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  • 99
    ATCC hek 293 cells
    Optogenetic activation of SthK channels by various PACs in cell lines. a Illustration of the split-PAC-K and fused-PAC-K construct design for SthK co-expression with PACs. b Photocurrents elicited by different split-PAC-K variants after 10 ms exposure to a 470 nm light pulse. Scale bars: 10 s, 0.5 nA. c Normalized integrals of photocurrents as a function of the intensity of a 10 ms light pulse. Arrows denote the EC 50 for current activation by light ( n = 3 cells, two cultures). d Combined whole-cell patch-clamp (black) and optical recording (gray) of a <t>HEK</t> 293 cell expressing fused-bPAC-K and a fluorescent cAMP sensor. bPAC was excited with two 1 ms light flashes (470 nm). The dotted line indicates the zero current level. Scale bars: 20 s, 20 pA (upper trace); 20 s, 2000 aU (lower trace). e Comparison of photocurrent amplitudes generated by bPAC or TpPAC and SthK as split or fused constructs. f Current density comparison at saturating photon exposures ( ** p
    Hek 293 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 6330 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore 293t cells
    Optogenetic activation of SthK channels by various PACs in cell lines. a Illustration of the split-PAC-K and fused-PAC-K construct design for SthK co-expression with PACs. b Photocurrents elicited by different split-PAC-K variants after 10 ms exposure to a 470 nm light pulse. Scale bars: 10 s, 0.5 nA. c Normalized integrals of photocurrents as a function of the intensity of a 10 ms light pulse. Arrows denote the EC 50 for current activation by light ( n = 3 cells, two cultures). d Combined whole-cell patch-clamp (black) and optical recording (gray) of a <t>HEK</t> 293 cell expressing fused-bPAC-K and a fluorescent cAMP sensor. bPAC was excited with two 1 ms light flashes (470 nm). The dotted line indicates the zero current level. Scale bars: 20 s, 20 pA (upper trace); 20 s, 2000 aU (lower trace). e Comparison of photocurrent amplitudes generated by bPAC or TpPAC and SthK as split or fused constructs. f Current density comparison at saturating photon exposures ( ** p
    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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    Thermo Fisher hek 293 cells
    Optogenetic activation of SthK channels by various PACs in cell lines. a Illustration of the split-PAC-K and fused-PAC-K construct design for SthK co-expression with PACs. b Photocurrents elicited by different split-PAC-K variants after 10 ms exposure to a 470 nm light pulse. Scale bars: 10 s, 0.5 nA. c Normalized integrals of photocurrents as a function of the intensity of a 10 ms light pulse. Arrows denote the EC 50 for current activation by light ( n = 3 cells, two cultures). d Combined whole-cell patch-clamp (black) and optical recording (gray) of a <t>HEK</t> 293 cell expressing fused-bPAC-K and a fluorescent cAMP sensor. bPAC was excited with two 1 ms light flashes (470 nm). The dotted line indicates the zero current level. Scale bars: 20 s, 20 pA (upper trace); 20 s, 2000 aU (lower trace). e Comparison of photocurrent amplitudes generated by bPAC or TpPAC and SthK as split or fused constructs. f Current density comparison at saturating photon exposures ( ** p
    Hek 293 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12989 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC hek 293t cells
    The PPI induced by EBNA1 and PAX5 is required for EBNA1/ oriP -mediated binding and transcription. (A) YN-FPAX5, yellow fluorescent protein (YFP)-C terminus-fused (YC)-FEBNA1 or its deletion mutants, or YC-FNCL control was cotransfected into <t>293T</t> cells. The resulting protein expression of each BiFC plasmid was identified by Western blotting (left). YN-FPAX5- and YC-FEBNA1-derived plasmids were cotransfected into 293T cells, and the BiFC images were analyzed by immunofluorescence confocal microscopy (middle). Schematic diagrams of FPAX5 and its deletion mutants are shown. The results obtained from the BiFC assays were summarized and presented (right). Y, positive fluorescence signal; N, negative fluorescence signal. (B) The FEBNA1 expression plasmid or each DBD-derived deletion mutant was transfected into BAJB cells. Flag-epitope (M2)-conjugated agarose was used to precipitate either FEBNA1 or its mutant derivatives. The amounts of M2-precipitated FEBNA1 or its mutants or coprecipitated endogenous PAX5 were identified