 hpt, the cells were further treated with CQ or the vehicle, DMSO, for 6 h. Then, cell lysates were collected and analyzed by WB for CD163 expression.

    Article Snippet: To explore which nsps of PRRSV were involved in the down-regulation of PCSK9 expression, several nsps including nsp1α, nsp1β, nsp2, nsp4, nsp9, nsp10, nsp11, and nsp12 of PRRSV were cloned and cotransfected with pCAGGS-PCSK9-Flag into HEK-293T cells.

    Techniques: Transfection, Plasmid Preparation, Isolation, Quantitative RT-PCR, Expressing, Western Blot, Immunoprecipitation, Staining

    PRRSV nsp11 could inhibit PCSK9 expression. ( A and B ) PRRSV non-structural protein sequences including nsp1α, nsp1β, nsp2, nsp4, nsp9, nsp10, nsp11, and nsp12 were cloned into p3X-Flag or pCAGGS vectors with Flag or MYC tag. HEK-293T cells were cotransfected with pCAGGS-PCSK9-Flag (2 μg) and different nsp constructs (2 μg). At 36 hpt, cell lysates were collected and analyzed by WB for PCSK9 expression and nsp expression. ( C ) PCSK9 expression construct pCAGGS-PCSK9-Flag was cotransfected with different doses of an expression vector encoding nsp11. At 36 hpt, cell lysates were collected and analyzed by Western blotting.

    Journal: Viruses

    Article Title: Porcine Reproductive and Respiratory Syndrome Virus Antagonizes PCSK9’s Antiviral Effect via Nsp11 Endoribonuclease Activity

    doi: 10.3390/v12060655

    Figure Lengend Snippet: PRRSV nsp11 could inhibit PCSK9 expression. ( A and B ) PRRSV non-structural protein sequences including nsp1α, nsp1β, nsp2, nsp4, nsp9, nsp10, nsp11, and nsp12 were cloned into p3X-Flag or pCAGGS vectors with Flag or MYC tag. HEK-293T cells were cotransfected with pCAGGS-PCSK9-Flag (2 μg) and different nsp constructs (2 μg). At 36 hpt, cell lysates were collected and analyzed by WB for PCSK9 expression and nsp expression. ( C ) PCSK9 expression construct pCAGGS-PCSK9-Flag was cotransfected with different doses of an expression vector encoding nsp11. At 36 hpt, cell lysates were collected and analyzed by Western blotting.

    Article Snippet: To explore which nsps of PRRSV were involved in the down-regulation of PCSK9 expression, several nsps including nsp1α, nsp1β, nsp2, nsp4, nsp9, nsp10, nsp11, and nsp12 of PRRSV were cloned and cotransfected with pCAGGS-PCSK9-Flag into HEK-293T cells.

    Techniques: Expressing, Clone Assay, Construct, Western Blot, Plasmid Preparation

    GDF11 promotes cohesion of transformed triple-negative cells before malignancy ) intraductal xenografts upon inducible knockdown of GDF11 at day 14. (B and C) Inducible GDF11 knockdown increases MCF10DCIS.com discohesion in vivo. Mammary lesions were stained with hematoxylin-eosin (B), and the relative proportion of acellular voids was quantified for paired shGDF11 and shGFP control glands (C). (D) Knockdown of ectopic proGDF11 by shRNA in doxycycline-treated 293T/17 cells. Extracts were immunoblotted for intracellular GDF11 by V5 epitope tag with vinculin, Hsp90, and p38 used as loading controls. (E) Inducible knockdown of GDF11 causes preexisting MCF10DCIS.com 3D spheroids to rupture and release single cells into the culture. (F and G) Inducible GDF11 knockdown reduces CDH1 mRNA and E-cadherin (E-cad) protein. RNA and protein extracts were collected after 16 days in culture. (H) Upregulated GDF11 mRNA in MCF10DCIS.com cells compared to MCF10A-5E cells in 3D culture (day 16). (I) Inducible knockdown of GDF11 does not alter the organization of MCF10A-5E 3D spheroids. Data are shown as the median ± 90% confidence interval for n = 10 mice (A) or the mean ± SEM for n = 4 (E, I) or the geometric mean ± log-transformed SEM for n = 8 (F, H) biological replicates. Representative immunoblots are shown from biological duplicates. For (E, I), shLuc was induced as a control shRNA. For (E–G, I), cultures were treated with 1 μg/ml doxycycline at day 4 and analyzed at day 16. Scale bar is 80 μm (B) and 200 μm (E, I). n.s., not significant ( p > 0.05 by two-sided Ward’s test). .

    Journal: Developmental cell

    Article Title: Tumor Suppressor Inactivation of GDF11 Occurs by Precursor Sequestration in Triple-Negative Breast Cancer

    doi: 10.1016/j.devcel.2017.10.027

    Figure Lengend Snippet: GDF11 promotes cohesion of transformed triple-negative cells before malignancy ) intraductal xenografts upon inducible knockdown of GDF11 at day 14. (B and C) Inducible GDF11 knockdown increases MCF10DCIS.com discohesion in vivo. Mammary lesions were stained with hematoxylin-eosin (B), and the relative proportion of acellular voids was quantified for paired shGDF11 and shGFP control glands (C). (D) Knockdown of ectopic proGDF11 by shRNA in doxycycline-treated 293T/17 cells. Extracts were immunoblotted for intracellular GDF11 by V5 epitope tag with vinculin, Hsp90, and p38 used as loading controls. (E) Inducible knockdown of GDF11 causes preexisting MCF10DCIS.com 3D spheroids to rupture and release single cells into the culture. (F and G) Inducible GDF11 knockdown reduces CDH1 mRNA and E-cadherin (E-cad) protein. RNA and protein extracts were collected after 16 days in culture. (H) Upregulated GDF11 mRNA in MCF10DCIS.com cells compared to MCF10A-5E cells in 3D culture (day 16). (I) Inducible knockdown of GDF11 does not alter the organization of MCF10A-5E 3D spheroids. Data are shown as the median ± 90% confidence interval for n = 10 mice (A) or the mean ± SEM for n = 4 (E, I) or the geometric mean ± log-transformed SEM for n = 8 (F, H) biological replicates. Representative immunoblots are shown from biological duplicates. For (E, I), shLuc was induced as a control shRNA. For (E–G, I), cultures were treated with 1 μg/ml doxycycline at day 4 and analyzed at day 16. Scale bar is 80 μm (B) and 200 μm (E, I). n.s., not significant ( p > 0.05 by two-sided Ward’s test). .

    Article Snippet: For the GDF11–EPR antibody, 293T/17 cells were transduced to stably express GDF11-V5 or LacZ-2×V5 control and either inducible shGDF11 or inducible shGFP control.

    Techniques: Transformation Assay, In Vivo, Staining, shRNA, Mouse Assay, Western Blot

    Physical interaction between Dnmt1 and the MutSα complex. ( A ) The potential for physical interaction between Dnmt1 and MutSα complex was examined by immunoprecipitation (IP) coupled with Western blotting (IB). Extracts from 293T cells co-expressing Dnmt1, HA-Msh6 and Flag-Msh2 were immunoprecipitated with control rabbit IgG, mouse IgG, anti-Dnmt1, anti-HA or anti-Flag, and then analyzed by IB using the latter three antibodies. The detection by IB of HA-Msh6 and Flag-Msh2 in the anti-Dnmt1 precipitate, as well as detection of Dnmt1 in the anti-HA (Msh6) or anti-Flag (Msh2) precipitate, indicates that Dnmt1 physically interacts with the MutSα complex. ( B ) Domain(s) of Dnmt1 interacting with the MutSα complex. The domain(s) was mapped by IP/IB assay of extracts from 293T cells co-expressing HA-Msh6, Flag-Msh2, and different fragments of Dnmt1. As summarized in the diagram, the IP/IB data (see Supplementary Fig. S4 ) show that Dnmt1 interacts with the MutSα complex through its central region (a.a. 446~1080), which is non-overlapping with the Np95-interacting domains of Dnmt1.

    Journal: Scientific Reports

    Article Title: Epigenetic Enhancement of the Post-replicative DNA Mismatch Repair of Mammalian Genomes by a Hemi-mCpG-Np95-Dnmt1 Axis

    doi: 10.1038/srep37490

    Figure Lengend Snippet: Physical interaction between Dnmt1 and the MutSα complex. ( A ) The potential for physical interaction between Dnmt1 and MutSα complex was examined by immunoprecipitation (IP) coupled with Western blotting (IB). Extracts from 293T cells co-expressing Dnmt1, HA-Msh6 and Flag-Msh2 were immunoprecipitated with control rabbit IgG, mouse IgG, anti-Dnmt1, anti-HA or anti-Flag, and then analyzed by IB using the latter three antibodies. The detection by IB of HA-Msh6 and Flag-Msh2 in the anti-Dnmt1 precipitate, as well as detection of Dnmt1 in the anti-HA (Msh6) or anti-Flag (Msh2) precipitate, indicates that Dnmt1 physically interacts with the MutSα complex. ( B ) Domain(s) of Dnmt1 interacting with the MutSα complex. The domain(s) was mapped by IP/IB assay of extracts from 293T cells co-expressing HA-Msh6, Flag-Msh2, and different fragments of Dnmt1. As summarized in the diagram, the IP/IB data (see Supplementary Fig. S4 ) show that Dnmt1 interacts with the MutSα complex through its central region (a.a. 446~1080), which is non-overlapping with the Np95-interacting domains of Dnmt1.

    Article Snippet: The 293T cell line was obtained from ATCC and cultured in Dulbecco’s Modified Eagle’s Medium plus 10% fetal bovine serum.