by Western blotting. Input (2%) of each protein from the lysate is shown. (C) The schematic diagrams of FPAX5 and its deletion mutants are shown. BJAB cells cotransfected with the expression plasmids of EBNA1 and FPAX5 or its mutant derivatives were used to perform M2 agarose-mediated co-IP assays. The immune blots for the indicated proteins are shown with 2% of input. (D) BJAB cells cotransfected with the expression plasmids of FPAX5 or two pair domain (PD) mutants, V 26 G and P 80 R, and that of EBNA1 were used to perform M2 agarose-mediated co-IP assays. The immune blots for EBNA1, FPAX5, p300, H3K4Me2, and H3K4Me3 are shown with 2% input as the loading control.
    Hek 293t Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 5118 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hek293t cells
    The PPI induced by EBNA1 and PAX5 is required for EBNA1/ oriP -mediated binding and transcription. (A) YN-FPAX5, yellow fluorescent protein (YFP)-C terminus-fused (YC)-FEBNA1 or its deletion mutants, or YC-FNCL control was cotransfected into <t>293T</t> cells. The resulting protein expression of each BiFC plasmid was identified by Western blotting (left). YN-FPAX5- and YC-FEBNA1-derived plasmids were cotransfected into 293T cells, and the BiFC images were analyzed by immunofluorescence confocal microscopy (middle). Schematic diagrams of FPAX5 and its deletion mutants are shown. The results obtained from the BiFC assays were summarized and presented (right). Y, positive fluorescence signal; N, negative fluorescence signal. (B) The FEBNA1 expression plasmid or each DBD-derived deletion mutant was transfected into BAJB cells. Flag-epitope (M2)-conjugated agarose was used to precipitate either FEBNA1 or its mutant derivatives. The amounts of M2-precipitated FEBNA1 or its mutants or coprecipitated endogenous PAX5 were identified by Western blotting. Input (2%) of each protein from the lysate is shown. (C) The schematic diagrams of FPAX5 and its deletion mutants are shown. BJAB cells cotransfected with the expression plasmids of EBNA1 and FPAX5 or its mutant derivatives were used to perform M2 agarose-mediated co-IP assays. The immune blots for the indicated proteins are shown with 2% of input. (D) BJAB cells cotransfected with the expression plasmids of FPAX5 or two pair domain (PD) mutants, V 26 G and P 80 R, and that of EBNA1 were used to perform M2 agarose-mediated co-IP assays. The immune blots for EBNA1, FPAX5, p300, H3K4Me2, and H3K4Me3 are shown with 2% input as the loading control.
    Hek293t Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 35596 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher human embryonic kidney hek 293 cells
    The PPI induced by EBNA1 and PAX5 is required for EBNA1/ oriP -mediated binding and transcription. (A) YN-FPAX5, yellow fluorescent protein (YFP)-C terminus-fused (YC)-FEBNA1 or its deletion mutants, or YC-FNCL control was cotransfected into <t>293T</t> cells. The resulting protein expression of each BiFC plasmid was identified by Western blotting (left). YN-FPAX5- and YC-FEBNA1-derived plasmids were cotransfected into 293T cells, and the BiFC images were analyzed by immunofluorescence confocal microscopy (middle). Schematic diagrams of FPAX5 and its deletion mutants are shown. The results obtained from the BiFC assays were summarized and presented (right). Y, positive fluorescence signal; N, negative fluorescence signal. (B) The FEBNA1 expression plasmid or each DBD-derived deletion mutant was transfected into BAJB cells. Flag-epitope (M2)-conjugated agarose was used to precipitate either FEBNA1 or its mutant derivatives. The amounts of M2-precipitated FEBNA1 or its mutants or coprecipitated endogenous PAX5 were identified by Western blotting. Input (2%) of each protein from the lysate is shown. (C) The schematic diagrams of FPAX5 and its deletion mutants are shown. BJAB cells cotransfected with the expression plasmids of EBNA1 and FPAX5 or its mutant derivatives were used to perform M2 agarose-mediated co-IP assays. The immune blots for the indicated proteins are shown with 2% of input. (D) BJAB cells cotransfected with the expression plasmids of FPAX5 or two pair domain (PD) mutants, V 26 G and P 80 R, and that of EBNA1 were used to perform M2 agarose-mediated co-IP assays. The immune blots for EBNA1, FPAX5, p300, H3K4Me2, and H3K4Me3 are shown with 2% input as the loading control.
    Human Embryonic Kidney Hek 293 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1827 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC hek293t cells
    The PPI induced by EBNA1 and PAX5 is required for EBNA1/ oriP -mediated binding and transcription. (A) YN-FPAX5, yellow fluorescent protein (YFP)-C terminus-fused (YC)-FEBNA1 or its deletion mutants, or YC-FNCL control was cotransfected into <t>293T</t> cells. The resulting protein expression of each BiFC plasmid was identified by Western blotting (left). YN-FPAX5- and YC-FEBNA1-derived plasmids were cotransfected into 293T cells, and the BiFC images were analyzed by immunofluorescence confocal microscopy (middle). Schematic diagrams of FPAX5 and its deletion mutants are shown. The results obtained from the BiFC assays were summarized and presented (right). Y, positive fluorescence signal; N, negative fluorescence signal. (B) The FEBNA1 expression plasmid or each DBD-derived deletion mutant was transfected into BAJB cells. Flag-epitope (M2)-conjugated agarose was used to precipitate either FEBNA1 or its mutant derivatives. The amounts of M2-precipitated FEBNA1 or its mutants or coprecipitated endogenous PAX5 were identified by Western blotting. Input (2%) of each protein from the lysate is shown. (C) The schematic diagrams of FPAX5 and its deletion mutants are shown. BJAB cells cotransfected with the expression plasmids of EBNA1 and FPAX5 or its mutant derivatives were used to perform M2 agarose-mediated co-IP assays. The immune blots for the indicated proteins are shown with 2% of input. (D) BJAB cells cotransfected with the expression plasmids of FPAX5 or two pair domain (PD) mutants, V 26 G and P 80 R, and that of EBNA1 were used to perform M2 agarose-mediated co-IP assays. The immune blots for EBNA1, FPAX5, p300, H3K4Me2, and H3K4Me3 are shown with 2% input as the loading control.
    Hek293t Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 14202 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Mirus Bio hek 293 cells
    TPβ Trp 334 is a major determinant for CCT7 interaction. (A) Schematic representation of TPβ and TPα C-termini. The TPβ region important for CCT7 interaction is underlined in blue. The green, linked amino acids represent residues showing similarity or identity between TPβ and TPα. Shown in red are TPβ Trp 334 and TPα Gln 333 . (B) Lysates of <t>HEK</t> 293 cells transiently expressing the indicated HA-TPβ constructs alone or with CCT7-MYC were immunoprecipitated with a HA-specific monoclonal antibody and analyzed by immunoblotting with MYC- and HA-specific HRP-conjugated antibodies. HA-TPβ 328, 337, 344, and 351 indicate that a stop codon was introduced after the corresponding amino acids. Densitometry on Western blots of four independent experiments are reported in the graphic and expressed as a relative ratio of CCT7 co-IP on receptors IP. (C) Lysates of HEK 293 cells transiently expressing HA-TPβ, HA-TPβ W334Q, HA-TPα, or HA-TPα Q333W alone or with CCT7-MYC were immunoprecipitated with a HA-specific monoclonal antibody and analyzed by immunoblotting with MYC- and HA-specific HRP-conjugated antibodies. Densitometry on Western blots of five independent experiments are reported in the graphic and expressed as a relative ratio of CCT7 co-IP on receptors’ IP. IB, immunoblotting; IP, immunoprecipitation.
    Hek 293 Cells, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 92/100, based on 428 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc 293t cells
    Deletion of the first cysteine triplet of ASP reduces multimer formation and autophagosome signals. (A) Schematic representation of the different domains of ASP and the deletion mutant of the first cysteine triplet ( 10 CCC 12 ). (B to D) COS-7 cells were transfected with expression vectors for wild-type or cysteine triplet-deleted ASP versus pcDNA3.1 (empty vector). After 48 h of transfection, using anti-Myc antibodies, cells were analyzed by flow cytometry (B), Western blotting (C), and confocal microscopy (60× objective with numerical aperture of 1.4) (D). In panel C, high-molecular-weight (including 200-kDa multimers) versus monomeric 20-kDa ASP signals are indicated. Cells analyzed by confocal microscopy were also stained for their nuclei with Hoechst (D). (E) <t>293T</t> cells were transfected with expression vectors for optimized (opt) NL4.3 ASP WT, ASPΔN1–15, ASPΔN1–30, and ρCCC versus pcDNA3.1 (mock) and analyzed by Western blotting using anti-ASP 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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    TaKaRa 293t cells