    Techniques: Immunoprecipitation, Western Blot, Expressing

    ExomiR-155 mediates the adipocyte metabolism by downregulating PPARγ. The adipocytes in 50 μg of exosomes purified from cancer-associated conditioned medium (CA-CM). ( a ) Exosomes originating from CA-CM viewed by electron microscopy (scale bar, 200 nm). ( b ) Exosomes from CA-CM were analysed by western blot. ( c ) NanoSight analysis of exosomes derived from CA-CM. ( d ) 4 T-1 were cocultivated in the presence or absence of adipocytes. After 3 days, exosomal miRNAs were further verified by qPCR. And RNA was extracted from the adipocytes and subjected to qPCR analysis with primers specific to mature miRNA. ( e ) The predicted miR-155 binding site in the 3’UTR of the PPARγ gene from TargetScan. ( f ) The GV272 vector containing the 3’UTR of the target gene harbouring wild-type (wt) or mutated (mt) miRNA binding sites was transfected into HEK 293 T cells stably expressing miRNA or empty vector (as a control). Luciferase activity was analysed at 48 h post-transfection, and the ratio of firefly luciferase activity to Renilla luciferase activity is shown. ( g ) Breast cancer cells were transfected with miRNA-155 pre-miRNA or miR-155 inhibitor, mature adipocytes were transfected with miR-155 mimic as the positive control and the control vector was applied as the negative control. Mature adipocytes cultured in the presence or absence of tumour exosomes for 3 days were stained with red oil, and Western blot analysis of related protein expression in different groups ( h ). Data are presented as the mean ± S.D. of at least three independent experiments. *  P

    Journal: Molecular Cancer

    Article Title: Tumour-originated exosomal miR-155 triggers cancer-associated cachexia to promote tumour progression

    doi: 10.1186/s12943-018-0899-5

    Figure Lengend Snippet: ExomiR-155 mediates the adipocyte metabolism by downregulating PPARγ. The adipocytes in 50 μg of exosomes purified from cancer-associated conditioned medium (CA-CM). ( a ) Exosomes originating from CA-CM viewed by electron microscopy (scale bar, 200 nm). ( b ) Exosomes from CA-CM were analysed by western blot. ( c ) NanoSight analysis of exosomes derived from CA-CM. ( d ) 4 T-1 were cocultivated in the presence or absence of adipocytes. After 3 days, exosomal miRNAs were further verified by qPCR. And RNA was extracted from the adipocytes and subjected to qPCR analysis with primers specific to mature miRNA. ( e ) The predicted miR-155 binding site in the 3’UTR of the PPARγ gene from TargetScan. ( f ) The GV272 vector containing the 3’UTR of the target gene harbouring wild-type (wt) or mutated (mt) miRNA binding sites was transfected into HEK 293 T cells stably expressing miRNA or empty vector (as a control). Luciferase activity was analysed at 48 h post-transfection, and the ratio of firefly luciferase activity to Renilla luciferase activity is shown. ( g ) Breast cancer cells were transfected with miRNA-155 pre-miRNA or miR-155 inhibitor, mature adipocytes were transfected with miR-155 mimic as the positive control and the control vector was applied as the negative control. Mature adipocytes cultured in the presence or absence of tumour exosomes for 3 days were stained with red oil, and Western blot analysis of related protein expression in different groups ( h ). Data are presented as the mean ± S.D. of at least three independent experiments. * P

    Article Snippet: The mouse breast cancer cell lines 4 T-1, C2C12 and HEK 293 T cells were obtained from American Type Culture Collection (ATCC, Shanghai) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% exosome-free foetal bovine serum (FBS, Shin Chin Industrial, SCI) and 1% penicillin–streptomycin (HyClone, Logan, UT, USA) in a humidified 37 °C incubator with 5% CO2 .

    Techniques: Purification, Electron Microscopy, Western Blot, Derivative Assay, Real-time Polymerase Chain Reaction, Binding Assay, Plasmid Preparation, Transfection, Stable Transfection, Expressing, Luciferase, Activity Assay, Positive Control, Negative Control, Cell Culture, Staining

    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

    USP7 interacts with ICN1. a HEK293T cells were transfected with plasmids encoding FLAG-tagged USP7 and/or Myc-tagged ICN1. Cell extracts were prepared and immunoprecipitated with anti-FLAG or anti-Myc antibodies. The protein interactions were analyzed by western blotting. b Whole-cell lysates from JURKAT and MOLT-4 cells were subjected to immunoprecipitation with a control IgG or an anti-ICN1 antibody. The immunoprecipitates were detected by western blotting. The input represented ~5% of the total protein extract used for immunoprecipitation. c The direct interaction between USP7 and ICN1 was detected using a GST pull-down assay, and the indicated proteins were examined by western blotting. d USP7 was co-localized with NOTCH1. CUTLL1 cells were fixed and immunostained with anti-USP7 (green) and anti-NOTCH1 (red) antibodies. The cell nuclei were counterstained with DAPI (blue). e Mapping of the ICN1-interacting domain in the USP7 protein. Top panel, a schematic representation of various USP7 truncated mutants. Bottom panel, HEK293T cells were co-transfected with constructs encoding FLAG-tagged ICN1 and GFP-tagged USP7 or truncated mutants. FLAG-tagged ICN1 proteins were immunoprecipitated with an anti-FLAG antibody, and the presence of USP7 protein and truncated mutants was examined by western blotting using an anti-GFP antibody

    Journal: Signal Transduction and Targeted Therapy

    Article Title: USP7 deubiquitinates and stabilizes NOTCH1 in T-cell acute lymphoblastic leukemia

    doi: 10.1038/s41392-018-0028-3

    Figure Lengend Snippet: USP7 interacts with ICN1. a HEK293T cells were transfected with plasmids encoding FLAG-tagged USP7 and/or Myc-tagged ICN1. Cell extracts were prepared and immunoprecipitated with anti-FLAG or anti-Myc antibodies. The protein interactions were analyzed by western blotting. b Whole-cell lysates from JURKAT and MOLT-4 cells were subjected to immunoprecipitation with a control IgG or an anti-ICN1 antibody. The immunoprecipitates were detected by western blotting. The input represented ~5% of the total protein extract used for immunoprecipitation. c The direct interaction between USP7 and ICN1 was detected using a GST pull-down assay, and the indicated proteins were examined by western blotting. d USP7 was co-localized with NOTCH1. CUTLL1 cells were fixed and immunostained with anti-USP7 (green) and anti-NOTCH1 (red) antibodies. The cell nuclei were counterstained with DAPI (blue). e Mapping of the ICN1-interacting domain in the USP7 protein. Top panel, a schematic representation of various USP7 truncated mutants. Bottom panel, HEK293T cells were co-transfected with constructs encoding FLAG-tagged ICN1 and GFP-tagged USP7 or truncated mutants. FLAG-tagged ICN1 proteins were immunoprecipitated with an anti-FLAG antibody, and the presence of USP7 protein and truncated mutants was examined by western blotting using an anti-GFP antibody

    Article Snippet: The human T-ALL cell lines JURKAT and MOLT-4 and human embryonic kidney (HEK293T) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

    Techniques: Transfection, Immunoprecipitation, Western Blot, Pull Down Assay, Construct

    Restriction of MoMLV and AKV by rat APOBEC3. (A) Amino acid alignment of rat and mouse APOBEC3 proteins. Yellow boxes indicate major amino acid differences; blue boxes indicate conservative amino acid changes. Residues making up exon 5 and the zinc coordination motif of the first and second protein domains are underlined. (B) Encapsidation of rat APOBEC3 by MoMLV and AKV. FLAG-tagged APOBEC3 proteins were detected in transfected cell lysates or in virions pelleted from culture supernatants by Western blot analysis using a horseradish peroxidase-conjugated anti-FLAG antibody. The lysate immunoblots were stripped and reprobed with an anti-tubulin antibody, whereas the virion blots were reprobed with an anti-p30 (Gag) antibody. hA2, human APOBEC2; hA3G, human APOBEC3G; mA3, mouse APOBEC3; ratA3, rat APOBEC3. (C) The infectivities of MoMLV or AKV particles that were produced by cotransfecting subconfluent 293T cells with 1 μg of either pMOV-eGFP or pAKV-NB-eGFP proviral DNA together with 0.2 μg of either the rat, mouse, or human FLAG-APOBEC3-expressing vector (or the human APOBEC2 control) were determined by transferring the virus-containing supernatants to NIH 3T3 cells at 36 h posttransfection and monitoring the percentages of cells displaying eGFP fluorescence after a further 48 h.

    Journal: Journal of Virology

    Article Title: The AKV Murine Leukemia Virus Is Restricted and Hypermutated by Mouse APOBEC3 ▿

    doi: 10.1128/JVI.01430-09

    Figure Lengend Snippet: Restriction of MoMLV and AKV by rat APOBEC3. (A) Amino acid alignment of rat and mouse APOBEC3 proteins. Yellow boxes indicate major amino acid differences; blue boxes indicate conservative amino acid changes. Residues making up exon 5 and the zinc coordination motif of the first and second protein domains are underlined. (B) Encapsidation of rat APOBEC3 by MoMLV and AKV. FLAG-tagged APOBEC3 proteins were detected in transfected cell lysates or in virions pelleted from culture supernatants by Western blot analysis using a horseradish peroxidase-conjugated anti-FLAG antibody. The lysate immunoblots were stripped and reprobed with an anti-tubulin antibody, whereas the virion blots were reprobed with an anti-p30 (Gag) antibody. hA2, human APOBEC2; hA3G, human APOBEC3G; mA3, mouse APOBEC3; ratA3, rat APOBEC3. (C) The infectivities of MoMLV or AKV particles that were produced by cotransfecting subconfluent 293T cells with 1 μg of either pMOV-eGFP or pAKV-NB-eGFP proviral DNA together with 0.2 μg of either the rat, mouse, or human FLAG-APOBEC3-expressing vector (or the human APOBEC2 control) were determined by transferring the virus-containing supernatants to NIH 3T3 cells at 36 h posttransfection and monitoring the percentages of cells displaying eGFP fluorescence after a further 48 h.

    Article Snippet: Infectious virus particles were produced by transfecting 293T cells at 70% confluence with either the pMOV-eGFP or pAKV-NB-eGFP plasmid, with or without FLAG-APOBEC3 expressing plasmids, using Genejuice transfection reagent (Novagen).

    Techniques: Transfection, Western Blot, Produced, Expressing, Plasmid Preparation, Transferring, Fluorescence

    Restriction of MoMLV and AKV by APOBEC3 proteins. (A) The infectivities of MoMLV or AKV particles that were produced by cotransfecting subconfluent 293T cells with 1 μg of either pMOV-eGFP or pAKV-NB-eGFP proviral DNA together with 0.2 μg of one of the FLAG-APOBEC3-expressing vectors were determined by transferring the virus-containing supernatants to NIH 3T3 cells at 36 h posttransfection and monitoring the percentages of cells displaying eGFP fluorescence after a further 48 h. The results are displayed as the level of infection relative to that obtained with cotransfection of the empty vector. Error bars represent the standard errors of the means for six independent experiments (as in panel B). hA2, human APOBEC2; mA3, mouse APOBEC3; hA3G, human APOBEC3G. (B) AKV infectivity, monitored by eGFP fluorescence of infected NIH 3T3 cells as a function of the amount of an APOBEC-expressing plasmid cotransfected with 1 μg of pAKV-NB-eGFP proviral DNA into subconfluent 293T cells during viral production. (C) Packaging of mouse APOBEC3 into AKV and MoMLV virions. Subconfluent 293T cells were cotransfected with 1 μg of either pMOV-eGFP or pAKV-NB-eGFP proviral DNA and 0.2 μg of each of the FLAG-APOBEC3-expressing vectors. After 36 h, virus was collected from the supernatants by ultracentrifugation. Western blot analysis with a horseradish peroxidase-conjugated anti-FLAG antibody was performed on transfected-cell lysates (top) or on virions pelleted from the supernatants (center). (Bottom) Virion encapsidation immunoblots were stripped and reprobed with an anti-p30 (Gag) antibody. CTRL, control. (D) Effects of different mouse APOBEC3 isoforms on AKV infectivity. Assays were performed and results presented as in panel A. +E5, with exon 5; −E5, without exon 5. (E) The expression of the various FLAG-tagged APOBEC proteins in the transfected 293T cells used in panel D was assayed by Western blot analysis using a horseradish peroxidase-conjugated anti-FLAG antibody on transfected-cell lysates. The immunoblot was then stripped and reprobed with an anti-tubulin antibody.

    Journal: Journal of Virology

    Article Title: The AKV Murine Leukemia Virus Is Restricted and Hypermutated by Mouse APOBEC3 ▿

    doi: 10.1128/JVI.01430-09

    Figure Lengend Snippet: Restriction of MoMLV and AKV by APOBEC3 proteins. (A) The infectivities of MoMLV or AKV particles that were produced by cotransfecting subconfluent 293T cells with 1 μg of either pMOV-eGFP or pAKV-NB-eGFP proviral DNA together with 0.2 μg of one of the FLAG-APOBEC3-expressing vectors were determined by transferring the virus-containing supernatants to NIH 3T3 cells at 36 h posttransfection and monitoring the percentages of cells displaying eGFP fluorescence after a further 48 h. The results are displayed as the level of infection relative to that obtained with cotransfection of the empty vector. Error bars represent the standard errors of the means for six independent experiments (as in panel B). hA2, human APOBEC2; mA3, mouse APOBEC3; hA3G, human APOBEC3G. (B) AKV infectivity, monitored by eGFP fluorescence of infected NIH 3T3 cells as a function of the amount of an APOBEC-expressing plasmid cotransfected with 1 μg of pAKV-NB-eGFP proviral DNA into subconfluent 293T cells during viral production. (C) Packaging of mouse APOBEC3 into AKV and MoMLV virions. Subconfluent 293T cells were cotransfected with 1 μg of either pMOV-eGFP or pAKV-NB-eGFP proviral DNA and 0.2 μg of each of the FLAG-APOBEC3-expressing vectors. After 36 h, virus was collected from the supernatants by ultracentrifugation. Western blot analysis with a horseradish peroxidase-conjugated anti-FLAG antibody was performed on transfected-cell lysates (top) or on virions pelleted from the supernatants (center). (Bottom) Virion encapsidation immunoblots were stripped and reprobed with an anti-p30 (Gag) antibody. CTRL, control. (D) Effects of different mouse APOBEC3 isoforms on AKV infectivity. Assays were performed and results presented as in panel A. +E5, with exon 5; −E5, without exon 5. (E) The expression of the various FLAG-tagged APOBEC proteins in the transfected 293T cells used in panel D was assayed by Western blot analysis using a horseradish peroxidase-conjugated anti-FLAG antibody on transfected-cell lysates. The immunoblot was then stripped and reprobed with an anti-tubulin antibody.

    Article Snippet: Infectious virus particles were produced by transfecting 293T cells at 70% confluence with either the pMOV-eGFP or pAKV-NB-eGFP plasmid, with or without FLAG-APOBEC3 expressing plasmids, using Genejuice transfection reagent (Novagen).

    Techniques: Produced, Expressing, Transferring, Fluorescence, Infection, Cotransfection, Plasmid Preparation, Western Blot, Transfection

    Restriction of AKV by endogenous mouse APOBEC3. (A, B, and C) Purified mouse splenocytes were infected with replicative MoMLV (A) or AKV (B and C) that had been produced by plasmid transfection into 293T cells. At 72 h postinfection, virus-containing culture supernatants were harvested from the cultured splenocytes and used to infect NIH 3T3 cells. Infection levels were determined by assessing the percentages of eGFP-positive cells 48 h later. Relative infection levels were established by setting the average percentage of eGFP-positive cells from the C57BL/6 mouse cohort to 1. Each point represents the mean of five independent infectivity measurements from a mouse splenocyte preparation. (D) Purified thymocytes from APOBEC3-deficient C57BL/6 mice were infected with AKV, and restriction of the virus was analyzed as for panel B. Each point represents the mean of four independent infectivity measurements from a mouse thymocyte preparation. (E) The pie chart depicts the proportions of sequences with the indicated numbers of mutations. The total number of clones sequenced is given at the center of the pie. A line drawing depicts the distribution of G→A mutations along the eGFP gene in the first 10 mutated sequences analyzed. mA3, mouse APOBEC3. (F) Local sequence preference for deamination by endogenous mouse APOBEC3, computed with respect to the deaminated cytidine (position zero) on the viral minus strand. “n” indicates the total number of mutations analyzed.

    Journal: Journal of Virology

    Article Title: The AKV Murine Leukemia Virus Is Restricted and Hypermutated by Mouse APOBEC3 ▿

    doi: 10.1128/JVI.01430-09

    Figure Lengend Snippet: Restriction of AKV by endogenous mouse APOBEC3. (A, B, and C) Purified mouse splenocytes were infected with replicative MoMLV (A) or AKV (B and C) that had been produced by plasmid transfection into 293T cells. At 72 h postinfection, virus-containing culture supernatants were harvested from the cultured splenocytes and used to infect NIH 3T3 cells. Infection levels were determined by assessing the percentages of eGFP-positive cells 48 h later. Relative infection levels were established by setting the average percentage of eGFP-positive cells from the C57BL/6 mouse cohort to 1. Each point represents the mean of five independent infectivity measurements from a mouse splenocyte preparation. (D) Purified thymocytes from APOBEC3-deficient C57BL/6 mice were infected with AKV, and restriction of the virus was analyzed as for panel B. Each point represents the mean of four independent infectivity measurements from a mouse thymocyte preparation. (E) The pie chart depicts the proportions of sequences with the indicated numbers of mutations. The total number of clones sequenced is given at the center of the pie. A line drawing depicts the distribution of G→A mutations along the eGFP gene in the first 10 mutated sequences analyzed. mA3, mouse APOBEC3. (F) Local sequence preference for deamination by endogenous mouse APOBEC3, computed with respect to the deaminated cytidine (position zero) on the viral minus strand. “n” indicates the total number of mutations analyzed.

    Article Snippet: Infectious virus particles were produced by transfecting 293T cells at 70% confluence with either the pMOV-eGFP or pAKV-NB-eGFP plasmid, with or without FLAG-APOBEC3 expressing plasmids, using Genejuice transfection reagent (Novagen).

    Techniques: Purification, Infection, Produced, Plasmid Preparation, Transfection, Cell Culture, Mouse Assay, Clone Assay, Sequencing

    Direct binding measurements of CD4-Ig and mabs follows neutralization sensitivity of Env+ pseudoviruses. LN8 wt, 375W and 380P Envs were expressed on 293T cells before measuring binding of CD4-Ig and mabs using flow cytometry. Boxed values in the right hand, top corner of each flow profile represents the neutralization titer for each reagent and shows that binding closely followed neutralization sensitivity.

    Journal: PLoS Pathogens

    Article Title: Saturation Mutagenesis of the HIV-1 Envelope CD4 Binding Loop Reveals Residues Controlling Distinct Trimer Conformations

    doi: 10.1371/journal.ppat.1005988

    Figure Lengend Snippet: Direct binding measurements of CD4-Ig and mabs follows neutralization sensitivity of Env+ pseudoviruses. LN8 wt, 375W and 380P Envs were expressed on 293T cells before measuring binding of CD4-Ig and mabs using flow cytometry. Boxed values in the right hand, top corner of each flow profile represents the neutralization titer for each reagent and shows that binding closely followed neutralization sensitivity.

    Article Snippet: Expression of Env trimers on 293T cells and mab binding HIV-1 Envs were expressed on 293T cells following transfection of Env expression vectors using Fugene6 following the manufacturer’s protocol.