    Deletion of the first cysteine triplet of ASP reduces multimer formation and autophagosome signals. (A) Schematic representation of the different domains of ASP and the deletion mutant of the first cysteine triplet ( 10 CCC 12 ). (B to D) COS-7 cells were transfected with expression vectors for wild-type or cysteine triplet-deleted ASP versus pcDNA3.1 (empty vector). After 48 h of transfection, using anti-Myc antibodies, cells were analyzed by flow cytometry (B), Western blotting (C), and confocal microscopy (60× objective with numerical aperture of 1.4) (D). In panel C, high-molecular-weight (including 200-kDa multimers) versus monomeric 20-kDa ASP signals are indicated. Cells analyzed by confocal microscopy were also stained for their nuclei with Hoechst (D). (E) <t>293T</t> cells were transfected with expression vectors for optimized (opt) NL4.3 ASP WT, ASPΔN1–15, ASPΔN1–30, and ρCCC versus pcDNA3.1 (mock) and analyzed by Western blotting using anti-ASP antibodies.
    293t Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 2066 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson 293t cells
    VPg-primed RNA synthesis in the presence of NoV RdRp. (A) Western blot analysis of VPg immunoprecipitated from HEK <t>293T</t> cells expressing the WT (NoV Rp) or mutant RdRp (Rp GAA ) and WT VPg (VPg) proteins. A plus symbol denotes addition of the corresponding plasmid. The samples were resolved on a 4-to-12% denaturing PAGE gel prior to Western blotting with anti-HA antibodies. The relevant masses from the standards are shown to the left of the Western blot image. The identities of the molecules are shown to the right of the Western blot image. VPg-RNA, VPg linked to RNA; VPg, free VPg. (B) Western blotting results for the total RNAs purified from HEK 293T cells that expressed either the WT or mutant RdRp and VPg proteins. The RNAs were electrophoresed in a 4-to-12% denaturing PAGE gel. A plus symbol in the rows labeled RNase A or RQ1 DNase denotes a 30-min treatment with a 10-μg final concentration or 5 units, respectively, of the appropriate enzyme prior to loading the sample for SDS-PAGE. VPg-RNA, VPg linked to RNA; VPg, free VPg. Lane 2 contains free VPg generated from cells transfected to produce only VPg and was loaded to serve as a molecular mass marker.
    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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    Addgene inc hek 293t cells
    TACI co-localizes with MyD88 and TRAF6 in the late endosomal compartment. (A) mCherry labeled TACI-S, TACI-L or TACI-S194X isoform transfected <t>HEK-293T</t> cells, were stained with rabbit Ab to MyD88 or TRAF6; nuclei were stained with DAPI. Merged images show that each TACI isoform co-stains with MyD88 (A) and TRAF6 (B) , but colocalization is absent for the S194X mutant. (C) HEK-293T cells transfected with mCherry labeled TACI-L and TACI-S were incubated with Alexa Fluor 647-conjugated transferrin (40 μg/ml Tfn-647) at 37°C for 5 min fixed with 4% paraformaldehyde. Merged images show that both TACI-L and TACI-S co-localize with Tfn-647 (white arrows). (D) In other experiments, transfected HEK-293T cells were stained with mAb Rab7 as a marker of late endosomes, also showing co-localization. Samples were examined by Leica SP5 DMI confocal microscopy, acquiring 3 different xy planes with 63×/1.4 NA objective lenses (Carl Zeiss) with optimal z spacing (~0.016 μm). Images were processed using Adobe Photoshop.
    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 293t cells
    Expression of MetYPCP of HEV ORF1 decreased signal transducer and activator of transcription protein (STAT)1 nuclear translocation upon IFN-β treatment. ( a ) Here, <t>293T</t> cells were transfected with a pCINeo-3xFLAG empty vector or a plasmid coding for MetYPCP, PCP or MV-V fused to a 3xFLAG tag. Twenty-four hours post-transfection, cells were stimulated or not for 30 min with 1000 IU/mL of IFN-β. Cells were then washed, fixed and stained with primary antibodies raised against STAT1 and FLAG, followed by fluorescent dye-conjugated secondary antibodies. Intracellular localization of 4,6-diamidine-2-phenylindole dihydrochloride (DAPI)-stained nuclei (blue), FLAG (green) and STAT1 (red) was visualized by microscopy (magnification, 630×). Scale bars: 10 μm. Cells showing diffuse cytoplasmic/nuclear localisation of STAT1 upon IFN-β treatment are indicated by arrows. ( b ) STAT1 localization was visualized after immunostaining as described in ( a ) in 293T cells transfected with a pCINeo-3xFLAG empty vector or a plasmid coding for MetYPCP, PCP or MV-V fused to a FLAG tag. For each condition, STAT1 localisation (predominant nuclear localisation or diffuse localisation within the cytoplasm and nucleus) was determined in 70 to 172 cells expressing the corresponding FLAG-tagged protein (except for the EV control, for which 356 to 384 cells were randomly assessed). The mean percentage (± standard deviation) of cells showing a predominant nuclear localization of STAT1 from three independent experiments is shown: ** p