    Techniques: Binding Assay, Neutralization, Flow Cytometry, Cytometry

    Exchangeable apolipoproteins redundantly participate in the formation of infectious HCV particles. (A) BE-KO1 cells infected with HCVcc at an MOI of 1 at 6 h post-transfection with siRNAs targeting ApoA1 (A1), ApoA2 (A2), ApoC1 (C1), ApoC2 (C2), ApoC3 (C3) and ApoH (H) and infectious titers in the culture supernatants were determined by focus-forming assay at 72 h post-infection. (B) ApoA1, ApoA2, ApoC1, ApoC2, ApoC3, ApoE and ApoH were exogenously expressed in BE-KO1 cells by infection with lentiviral vectors, and then infected with HCVcc at an MOI of 1. Expression of the apolipoproteins was determined by immunoblot analysis (upper), and infectious titers in the culture supernatants were determined at 72 h post-infection by focus-forming assay (lower). (C) Extracellular and intracellular HCV RNA in BE-KO1 cells expressing apolipoproteins and infected with HCVcc were determined at 72 h post-infection by qRT-PCR. (D) Specific infectivity was calculated as extracellular infectious titers/extracellular HCV RNA copies in BE-KO1 cells expressing apolipoproteins at 72 h post-infection. (E) 293T cells stably expressing CLDN1 and miR-122 (293T-CLDN/miR-122 cells) were infected with the lentiviral vectors, and the expressions of the apolipoproteins were determined by immunoblot analysis (upper). These cells were infected with HCVcc at an MOI of 1, and infectious titers in the supernatants were determined at 72 h post-infection by focus-forming assay (lower). In all cases, asterisks indicate significant differences (*, P

    Journal: PLoS Pathogens

    Article Title: Amphipathic α-Helices in Apolipoproteins Are Crucial to the Formation of Infectious Hepatitis C Virus Particles

    doi: 10.1371/journal.ppat.1004534

    Figure Lengend Snippet: Exchangeable apolipoproteins redundantly participate in the formation of infectious HCV particles. (A) BE-KO1 cells infected with HCVcc at an MOI of 1 at 6 h post-transfection with siRNAs targeting ApoA1 (A1), ApoA2 (A2), ApoC1 (C1), ApoC2 (C2), ApoC3 (C3) and ApoH (H) and infectious titers in the culture supernatants were determined by focus-forming assay at 72 h post-infection. (B) ApoA1, ApoA2, ApoC1, ApoC2, ApoC3, ApoE and ApoH were exogenously expressed in BE-KO1 cells by infection with lentiviral vectors, and then infected with HCVcc at an MOI of 1. Expression of the apolipoproteins was determined by immunoblot analysis (upper), and infectious titers in the culture supernatants were determined at 72 h post-infection by focus-forming assay (lower). (C) Extracellular and intracellular HCV RNA in BE-KO1 cells expressing apolipoproteins and infected with HCVcc were determined at 72 h post-infection by qRT-PCR. (D) Specific infectivity was calculated as extracellular infectious titers/extracellular HCV RNA copies in BE-KO1 cells expressing apolipoproteins at 72 h post-infection. (E) 293T cells stably expressing CLDN1 and miR-122 (293T-CLDN/miR-122 cells) were infected with the lentiviral vectors, and the expressions of the apolipoproteins were determined by immunoblot analysis (upper). These cells were infected with HCVcc at an MOI of 1, and infectious titers in the supernatants were determined at 72 h post-infection by focus-forming assay (lower). In all cases, asterisks indicate significant differences (*, P

    Article Snippet: Lipofection and lentiviral gene transduction The lentiviral vectors and ViraPower Lentiviral Packaging Mix (Life Technologies) were co-transfected into 293T cells by Trans IT LT-1 (Mirus), and the supernatants were recovered at 48 h post-transfection.

    Techniques: Infection, Transfection, Focus Forming Assay, Expressing, Quantitative RT-PCR, Stable Transfection

    Residual enzymatic conversion of 5-formyl-THF to methenyl-THF in lysates (a) Reaction schematic for methenyl-THF, 10-formyl- and 5-formyl-THF interconversion. The proton highlighted in red can exchange with H2O. (b) Labeling kinetics of freshly dissolved unlabeled methenyl-THF standard incubated in D2O phosphate buffer (pH 7) at 4°C. To verify that 5-formyl-THF, unlike 10-formyl-THF, does not interconvert with methenyl-THF under these conditions, an identical experiment was performed with 5-formyl-THF standard (Mean ± SD, N = 3). (c) Folate and metabolite labeling patterns after 24 h incubation of in HEK293T cells with 1 mM [13C,2H]formate in the growth media (Mean ± SD, N ≥ 2). (d) Conversion of 5-formyl-THF to methenyl-THF in cell extracts. HEK293T cell extracts were prepared as described in method, and optionally heated to 60°C for 5 min after addition (when indicated) of 4 pmol 5-formyl-THF standard (Mean ± SD, N = 3).

    Journal: Analytical and bioanalytical chemistry

    Article Title: An LC-MS Chemical Derivatization Method for the Measurement of Five Different One-carbon States of Cellular Tetrahydrofolate

    doi: 10.1007/s00216-017-0514-4

    Figure Lengend Snippet: Residual enzymatic conversion of 5-formyl-THF to methenyl-THF in lysates (a) Reaction schematic for methenyl-THF, 10-formyl- and 5-formyl-THF interconversion. The proton highlighted in red can exchange with H2O. (b) Labeling kinetics of freshly dissolved unlabeled methenyl-THF standard incubated in D2O phosphate buffer (pH 7) at 4°C. To verify that 5-formyl-THF, unlike 10-formyl-THF, does not interconvert with methenyl-THF under these conditions, an identical experiment was performed with 5-formyl-THF standard (Mean ± SD, N = 3). (c) Folate and metabolite labeling patterns after 24 h incubation of in HEK293T cells with 1 mM [13C,2H]formate in the growth media (Mean ± SD, N ≥ 2). (d) Conversion of 5-formyl-THF to methenyl-THF in cell extracts. HEK293T cell extracts were prepared as described in method, and optionally heated to 60°C for 5 min after addition (when indicated) of 4 pmol 5-formyl-THF standard (Mean ± SD, N = 3).

    Article Snippet: HEK293T cells (ATCC, CRL-11268) were grown in DMEM (Cellgro, 10-017) without pyruvate and supplemented with 10% FBS (Sigma) in an incubator with 5% CO2 and ambient oxygen at 37°C.

    Techniques: Labeling, Incubation

    Methylene-THF’s 1C unit exchanges with formaldehyde (a) Reaction schematic for THF and methylene-THF interconversion. The methylene group highlighted in red can exchange with formaldehyde. (b) Labeling patterns in HEK293T cells after 24 h incubation with [3-13C]serine. Note the lack of labeling of methylene-THF, despite labeling of its downstream products, indicative of label loss during the sample preparation process (Mean ± SD, N = 3). (c) Labeling patterns in HEK293T cells after 24 h incubation with 1 mM [13C,2H]formate (Mean ± SD, N = 3). (d) Labeling kinetics of freshly dissolved unlabeled THF or methylene-THF standard incubated in 1:1 H2O:[U-2H]MeOH at 4°C. (e) Labeling pattern of freshly dissolved unlabeled methylene-THF standard incubated at 4°C for 2 h in unlabeled H2O:MeOH with the indicated concentration of deuterated formaldehyde.

    Journal: Analytical and bioanalytical chemistry

    Article Title: An LC-MS Chemical Derivatization Method for the Measurement of Five Different One-carbon States of Cellular Tetrahydrofolate

    doi: 10.1007/s00216-017-0514-4

    Figure Lengend Snippet: Methylene-THF’s 1C unit exchanges with formaldehyde (a) Reaction schematic for THF and methylene-THF interconversion. The methylene group highlighted in red can exchange with formaldehyde. (b) Labeling patterns in HEK293T cells after 24 h incubation with [3-13C]serine. Note the lack of labeling of methylene-THF, despite labeling of its downstream products, indicative of label loss during the sample preparation process (Mean ± SD, N = 3). (c) Labeling patterns in HEK293T cells after 24 h incubation with 1 mM [13C,2H]formate (Mean ± SD, N = 3). (d) Labeling kinetics of freshly dissolved unlabeled THF or methylene-THF standard incubated in 1:1 H2O:[U-2H]MeOH at 4°C. (e) Labeling pattern of freshly dissolved unlabeled methylene-THF standard incubated at 4°C for 2 h in unlabeled H2O:MeOH with the indicated concentration of deuterated formaldehyde.

    Article Snippet: HEK293T cells (ATCC, CRL-11268) were grown in DMEM (Cellgro, 10-017) without pyruvate and supplemented with 10% FBS (Sigma) in an incubator with 5% CO2 and ambient oxygen at 37°C.

    Techniques: Labeling, Incubation, Sample Prep, Concentration Assay

    Acute effect of methotrexate on cellular folates. HEK293T cells were incubated with 1 μM methotrexate for 2 h before extraction. Pool sizes are normalized to DMSO-treated control cells (Mean ± SD, N ≥ 3).

    Journal: Analytical and bioanalytical chemistry

    Article Title: An LC-MS Chemical Derivatization Method for the Measurement of Five Different One-carbon States of Cellular Tetrahydrofolate

    doi: 10.1007/s00216-017-0514-4

    Figure Lengend Snippet: Acute effect of methotrexate on cellular folates. HEK293T cells were incubated with 1 μM methotrexate for 2 h before extraction. Pool sizes are normalized to DMSO-treated control cells (Mean ± SD, N ≥ 3).

    Article Snippet: HEK293T cells (ATCC, CRL-11268) were grown in DMEM (Cellgro, 10-017) without pyruvate and supplemented with 10% FBS (Sigma) in an incubator with 5% CO2 and ambient oxygen at 37°C.

    Techniques: Incubation

    Posttranslational modifications for bovine BST-2s. (A) Expression plasmid for FLAG-tagged bBST-2A1, bBST-2A2 or bBST-2B was transfected into HEK 293T cells. Cells were dissolved 24 h after transfection and subjected to 12.5% SDS-PAGE. Western blotting was performed with anti-FLAG or anti-Actin antibody. (B) Bovine BST-2s expressed in HEK 293T cells were treated with (+) or without (−)PNGase F for de- N -glycosylated reaction, and then subjected to 12.5% SDS-PAGE. Reaction conditions with PNGase F were as described in the Materials and Methods. BST-2 was detected with anti-FLAG antibody by Western blotting analysis.

    Journal: PLoS ONE

    Article Title: Identification and Functional Analysis of Three Isoforms of Bovine BST-2

    doi: 10.1371/journal.pone.0041483

    Figure Lengend Snippet: Posttranslational modifications for bovine BST-2s. (A) Expression plasmid for FLAG-tagged bBST-2A1, bBST-2A2 or bBST-2B was transfected into HEK 293T cells. Cells were dissolved 24 h after transfection and subjected to 12.5% SDS-PAGE. Western blotting was performed with anti-FLAG or anti-Actin antibody. (B) Bovine BST-2s expressed in HEK 293T cells were treated with (+) or without (−)PNGase F for de- N -glycosylated reaction, and then subjected to 12.5% SDS-PAGE. Reaction conditions with PNGase F were as described in the Materials and Methods. BST-2 was detected with anti-FLAG antibody by Western blotting analysis.

    Article Snippet: Human embryonic kidney (HEK) 293T cells (ATCC CRL-11268), HeLa cells (ATCC CCL-2), and fetal lamb kidney cells constitutively producing bovine leukemia virus (BLV), FLK-BLV cells , were maintained in Dulbecco's modified Eagle's medium (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum and penicillin/streptomycin.