    293t Cells, supplied by Polyplus Transfection, used in various techniques. Bioz Stars score: 93/100, based on 784 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene 293t cells
    Cell Surface Shed and Secreted, Soluble HLA-G Is Endocytosed into KIR2DL4-Containing Vesicles (A) Endocytosis of soluble HLA-G in resting NK cells. The 221 cells and 221 cells transfected with HLA-Cw3 (221-Cw3) were fixed, permeabilized, and stained with mAb F4/326. Resting NK cells were incubated at 37 °C for 120 min with soluble, refolded HLA-C or HLA-G. Cells were then fixed, permeabilized, and stained with reagents to detect HLA-C (F4/326) or HLA-G (G233) as indicated. (B) The NK cell line YTS-2DL4-gfp was loaded at 37 °C for 120 min with refolded HLA-G. Cells were fixed, permeabilized, and stained with mAb G233 to detect co-localization of soluble HLA-G with gfp-tagged KIR2DL4. (C) Recombinant soluble molecules of HLA-G but not HLA-C are endocytosed into <t>293T-2DL4-gfp</t> cells. Refolded HLA-G and HLA-C were incubated with 293T-2DL4-gfp cells for 2 h. Cells were then fixed, permeabilized, and stained with either mAb G233 (to detect endocytosed HLA-G; upper) or mAb F4/326 (to detect endocytosed HLA-C; middle). (D) The 293T-2DL4-gfp cells were co-cultured with an equal number of 221 cells, 221 cells expressing transmembrane HLA-G (221-G), and 221 cells expressing a soluble isoform of HLA-G (221-sG) for 48 h. Adherent 293T-2DL4-gfp cells were fixed, permeabilized, and stained with mAb G233 followed by Alexa-568–conjugated secondary antibodies prior to acquisition of confocal images. Two 221-G cells are visible in the middle panel.
    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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    Corning Life Sciences 293t cells
    Micrographs of <t>293T</t> cells transfected with pEGFP-N1 or pEGFP-N1-BDNF after transfection at the various time points by fluorescence microscopy (× 200). (A1, A2, B1, B2, C1, C2, D1 and D2): 293T cells transfected with pEGFP-N1-BDNF 24–96 hours (h) after transfection under fluorescence and light microscopes. A2, B2, C2, D2 are light micrographs of the same visual field as A1, B1, C1, D1, respectively. With increasing time after transfection, EGFP signals (green) increased, suggesting increased expression of EGFP-BDNF protein. (A3, A4, B3, B4, C3, C4, D3 and D4) 293T cells transfected with pEGFP-N1 24–96 h after transfection under fluorescence and light microscopes. A4, B4, C4, D4 are light micrographs of the same visual fields as A3, B3, C3, D3, respectively. With increasing time after transfection, EGFP signals increased, suggesting increased expression of EGFP protein. Scale bars: 250 μm. EGFP: Enhanced green fluorescent protein; BDNF: brain-derived neurotrophic factor.
    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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    Optogenetic activation of SthK channels by various PACs in cell lines. a Illustration of the split-PAC-K and fused-PAC-K construct design for SthK co-expression with PACs. b Photocurrents elicited by different split-PAC-K variants after 10 ms exposure to a 470 nm light pulse. Scale bars: 10 s, 0.5 nA. c Normalized integrals of photocurrents as a function of the intensity of a 10 ms light pulse. Arrows denote the EC 50 for current activation by light ( n = 3 cells, two cultures). d Combined whole-cell patch-clamp (black) and optical recording (gray) of a HEK 293 cell expressing fused-bPAC-K and a fluorescent cAMP sensor. bPAC was excited with two 1 ms light flashes (470 nm). The dotted line indicates the zero current level. Scale bars: 20 s, 20 pA (upper trace); 20 s, 2000 aU (lower trace). e Comparison of photocurrent amplitudes generated by bPAC or TpPAC and SthK as split or fused constructs. f Current density comparison at saturating photon exposures ( ** p