    Techniques: Expressing, Plasmid Preparation, Transfection, SDS Page, Western Blot

    Analysis of Env incorporation into virions. HEK293T/17 cells were transfected with DNA encoding the HIV-1 virus. Three days after transfection the viral supernatants were pelleted by ultracentrifugation and separated by SDS-PAGE on a 4–12% bis-tris

    Journal: The Journal of Biological Chemistry

    Article Title: Unique Functional Properties of Conserved Arginine Residues in the Lentivirus Lytic Peptide Domains of the C-terminal Tail of HIV-1 gp41 *

    doi: 10.1074/jbc.M113.529339

    Figure Lengend Snippet: Analysis of Env incorporation into virions. HEK293T/17 cells were transfected with DNA encoding the HIV-1 virus. Three days after transfection the viral supernatants were pelleted by ultracentrifugation and separated by SDS-PAGE on a 4–12% bis-tris

    Article Snippet: HEK293T/17 cells were transfected with pUC19–89.6 wild type or mutated DNA in a 6-well plate, and after 24 h the transfected cells were harvested using 1× citric saline for 4 min at 37 °C and diluted with 1 volume of 1× PBS.

    Techniques: Transfection, SDS Page

    Cell-to-cell fusion mediated by Env at the cell surface. A, cell-free supernatant from donor-transfected HEK293T/17 cells was added to the TZMbl target cells for 6 h in the presence of AZT. Luciferase activity is expressed as RLUs. Data represent one

    Journal: The Journal of Biological Chemistry

    Article Title: Unique Functional Properties of Conserved Arginine Residues in the Lentivirus Lytic Peptide Domains of the C-terminal Tail of HIV-1 gp41 *

    doi: 10.1074/jbc.M113.529339

    Figure Lengend Snippet: Cell-to-cell fusion mediated by Env at the cell surface. A, cell-free supernatant from donor-transfected HEK293T/17 cells was added to the TZMbl target cells for 6 h in the presence of AZT. Luciferase activity is expressed as RLUs. Data represent one

    Article Snippet: HEK293T/17 cells were transfected with pUC19–89.6 wild type or mutated DNA in a 6-well plate, and after 24 h the transfected cells were harvested using 1× citric saline for 4 min at 37 °C and diluted with 1 volume of 1× PBS.

    Techniques: Transfection, Luciferase, Activity Assay

    Functional analysis of components of K11/K48-specific quality control A. UBR4 and UBR5 assemble most K11/K48-linked chains during quality control. 293T cells were co- depleted of UBR4 and UBR5, treated with MG132, and analyzed for ubiquitin chains of different topologies using Western blotting. B. UBR4 and UBR5 target puromycylated proteins for degradation. 293T cells were co-depleted of UBR4 and UBR5, treated with puromycin, and the stability of puromycylated proteins was determined by cycloheximide chase and Western blotting. C. UBR4 and UBR5 complexes synthesize K11/K48-linked chains in vitro . Endogenous FLAG UBR4 and FLAG UBR5 were affinity-purified from CRISPR/Cas9-edited 293T cells, incubated with E1, the E2 UBE2D3, and ATP (as indicated). Reactions were supplemented with wild-type ubiquitin (WT); a mixture of ubiquitin K11R and ubiquitin K48R (K11R+K48R) or double mutant ubiquitin K11R/K48R (K11R/K48R). Reactions were analyzed for K11/K48-linked chains and the respective E3 enzymes by Western blotting. D. UBR5 assembles branched ubiquitin chains. Affinity-purified FLAG UBR5 were incubated with E1, UBE2D3, and ubiquitin FLAG/TEV . Conjugates were treated with TEV-protease as indicated and analyzed by αFLAG immunoblotting. The presence of two or more FLAG-positive stamps indicates branching. As control, ubiquitylation reactions were performed with UBE2G2/gp78, which only assemble homotypic K48-linked chains. E. UBR5 produces branched ubiquitin trimers. UBR5 and the K11-specific UBE2S were incubated with ubiquitin ∆GG (a mutant that can be modified, but not used as a modifier) and methyl-ubiquitin (which can be transferred to ubiquitin ∆GG , but not further modified), as indicated. Formation of K11- or K48-positive ubiquitin conjugates was analyzed by linkage-specific Western blotting. The presence of both UBE2S and UBR5 leads to formation of branched ubiquitin trimers. F. UBR5 strongly prefers K48-linkages. Endogenous UBR5 complexes were incubated with indicated single-Lys ubiquitin mutants and analyzed by αUbiquitin immunoblotting. G. K11/K48-branched ubiquitin chains produced by UBR5 show strongly increased affinity to the p97/VCP adaptor NSFL1/p47 than homotypic K48-linked chains. K11/K48-branched or K48-linked chains were assembled by UBR5 using wt-ubiquitin or ubiquitin K48 and incubated with immobilized p97-NSFL1 complexes. Binding reactions were stopped at the indicated times and analyzed by αUbiquitin immunoblotting. H. The proteasome targets puromycylated and K11/K48-labeled proteins for degradation. The stability of puromycylated proteins was analyzed in cells treated with DMSO or MG132, and puromycylated proteins or K11/K48-linked chains were detected by Western blotting using specific antibodies.

    Journal: Cell

    Article Title: Assembly and Function of Heterotypic Ubiquitin Chains in Cell Cycle and Protein Quality Control

    doi: 10.1016/j.cell.2017.09.040

    Figure Lengend Snippet: Functional analysis of components of K11/K48-specific quality control A. UBR4 and UBR5 assemble most K11/K48-linked chains during quality control. 293T cells were co- depleted of UBR4 and UBR5, treated with MG132, and analyzed for ubiquitin chains of different topologies using Western blotting. B. UBR4 and UBR5 target puromycylated proteins for degradation. 293T cells were co-depleted of UBR4 and UBR5, treated with puromycin, and the stability of puromycylated proteins was determined by cycloheximide chase and Western blotting. C. UBR4 and UBR5 complexes synthesize K11/K48-linked chains in vitro . Endogenous FLAG UBR4 and FLAG UBR5 were affinity-purified from CRISPR/Cas9-edited 293T cells, incubated with E1, the E2 UBE2D3, and ATP (as indicated). Reactions were supplemented with wild-type ubiquitin (WT); a mixture of ubiquitin K11R and ubiquitin K48R (K11R+K48R) or double mutant ubiquitin K11R/K48R (K11R/K48R). Reactions were analyzed for K11/K48-linked chains and the respective E3 enzymes by Western blotting. D. UBR5 assembles branched ubiquitin chains. Affinity-purified FLAG UBR5 were incubated with E1, UBE2D3, and ubiquitin FLAG/TEV . Conjugates were treated with TEV-protease as indicated and analyzed by αFLAG immunoblotting. The presence of two or more FLAG-positive stamps indicates branching. As control, ubiquitylation reactions were performed with UBE2G2/gp78, which only assemble homotypic K48-linked chains. E. UBR5 produces branched ubiquitin trimers. UBR5 and the K11-specific UBE2S were incubated with ubiquitin ∆GG (a mutant that can be modified, but not used as a modifier) and methyl-ubiquitin (which can be transferred to ubiquitin ∆GG , but not further modified), as indicated. Formation of K11- or K48-positive ubiquitin conjugates was analyzed by linkage-specific Western blotting. The presence of both UBE2S and UBR5 leads to formation of branched ubiquitin trimers. F. UBR5 strongly prefers K48-linkages. Endogenous UBR5 complexes were incubated with indicated single-Lys ubiquitin mutants and analyzed by αUbiquitin immunoblotting. G. K11/K48-branched ubiquitin chains produced by UBR5 show strongly increased affinity to the p97/VCP adaptor NSFL1/p47 than homotypic K48-linked chains. K11/K48-branched or K48-linked chains were assembled by UBR5 using wt-ubiquitin or ubiquitin K48 and incubated with immobilized p97-NSFL1 complexes. Binding reactions were stopped at the indicated times and analyzed by αUbiquitin immunoblotting. H. The proteasome targets puromycylated and K11/K48-labeled proteins for degradation. The stability of puromycylated proteins was analyzed in cells treated with DMSO or MG132, and puromycylated proteins or K11/K48-linked chains were detected by Western blotting using specific antibodies.

    Article Snippet: Lentiviruses were produced in 293T cells by cotransfection of lentiviral constructs with packaging plasmids (Addgene) forand was harvested 48 and 72 hours post-transfection.

    Techniques: Functional Assay, Western Blot, In Vitro, Affinity Purification, CRISPR, Incubation, Mutagenesis, Modification, Produced, Binding Assay, Labeling

    Cells respond to proteotoxic stress by assembling K11/K48-branched chains A. 293T cells were treated with proteasome- (red), HSP70- (yellow), HSP90-inhibitors (blue), or other stressors, and formation of ubiquitin chains with the indicated topologies was monitored by Western blotting using specific antibodies. B. Accumulation of misfolded proteins results in formation of K11/K48-branched chains. 293T cells expressing ubiquitin TEV/FLAG were grown in the presence of MG132 as indicated. K11/K48-linked chains were affinity-purified using the K11/K48-bispecific antibody and subjected to TEV cleavage. As each attached ubiquitin TEV/FLAG ). Only the relevant MW region of the αFLAG Western blot is shown. C. Aggregating newly synthesized proteins are strongly labeled with K11/K48-linked chains. Cells were treated with either DMSO or the HSP70 inhibitor pifithrin µ, and then stained for K11/K48-linked chains by using fluorescently labeled K11/K48-bispecific antibodies (yellow). DNA was stained with Hoechst (blue). Inhibition of mRNA translation (cycloheximide, emetine, harringtonine), but not inhibition of transcription (α-amanitine), prevented formation of K11/K48-positive aggregates. D. Production of K11/K48-linked chains in cells experiencing proteotoxic stress relies on new protein synthesis. 293T cells were treated with the HSP70-inhibitor pifithrin µ, and various inhibitors of mRNA translation or transcription, as described above. K11/K48-linked chains were detected by Western blotting using bispecific antibodies. E. Nascent, misfolded proteins are decorated with K11/K48-linked chains. Ubiquitin conjugates from puromycin-treated 293T cells were purified under denaturing conditions using the indicated antibodies, and modified puromycylated proteins were detected with an antibody against puromycin. F. Puromycylated proteins accumulate in K11/K48-positive aggregates. HeLa cells were treated with puromycin for 2h and analyzed by immunofluorescence microscopy against puromycin (green), K11/K48-linked chains (red), and DNA (blue). The right panel depicts a merged image of the same experiment performed in the presence of cycloheximide.

    Journal: Cell

    Article Title: Assembly and Function of Heterotypic Ubiquitin Chains in Cell Cycle and Protein Quality Control

    doi: 10.1016/j.cell.2017.09.040

    Figure Lengend Snippet: Cells respond to proteotoxic stress by assembling K11/K48-branched chains A. 293T cells were treated with proteasome- (red), HSP70- (yellow), HSP90-inhibitors (blue), or other stressors, and formation of ubiquitin chains with the indicated topologies was monitored by Western blotting using specific antibodies. B. Accumulation of misfolded proteins results in formation of K11/K48-branched chains. 293T cells expressing ubiquitin TEV/FLAG were grown in the presence of MG132 as indicated. K11/K48-linked chains were affinity-purified using the K11/K48-bispecific antibody and subjected to TEV cleavage. As each attached ubiquitin TEV/FLAG ). Only the relevant MW region of the αFLAG Western blot is shown. C. Aggregating newly synthesized proteins are strongly labeled with K11/K48-linked chains. Cells were treated with either DMSO or the HSP70 inhibitor pifithrin µ, and then stained for K11/K48-linked chains by using fluorescently labeled K11/K48-bispecific antibodies (yellow). DNA was stained with Hoechst (blue). Inhibition of mRNA translation (cycloheximide, emetine, harringtonine), but not inhibition of transcription (α-amanitine), prevented formation of K11/K48-positive aggregates. D. Production of K11/K48-linked chains in cells experiencing proteotoxic stress relies on new protein synthesis. 293T cells were treated with the HSP70-inhibitor pifithrin µ, and various inhibitors of mRNA translation or transcription, as described above. K11/K48-linked chains were detected by Western blotting using bispecific antibodies. E. Nascent, misfolded proteins are decorated with K11/K48-linked chains. Ubiquitin conjugates from puromycin-treated 293T cells were purified under denaturing conditions using the indicated antibodies, and modified puromycylated proteins were detected with an antibody against puromycin. F. Puromycylated proteins accumulate in K11/K48-positive aggregates. HeLa cells were treated with puromycin for 2h and analyzed by immunofluorescence microscopy against puromycin (green), K11/K48-linked chains (red), and DNA (blue). The right panel depicts a merged image of the same experiment performed in the presence of cycloheximide.

    Article Snippet: Lentiviruses were produced in 293T cells by cotransfection of lentiviral constructs with packaging plasmids (Addgene) forand was harvested 48 and 72 hours post-transfection.

    Techniques: Western Blot, Expressing, Affinity Purification, Synthesized, Labeling, Staining, Inhibition, Purification, Modification, Immunofluorescence, Microscopy

    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

    Quantitative analysis of TGF-β pathway activation by aqueous humor of patients and controls. Box and whisker plot showing that the aqueous humor of naïve nAMD patients induces less TGF-β pathway activation in Lenti-X 293T cells. Cells were co-transfected with the plasmids pNL[NlucP/SBE], expressing NanoLuc luciferase under the control of three SMAD3 binding elements, and pGL4.54[luc2/TK], expressing the Firefly luciferase luc2 (used as transfection normalizer) under the control of the constitutive HSV-TK promoter. Transfected cells were treated with 10 μl of aqueous humor of the 20 nAMD patients, naïve or treated (once or twice, Treat.1 and Treat.2, respectively), or of the 20 control samples (Cntr.) described in Table 1 . Data are presented as the ratio between NanoLuc and luc2 luciferase activity, expressed as Relative Light Units (RLU) (number of replicates for each sample = 3). Asterisks indicate significant differences (**P

    Journal: Scientific Reports

    Article Title: TGF-β concentrations and activity are down-regulated in the aqueous humor of patients with neovascular age-related macular degeneration

    doi: 10.1038/s41598-018-26442-0

    Figure Lengend Snippet: Quantitative analysis of TGF-β pathway activation by aqueous humor of patients and controls. Box and whisker plot showing that the aqueous humor of naïve nAMD patients induces less TGF-β pathway activation in Lenti-X 293T cells. Cells were co-transfected with the plasmids pNL[NlucP/SBE], expressing NanoLuc luciferase under the control of three SMAD3 binding elements, and pGL4.54[luc2/TK], expressing the Firefly luciferase luc2 (used as transfection normalizer) under the control of the constitutive HSV-TK promoter. Transfected cells were treated with 10 μl of aqueous humor of the 20 nAMD patients, naïve or treated (once or twice, Treat.1 and Treat.2, respectively), or of the 20 control samples (Cntr.) described in Table 1 . Data are presented as the ratio between NanoLuc and luc2 luciferase activity, expressed as Relative Light Units (RLU) (number of replicates for each sample = 3). Asterisks indicate significant differences (**P

    Article Snippet: Analysis of TGF-β pathway activation The luciferase-based reporter system used to analyze activation of the TGF-β pathway consisted of Lenti-X 293T cells (#632180; Takara Bio, CA) transiently transfected with the plasmid pNL[NlucP/SBE] (Promega Corp., Madison, WI), carrying three copies of the SMAD binding element (SBE) (AGTATGTCTAGACTGA) which represent specific targets of for phospho-SMAD3 and drive the expression of a reporter gene encoding NanoLuc® Luciferase.

    Techniques: Activation Assay, Whisker Assay, Transfection, Expressing, Luciferase, Binding Assay, Activity Assay

    Functional analysis of ENPP1 in MCF7-luc cells. ( a ) Flow cytometric analysis of the SP fractions of MCF7-luc cells overexpressing ENPP1-MF or GFP as a control, in the presence and absence of Ko143. ( b ) Quantification of the SP fractions shown in a , determined as the difference between the level of Hoechst 33342 staining in the presence and absence of Ko143. Data are represented as the mean±s.d. of n =3 replicates. ( c ) Flow cytometric analysis showing the cell surface localization of ABCG2 in the indicated 293T co-transfectants. ( d ) Flow cytometric analyses of the cell surface localization of ABCG2 in MCF7-luc anti-miR-27b cells transfected with a control (shNC) or ENPP1-specific (shENPP1) shRNA. ( e ) Dose–response curves of docetaxel-treated MCF7-luc anti-miR-27b-DR cells transfected with shNC or shENPP1. Cell viability was normalized to that of the corresponding cells treated with dimethylsulphoxide (DMSO). The red dashed line indicates the IC 50 value. Data are represented as the mean±s.d. of n =3 replicates. ( f ) Proximity ligation assay using MCF7-luc anti-NC or MCF7-luc anti-miR-27b cells transiently expressing ABCG2-HA. Scale bar, 50 μm. ( g ) In vitro binding assay using C-terminally Flag-tagged GFP or C-terminally Myc- and Flag-tagged ENPP1 purified from 293T cells and C-terminally HA-tagged ABCG2 purified from Sf21 insect cell extracts.

    Journal: Nature Communications

    Article Title: Loss of microRNA-27b contributes to breast cancer stem cell generation by activating ENPP1

    doi: 10.1038/ncomms8318

    Figure Lengend Snippet: Functional analysis of ENPP1 in MCF7-luc cells. ( a ) Flow cytometric analysis of the SP fractions of MCF7-luc cells overexpressing ENPP1-MF or GFP as a control, in the presence and absence of Ko143. ( b ) Quantification of the SP fractions shown in a , determined as the difference between the level of Hoechst 33342 staining in the presence and absence of Ko143. Data are represented as the mean±s.d. of n =3 replicates. ( c ) Flow cytometric analysis showing the cell surface localization of ABCG2 in the indicated 293T co-transfectants. ( d ) Flow cytometric analyses of the cell surface localization of ABCG2 in MCF7-luc anti-miR-27b cells transfected with a control (shNC) or ENPP1-specific (shENPP1) shRNA. ( e ) Dose–response curves of docetaxel-treated MCF7-luc anti-miR-27b-DR cells transfected with shNC or shENPP1. Cell viability was normalized to that of the corresponding cells treated with dimethylsulphoxide (DMSO). The red dashed line indicates the IC 50 value. Data are represented as the mean±s.d. of n =3 replicates. ( f ) Proximity ligation assay using MCF7-luc anti-NC or MCF7-luc anti-miR-27b cells transiently expressing ABCG2-HA. Scale bar, 50 μm. ( g ) In vitro binding assay using C-terminally Flag-tagged GFP or C-terminally Myc- and Flag-tagged ENPP1 purified from 293T cells and C-terminally HA-tagged ABCG2 purified from Sf21 insect cell extracts.

    Article Snippet: Cell culture MCF7, ZR75-1 and MDA-MB-231 cells were obtained from American Type Culture Collection, and 293T cells were obtained from Clontech.

    Techniques: Functional Assay, Flow Cytometry, Staining, Transfection, shRNA, Proximity Ligation Assay, Expressing, In Vitro, Binding Assay, Purification

    Targeted transduction and delivery of PSCA antigen gene into dendritic cells (DCs) by DCLV-PSCA. (A) 293T cells were transfected transiently with plasmids FUW-Null (mock control, blue line) or FUW-PSCA (red line). Two days later, cells were collected and stained for PSCA expression analyzed by flow cytometry. 293T cells stained with the isotype antibody were included as a control (grey shade area). (B) 293T cells were transfected transiently with plasmids FUW-PSCA, SVGmu, and other necessary lentiviral packaging plasmids to produce DCLV-PSCA vectors. Fresh virus supernatant was used to transduce 293T cells (blue line) or 293T.hDC-SIGN cells (red line) with MOI = 10. PSCA expression was analyzed by flow cytometry 3 days post-transduction. (C) Bone marrow-derived DCs were transduced with a mock vector DC-LV-Null or DC-LV-PSCA vector. Five days later, CD11c and PSCA expression were assessed by flow cytometric analysis. All experiments were repeated three times and the representative data is shown.