    Journal: Nature Communications

    Article Title: Potassium channel-based optogenetic silencing

    doi: 10.1038/s41467-018-07038-8

    Figure Lengend Snippet: Optogenetic activation of SthK channels by various PACs in cell lines. a Illustration of the split-PAC-K and fused-PAC-K construct design for SthK co-expression with PACs. b Photocurrents elicited by different split-PAC-K variants after 10 ms exposure to a 470 nm light pulse. Scale bars: 10 s, 0.5 nA. c Normalized integrals of photocurrents as a function of the intensity of a 10 ms light pulse. Arrows denote the EC 50 for current activation by light ( n = 3 cells, two cultures). d Combined whole-cell patch-clamp (black) and optical recording (gray) of a HEK 293 cell expressing fused-bPAC-K and a fluorescent cAMP sensor. bPAC was excited with two 1 ms light flashes (470 nm). The dotted line indicates the zero current level. Scale bars: 20 s, 20 pA (upper trace); 20 s, 2000 aU (lower trace). e Comparison of photocurrent amplitudes generated by bPAC or TpPAC and SthK as split or fused constructs. f Current density comparison at saturating photon exposures ( ** p

    Article Snippet: Briefly, HEK 293 cells (ATCC CRL-1573) were transfected with a mix of pAD deltaF6, pAAV2/9, or pAAV 2/1, and the AAV expression vector.

    Techniques: Activation Assay, Construct, Expressing, Mass Spectrometry, Patch Clamp, Generated

    The PPI induced by EBNA1 and PAX5 is required for EBNA1/ oriP -mediated binding and transcription. (A) YN-FPAX5, yellow fluorescent protein (YFP)-C terminus-fused (YC)-FEBNA1 or its deletion mutants, or YC-FNCL control was cotransfected into 293T cells. The resulting protein expression of each BiFC plasmid was identified by Western blotting (left). YN-FPAX5- and YC-FEBNA1-derived plasmids were cotransfected into 293T cells, and the BiFC images were analyzed by immunofluorescence confocal microscopy (middle). Schematic diagrams of FPAX5 and its deletion mutants are shown. The results obtained from the BiFC assays were summarized and presented (right). Y, positive fluorescence signal; N, negative fluorescence signal. (B) The FEBNA1 expression plasmid or each DBD-derived deletion mutant was transfected into BAJB cells. Flag-epitope (M2)-conjugated agarose was used to precipitate either FEBNA1 or its mutant derivatives. The amounts of M2-precipitated FEBNA1 or its mutants or coprecipitated endogenous PAX5 were identified by Western blotting. Input (2%) of each protein from the lysate is shown. (C) The schematic diagrams of FPAX5 and its deletion mutants are shown. BJAB cells cotransfected with the expression plasmids of EBNA1 and FPAX5 or its mutant derivatives were used to perform M2 agarose-mediated co-IP assays. The immune blots for the indicated proteins are shown with 2% of input. (D) BJAB cells cotransfected with the expression plasmids of FPAX5 or two pair domain (PD) mutants, V 26 G and P 80 R, and that of EBNA1 were used to perform M2 agarose-mediated co-IP assays. The immune blots for EBNA1, FPAX5, p300, H3K4Me2, and H3K4Me3 are shown with 2% input as the loading control.

    Journal: Journal of Virology

    Article Title: B Cell-Specific Transcription Activator PAX5 Recruits p300 To Support EBNA1-Driven Transcription

    doi: 10.1128/JVI.02028-19

    Figure Lengend Snippet: The PPI induced by EBNA1 and PAX5 is required for EBNA1/ oriP -mediated binding and transcription. (A) YN-FPAX5, yellow fluorescent protein (YFP)-C terminus-fused (YC)-FEBNA1 or its deletion mutants, or YC-FNCL control was cotransfected into 293T cells. The resulting protein expression of each BiFC plasmid was identified by Western blotting (left). YN-FPAX5- and YC-FEBNA1-derived plasmids were cotransfected into 293T cells, and the BiFC images were analyzed by immunofluorescence confocal microscopy (middle). Schematic diagrams of FPAX5 and its deletion mutants are shown. The results obtained from the BiFC assays were summarized and presented (right). Y, positive fluorescence signal; N, negative fluorescence signal. (B) The FEBNA1 expression plasmid or each DBD-derived deletion mutant was transfected into BAJB cells. Flag-epitope (M2)-conjugated agarose was used to precipitate either FEBNA1 or its mutant derivatives. The amounts of M2-precipitated FEBNA1 or its mutants or coprecipitated endogenous PAX5 were identified by Western blotting. Input (2%) of each protein from the lysate is shown. (C) The schematic diagrams of FPAX5 and its deletion mutants are shown. BJAB cells cotransfected with the expression plasmids of EBNA1 and FPAX5 or its mutant derivatives were used to perform M2 agarose-mediated co-IP assays. The immune blots for the indicated proteins are shown with 2% of input. (D) BJAB cells cotransfected with the expression plasmids of FPAX5 or two pair domain (PD) mutants, V 26 G and P 80 R, and that of EBNA1 were used to perform M2 agarose-mediated co-IP assays. The immune blots for EBNA1, FPAX5, p300, H3K4Me2, and H3K4Me3 are shown with 2% input as the loading control.

    Article Snippet: 293T is a human embryonic kidney cell line (ATCC CRL-3216).

    Techniques: Binding Assay, Expressing, Bimolecular Fluorescence Complementation Assay, Plasmid Preparation, Western Blot, Derivative Assay, Immunofluorescence, Confocal Microscopy, Fluorescence, Mutagenesis, Transfection, FLAG-tag, Co-Immunoprecipitation Assay

    TPβ Trp 334 is a major determinant for CCT7 interaction. (A) Schematic representation of TPβ and TPα C-termini. The TPβ region important for CCT7 interaction is underlined in blue. The green, linked amino acids represent residues showing similarity or identity between TPβ and TPα. Shown in red are TPβ Trp 334 and TPα Gln 333 . (B) Lysates of HEK 293 cells transiently expressing the indicated HA-TPβ constructs alone or with CCT7-MYC were immunoprecipitated with a HA-specific monoclonal antibody and analyzed by immunoblotting with MYC- and HA-specific HRP-conjugated antibodies. HA-TPβ 328, 337, 344, and 351 indicate that a stop codon was introduced after the corresponding amino acids. Densitometry on Western blots of four independent experiments are reported in the graphic and expressed as a relative ratio of CCT7 co-IP on receptors IP. (C) Lysates of HEK 293 cells transiently expressing HA-TPβ, HA-TPβ W334Q, HA-TPα, or HA-TPα Q333W alone or with CCT7-MYC were immunoprecipitated with a HA-specific monoclonal antibody and analyzed by immunoblotting with MYC- and HA-specific HRP-conjugated antibodies. Densitometry on Western blots of five independent experiments are reported in the graphic and expressed as a relative ratio of CCT7 co-IP on receptors’ IP. IB, immunoblotting; IP, immunoprecipitation.

    Journal: Molecular Biology of the Cell

    Article Title: Regulation of GPCR expression through an interaction with CCT7, a subunit of the CCT/TRiC complex

    doi: 10.1091/mbc.E16-04-0224

    Figure Lengend Snippet: TPβ Trp 334 is a major determinant for CCT7 interaction. (A) Schematic representation of TPβ and TPα C-termini. The TPβ region important for CCT7 interaction is underlined in blue. The green, linked amino acids represent residues showing similarity or identity between TPβ and TPα. Shown in red are TPβ Trp 334 and TPα Gln 333 . (B) Lysates of HEK 293 cells transiently expressing the indicated HA-TPβ constructs alone or with CCT7-MYC were immunoprecipitated with a HA-specific monoclonal antibody and analyzed by immunoblotting with MYC- and HA-specific HRP-conjugated antibodies. HA-TPβ 328, 337, 344, and 351 indicate that a stop codon was introduced after the corresponding amino acids. Densitometry on Western blots of four independent experiments are reported in the graphic and expressed as a relative ratio of CCT7 co-IP on receptors IP. (C) Lysates of HEK 293 cells transiently expressing HA-TPβ, HA-TPβ W334Q, HA-TPα, or HA-TPα Q333W alone or with CCT7-MYC were immunoprecipitated with a HA-specific monoclonal antibody and analyzed by immunoblotting with MYC- and HA-specific HRP-conjugated antibodies. Densitometry on Western blots of five independent experiments are reported in the graphic and expressed as a relative ratio of CCT7 co-IP on receptors’ IP. IB, immunoblotting; IP, immunoprecipitation.