    Journal: PLoS ONE

    Article Title: Dendritic Cell-Directed Vaccination with a Lentivector Encoding PSCA for Prostate Cancer in Mice

    doi: 10.1371/journal.pone.0048866

    Figure Lengend Snippet: Targeted transduction and delivery of PSCA antigen gene into dendritic cells (DCs) by DCLV-PSCA. (A) 293T cells were transfected transiently with plasmids FUW-Null (mock control, blue line) or FUW-PSCA (red line). Two days later, cells were collected and stained for PSCA expression analyzed by flow cytometry. 293T cells stained with the isotype antibody were included as a control (grey shade area). (B) 293T cells were transfected transiently with plasmids FUW-PSCA, SVGmu, and other necessary lentiviral packaging plasmids to produce DCLV-PSCA vectors. Fresh virus supernatant was used to transduce 293T cells (blue line) or 293T.hDC-SIGN cells (red line) with MOI = 10. PSCA expression was analyzed by flow cytometry 3 days post-transduction. (C) Bone marrow-derived DCs were transduced with a mock vector DC-LV-Null or DC-LV-PSCA vector. Five days later, CD11c and PSCA expression were assessed by flow cytometric analysis. All experiments were repeated three times and the representative data is shown.

    Article Snippet: Briefly, 293T cells cultured in a 15-cm tissue culture plate (BD Biosciences, San Jose, CA, USA) were transfected via a standard calcium phosphate precipitation method with the following plasmids: the lentiviral backbone plasmid FUW-PSCA (37.5 µg, ), the plasmid encoding the mutant Sindbis virus glycoprotein (SVGmu, 18.75 µg, ), and the packaging plasmids (pMDLg/pRRE and pRSV-Rev, 18.75 µg each).

    Techniques: Transduction, Transfection, Staining, Expressing, Flow Cytometry, Cytometry, Derivative Assay, Plasmid Preparation

    PSCA-specific T cell response after a single dose of in vivo immunization with DCLV-PSCA. (A) Male C57BL/6 mice were immunized with 6×10 7 TU of DCLV-PSCA through different administration routes: intraperitoneal space (i.p.), subcutaneous area (s.c.), intramuscular area (i.m.), footpad (f.p.), or intradermal (the base of tail, i.d.). One immunization group was included as a negative control. Two weeks after immunization, splenocytes from mice were harvested and analyzed for the presence of PSCA-specific CD8 + T cells by restimulating splenocytes with a PSCA peptide (PSCA 83-91 ), followed by intracellular staining for IFN-γ and surface staining for CD8. Percentage of IFN-γ-secreting CD8 + T cells is indicated. (B) Statistical comparison of immunization elicited by administration of DCLV-PSCA among different administration routes. (C) Male C57BL/6 mice were immunized with different doses of DCLV-PSCA vectors (0, 2, 10, 40 and 80 million TU) at the base of tail. Two weeks post-vaccination, PSCA-specific CD8 + T cells from the spleen were analyzed by restimulating with the peptide PSCA 83-91 , followed by intracellular staining for IFN-γ. (D) Production of PSCA-specific IFN-γ-secreting cells from both spleen (SP) and inguinal lymph node (LN) was evaluated by restimulation with the PSCA 83-91 peptide, followed by ELISPOT analysis for IFN-γ. (E) Production of PSCA-specific IL-2 from splenocytes (with CD8 + T cells depleted) was measured by restimulation with 293T cell lysate transfected to express PSCA, followed by the ELISPOT analysis for IL-2. (**: P

    Journal: PLoS ONE

    Article Title: Dendritic Cell-Directed Vaccination with a Lentivector Encoding PSCA for Prostate Cancer in Mice

    doi: 10.1371/journal.pone.0048866

    Figure Lengend Snippet: PSCA-specific T cell response after a single dose of in vivo immunization with DCLV-PSCA. (A) Male C57BL/6 mice were immunized with 6×10 7 TU of DCLV-PSCA through different administration routes: intraperitoneal space (i.p.), subcutaneous area (s.c.), intramuscular area (i.m.), footpad (f.p.), or intradermal (the base of tail, i.d.). One immunization group was included as a negative control. Two weeks after immunization, splenocytes from mice were harvested and analyzed for the presence of PSCA-specific CD8 + T cells by restimulating splenocytes with a PSCA peptide (PSCA 83-91 ), followed by intracellular staining for IFN-γ and surface staining for CD8. Percentage of IFN-γ-secreting CD8 + T cells is indicated. (B) Statistical comparison of immunization elicited by administration of DCLV-PSCA among different administration routes. (C) Male C57BL/6 mice were immunized with different doses of DCLV-PSCA vectors (0, 2, 10, 40 and 80 million TU) at the base of tail. Two weeks post-vaccination, PSCA-specific CD8 + T cells from the spleen were analyzed by restimulating with the peptide PSCA 83-91 , followed by intracellular staining for IFN-γ. (D) Production of PSCA-specific IFN-γ-secreting cells from both spleen (SP) and inguinal lymph node (LN) was evaluated by restimulation with the PSCA 83-91 peptide, followed by ELISPOT analysis for IFN-γ. (E) Production of PSCA-specific IL-2 from splenocytes (with CD8 + T cells depleted) was measured by restimulation with 293T cell lysate transfected to express PSCA, followed by the ELISPOT analysis for IL-2. (**: P

    Article Snippet: Briefly, 293T cells cultured in a 15-cm tissue culture plate (BD Biosciences, San Jose, CA, USA) were transfected via a standard calcium phosphate precipitation method with the following plasmids: the lentiviral backbone plasmid FUW-PSCA (37.5 µg, ), the plasmid encoding the mutant Sindbis virus glycoprotein (SVGmu, 18.75 µg, ), and the packaging plasmids (pMDLg/pRRE and pRSV-Rev, 18.75 µg each).

    Techniques: In Vivo, Mouse Assay, Negative Control, Staining, Enzyme-linked Immunospot, Transfection

    Identification and analysis of global Ca 2+ signals from multiple, individual cells. ( A ) The panel shows a Gaussian blurred image of resting fluorescence of Cal-520 loaded HEK-293 WT cells, averaged over ~100 frames prior to stimulation. Imaging was performed by wide-field epifluorescence microscopy at low (10x) magnification. Scale bar = 20 µm ( B ) Binarized image of A, with yellow circles illustrating the identification of individual cells performed by the ‘generate ROIs’ operation in Flika. ( C ) ROIs overlaid on a fluorescence ratio (F/F 0 ) image from the same image sequence as A, at a time when nearly all cells exhibited a large increase in cytosolic Ca 2+ following stimulation by the muscarinic receptor agonist carbachol (CCH). ( D ) Fluorescence traces (ΔF/F 0 ) corresponding to the average change in fluorescence over time from within each ROI shown in C (x axis labels denote frames, acquired at 100 ms intervals).

    Journal: Biochimica et biophysica acta. Molecular cell research

    Article Title: Applications of FLIKA, a Python-based image processing and analysis platform, for studying local events of cellular calcium signaling

    doi: 10.1016/j.bbamcr.2018.11.012

    Figure Lengend Snippet: Identification and analysis of global Ca 2+ signals from multiple, individual cells. ( A ) The panel shows a Gaussian blurred image of resting fluorescence of Cal-520 loaded HEK-293 WT cells, averaged over ~100 frames prior to stimulation. Imaging was performed by wide-field epifluorescence microscopy at low (10x) magnification. Scale bar = 20 µm ( B ) Binarized image of A, with yellow circles illustrating the identification of individual cells performed by the ‘generate ROIs’ operation in Flika. ( C ) ROIs overlaid on a fluorescence ratio (F/F 0 ) image from the same image sequence as A, at a time when nearly all cells exhibited a large increase in cytosolic Ca 2+ following stimulation by the muscarinic receptor agonist carbachol (CCH). ( D ) Fluorescence traces (ΔF/F 0 ) corresponding to the average change in fluorescence over time from within each ROI shown in C (x axis labels denote frames, acquired at 100 ms intervals).

    Article Snippet: Cell Culture and loading HEK-293 cell lines were cultured in EMEM (ATCC #30–2003) supplemented with 10% FBS.

    Techniques: Fluorescence, Imaging, Epifluorescence Microscopy, Sequencing

    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

    Simiate is vital to cells. A) Chariot reagent shuttled rbαSimiate (0.5µg) detects FLAG-Simiate in transfected HEK-293 cells. The nuclei are visualized by DAPI staining. The arrows indicate clusters of rbαSimiate and FLAG-Simiate immunofluorescence inside the nucleus. B-D) Apoptosis in rbαSimiate and αrbAlexa568 treated HEK-293 cells. B) 0.25µg rbαSimiate, C) 1.0µg rbαSimiate and D) 1.0µg αrbAlexa568 as negative control. TUNEL staining (in green) served to identify apoptotic cells, while nuclear speckles were outlined with SC35 (in red). E) Quantification of the amount of endogenous Simiate epitopes not targeted by antibodies. F) Quantification of apoptotic cells. The increase in the percentage of TUNEL positive cells after rbαSimiate treatment is extremely significant (Chi 2 : p

    Journal: PLoS ONE

    Article Title: Identification and Characterisation of Simiate, a Novel Protein Linked to the Fragile X Syndrome

    doi: 10.1371/journal.pone.0083007

    Figure Lengend Snippet: Simiate is vital to cells. A) Chariot reagent shuttled rbαSimiate (0.5µg) detects FLAG-Simiate in transfected HEK-293 cells. The nuclei are visualized by DAPI staining. The arrows indicate clusters of rbαSimiate and FLAG-Simiate immunofluorescence inside the nucleus. B-D) Apoptosis in rbαSimiate and αrbAlexa568 treated HEK-293 cells. B) 0.25µg rbαSimiate, C) 1.0µg rbαSimiate and D) 1.0µg αrbAlexa568 as negative control. TUNEL staining (in green) served to identify apoptotic cells, while nuclear speckles were outlined with SC35 (in red). E) Quantification of the amount of endogenous Simiate epitopes not targeted by antibodies. F) Quantification of apoptotic cells. The increase in the percentage of TUNEL positive cells after rbαSimiate treatment is extremely significant (Chi 2 : p

    Article Snippet: Representative immunofluorescence stainings of HEK-293 cells, which expressed a FLAG-Simiate construct for 24h (A) and 48h (B) or FLAG alone for 48h (C).

    Techniques: Transfection, Staining, Immunofluorescence, Negative Control, TUNEL Assay

    Simiate in the nucleus. A) A picture showing the nucleus of a Purkinje cell in the Cerebellum of an adult mouse brain along with a 3D reconstruction of Simiate and heterochromatin clusters. Please refer to the red and yellow arrows for orientation. B) Four virtual slices taken through the same nucleus. The blue arrows indicate areas displaying a colocalisation of Simiate and heterochromatin. C) Another Purkinje cell was treated as in A) and additionally labelled with SC35 to visualize nuclear speckles. D) Four nuclei from the CA1 pyramidal cell layer of the Hippocampus with different amounts of Simiate. The absence of Simiate coincides with the absence of nuclear speckles. Nuclear speckles of different sizes are labelled with blue arrows to illustrate the colocalisation of Simiate and SC35. For convenience, dotted circles are used to outline nuclei in all graphs not displaying DAPI. E) HEK-293 cells also express endogenous Simiate. The somata is indicated by a dotted line.

    Journal: PLoS ONE

    Article Title: Identification and Characterisation of Simiate, a Novel Protein Linked to the Fragile X Syndrome

    doi: 10.1371/journal.pone.0083007

    Figure Lengend Snippet: Simiate in the nucleus. A) A picture showing the nucleus of a Purkinje cell in the Cerebellum of an adult mouse brain along with a 3D reconstruction of Simiate and heterochromatin clusters. Please refer to the red and yellow arrows for orientation. B) Four virtual slices taken through the same nucleus. The blue arrows indicate areas displaying a colocalisation of Simiate and heterochromatin. C) Another Purkinje cell was treated as in A) and additionally labelled with SC35 to visualize nuclear speckles. D) Four nuclei from the CA1 pyramidal cell layer of the Hippocampus with different amounts of Simiate. The absence of Simiate coincides with the absence of nuclear speckles. Nuclear speckles of different sizes are labelled with blue arrows to illustrate the colocalisation of Simiate and SC35. For convenience, dotted circles are used to outline nuclei in all graphs not displaying DAPI. E) HEK-293 cells also express endogenous Simiate. The somata is indicated by a dotted line.

    Article Snippet: Representative immunofluorescence stainings of HEK-293 cells, which expressed a FLAG-Simiate construct for 24h (A) and 48h (B) or FLAG alone for 48h (C).

    Techniques:

    Activation of JNK by v-Crk. ( A ) JNK activities were measured by in vitro kinase assay using GST-c-Jun (1–79) as a substrate with lysates from NIH 3T3 cells and v-Crk NIH 3T3 cells after 24 h serum starvation followed by either serum stimulation for 20 min or not. UV-irradiated NIH 3T3 cells were used as a positive control. The activities of MAPK were also measured in vitro using myelin basic protein (MBP) as a substrate. The levels of endogenous JNK1 and ERKs in total cell lysates were examined by immunoblotting (IB) using anti-JNK1 and mixture of anti-ERK1 and ERK2 antibodies (Santa Cruz Biotechnology). ( B ) JNK activities were measured in NIH 3T3 and v-Crk NIH 3T3 at different growth stages. Cell densities were roughly determined by the percentage of the confluence as 40% (lanes 1 and 6), 60% (lanes 2 and 7), 80% (lanes 3 and 8), 100% (lanes 4 and 9), and 24 h after 100% (lanes 5 and 10). ( C Upper ) JNK activities were assayed in chicken embryo fibroblasts (CEF) uninfected (lane 1) and infected with avian CT10 virus encoding v-Crk (lane 2). Lane 3, CEF with UV irradiation as a control. ( Lower ) The endogenous levels of JNKs were determined by immunoblotting using anti-JNK1 and anti-JNK2 antibodies. Lanes: 1, CEF; 2, CEF infected with CT10 virus; 3, CEF with UV irradiation. HA-JNK1 expressed in mammalian cells (lane 4) and bacterially expressed His-JNK2 (lane 5) were used as positive controls. ( D ) JNK activities in human embryo kidney cells 293T that were transiently coexpressing HA-JNK1 with v-Crk, c-Crk-I, or c-Crk-II.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Downstream of Crk adaptor signaling pathway: Activation of Jun kinase by v-Crk through the guanine nucleotide exchange protein C3G

    doi:

    Figure Lengend Snippet: Activation of JNK by v-Crk. ( A ) JNK activities were measured by in vitro kinase assay using GST-c-Jun (1–79) as a substrate with lysates from NIH 3T3 cells and v-Crk NIH 3T3 cells after 24 h serum starvation followed by either serum stimulation for 20 min or not. UV-irradiated NIH 3T3 cells were used as a positive control. The activities of MAPK were also measured in vitro using myelin basic protein (MBP) as a substrate. The levels of endogenous JNK1 and ERKs in total cell lysates were examined by immunoblotting (IB) using anti-JNK1 and mixture of anti-ERK1 and ERK2 antibodies (Santa Cruz Biotechnology). ( B ) JNK activities were measured in NIH 3T3 and v-Crk NIH 3T3 at different growth stages. Cell densities were roughly determined by the percentage of the confluence as 40% (lanes 1 and 6), 60% (lanes 2 and 7), 80% (lanes 3 and 8), 100% (lanes 4 and 9), and 24 h after 100% (lanes 5 and 10). ( C Upper ) JNK activities were assayed in chicken embryo fibroblasts (CEF) uninfected (lane 1) and infected with avian CT10 virus encoding v-Crk (lane 2). Lane 3, CEF with UV irradiation as a control. ( Lower ) The endogenous levels of JNKs were determined by immunoblotting using anti-JNK1 and anti-JNK2 antibodies. Lanes: 1, CEF; 2, CEF infected with CT10 virus; 3, CEF with UV irradiation. HA-JNK1 expressed in mammalian cells (lane 4) and bacterially expressed His-JNK2 (lane 5) were used as positive controls. ( D ) JNK activities in human embryo kidney cells 293T that were transiently coexpressing HA-JNK1 with v-Crk, c-Crk-I, or c-Crk-II.

    Article Snippet: A total of 1.5 μg of pcDNA3-HA-JNK1 (HA, hemagglutinin); 5 μg of pMex-v-Crk, pMex-hCrk-I, pMex-hCrk-II; and 3 μg of pCAGGS-C3G, pCAGGS-C3G-F, pEBG-Sek1, pEBG-Sek1-KR, pcDNA3-HA-Sek-AL DNAs were used for transfection of 293T cells cultured in a 60-mm-diameter dish by a modified calcium phosphate transfection system (Stratagene).

    Techniques: Activation Assay, In Vitro, Kinase Assay, Irradiation, Positive Control, Infection

    Analysis of proteolytic activity of viral VP4 protein. (A) 293T cells were transfected with the recombinant wild-type A-segment plasmid or the Ala or Asp substituted A-segment plasmids at sites pSer538, pTyr611 and pThr674 within VP4. At 24 h post-transfection, cell samples were harvested and electrophoresed on 12% SDS-PAGE gels for Western blot analysis with mAbs specific to VP3 and VP4 proteins. GAPDH was used as a loading control. (B) Recombinant plasmids used in (A) were translated with the TNT T7 Quick Coupled Transcription/Translation System, and expressed proteins were detected with mAbs specific for VP3 and VP4 proteins. (C) Analysis of co-localization between IBDV-encoding proteins within segment A. 293T cells were transfected with the IBDV A-segment mutant with the single dephospho- and phospho-mimicking VP4 gene. Wild-type IBDV A-segment transfected cells were used as a positive control. At 24 h post-transfection, the cells were fixed and probed with chicken anti-VP2 pAb, mouse anti-VP3 mAb and rabbit anti-VP4 pAb followed by FITC-conjugated goat anti-chicken IgG (green), Alexa Fluor 647 donkey anti-mouse IgG (blue) and Alexa Fluor 546 donkey ant-rabbit IgG (red). Nuclei were counterstained with DAPI (grey). The cells were observed with a laser Zeiss LSM510 laser confocal microscope. Cells transfected with the A segment with the Tyr611Asp and Thr674 Ala/Asp substitutions revealed co-localization between the IBDV-encoded proteins.

    Journal: PLoS ONE

    Article Title: Avibirnavirus VP4 Protein Is a Phosphoprotein and Partially Contributes to the Cleavage of Intermediate Precursor VP4-VP3 Polyprotein

    doi: 10.1371/journal.pone.0128828

    Figure Lengend Snippet: Analysis of proteolytic activity of viral VP4 protein. (A) 293T cells were transfected with the recombinant wild-type A-segment plasmid or the Ala or Asp substituted A-segment plasmids at sites pSer538, pTyr611 and pThr674 within VP4. At 24 h post-transfection, cell samples were harvested and electrophoresed on 12% SDS-PAGE gels for Western blot analysis with mAbs specific to VP3 and VP4 proteins. GAPDH was used as a loading control. (B) Recombinant plasmids used in (A) were translated with the TNT T7 Quick Coupled Transcription/Translation System, and expressed proteins were detected with mAbs specific for VP3 and VP4 proteins. (C) Analysis of co-localization between IBDV-encoding proteins within segment A. 293T cells were transfected with the IBDV A-segment mutant with the single dephospho- and phospho-mimicking VP4 gene. Wild-type IBDV A-segment transfected cells were used as a positive control. At 24 h post-transfection, the cells were fixed and probed with chicken anti-VP2 pAb, mouse anti-VP3 mAb and rabbit anti-VP4 pAb followed by FITC-conjugated goat anti-chicken IgG (green), Alexa Fluor 647 donkey anti-mouse IgG (blue) and Alexa Fluor 546 donkey ant-rabbit IgG (red). Nuclei were counterstained with DAPI (grey). The cells were observed with a laser Zeiss LSM510 laser confocal microscope. Cells transfected with the A segment with the Tyr611Asp and Thr674 Ala/Asp substitutions revealed co-localization between the IBDV-encoded proteins.

    Article Snippet: DF-1 cells or 293T cells were seeded in 96-well plates (Corning, New York, NY) or 35-mm glass bottom dishes (Shengyou Biotechnology, China), infected with IBDV or transfected with wild-type A segment, with wild-type VP4 or Ala/Asp VP4 mutants.

    Techniques: Activity Assay, Transfection, Recombinant, Plasmid Preparation, SDS Page, Western Blot, Mutagenesis, Positive Control, Microscopy