    Article Snippet: Transient transfection of HEK 293 cells grown to 50–70% co nfluence was performed using the Trans IT-LT1 Reagent (Mirus, Madison, WI) according to the manufacturer’s instructions.

    Techniques: Expressing, Construct, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay

    CCT7 depletion causes redistribution of receptors in aggresomes. (A) HEK 293 cells stably expressing HA-TPβ transfected with CCT7 DsiRNA were fixed, permeabilized, and labeled with a rabbit anti-HA IgG and a mouse anti-GM130. Alexa Fluor 488–conjugated anti-rabbit IgG and Alexa Fluor 633–conjugated anti-mouse IgG were used as secondary antibodies. The fourth panel (d) represents a merge image of the blue (a), green (b), and red (c) signals. High degree of colocalization between the red and green signals appears in yellow. HEK 293 cells stably expressing HA-TPβ (B) or HA-β 2 AR (D) were treated with control or CCT7 DsiRNAs. The cells were fixed, permeabilized, and labeled with mouse anti-HA IgG and stained with PROTEOSTAT aggresome dye. We used Alexa Fluor 633–conjugated anti-mouse IgG as secondary antibody. The third image on the right represents a merged image (c and f) of the green and red signals where the areas with high degree of colocalization between the green signal of the receptors (a and d) and red signal of the aggresome (b and e) appear yellow. Scale bars: 10 μm. Images shown are single confocal slices representative of at least four independent experiments and more than 250 observed cells. (C, E) Mander’s colocalization coefficients represent the ratio of the green signal of the receptors overlapping the red signal of the aggresome and were calculated from at least 100 cells per condition. Results are presented as mean ± SEM.

    Journal: Molecular Biology of the Cell

    Article Title: Regulation of GPCR expression through an interaction with CCT7, a subunit of the CCT/TRiC complex

    doi: 10.1091/mbc.E16-04-0224

    Figure Lengend Snippet: CCT7 depletion causes redistribution of receptors in aggresomes. (A) HEK 293 cells stably expressing HA-TPβ transfected with CCT7 DsiRNA were fixed, permeabilized, and labeled with a rabbit anti-HA IgG and a mouse anti-GM130. Alexa Fluor 488–conjugated anti-rabbit IgG and Alexa Fluor 633–conjugated anti-mouse IgG were used as secondary antibodies. The fourth panel (d) represents a merge image of the blue (a), green (b), and red (c) signals. High degree of colocalization between the red and green signals appears in yellow. HEK 293 cells stably expressing HA-TPβ (B) or HA-β 2 AR (D) were treated with control or CCT7 DsiRNAs. The cells were fixed, permeabilized, and labeled with mouse anti-HA IgG and stained with PROTEOSTAT aggresome dye. We used Alexa Fluor 633–conjugated anti-mouse IgG as secondary antibody. The third image on the right represents a merged image (c and f) of the green and red signals where the areas with high degree of colocalization between the green signal of the receptors (a and d) and red signal of the aggresome (b and e) appear yellow. Scale bars: 10 μm. Images shown are single confocal slices representative of at least four independent experiments and more than 250 observed cells. (C, E) Mander’s colocalization coefficients represent the ratio of the green signal of the receptors overlapping the red signal of the aggresome and were calculated from at least 100 cells per condition. Results are presented as mean ± SEM.

    Article Snippet: Transient transfection of HEK 293 cells grown to 50–70% co nfluence was performed using the Trans IT-LT1 Reagent (Mirus, Madison, WI) according to the manufacturer’s instructions.

    Techniques: Stable Transfection, Expressing, Transfection, Labeling, Staining

    TPβ and β 2 AR interact with CCT7. (A) A yeast two-hybrid screen was performed using the TPβ C-terminus portion as bait on a human HeLa cell MATCHMAKER cDNA library. The interaction between CCT7 and the TPβ C-terminus was confirmed by the use of the selective yeast medium Trp − , Leu − , His − , and Ade − . (B) Lysates of HEK 293 cells transiently expressing HA-TPβ and CCT7-MYC alone or together were immunoprecipitated with HA-specific monoclonal antibody, and immunoblotting was performed with HA-specific HRP and MYC-specific HRP-conjugated antibodies. (C) Lysates of HEK 293 cells transiently expressing FLAG-β 2 AR and CCT7-MYC alone or together were immunoprecipitated with FLAG-specific monoclonal antibody, and immunoblotting was performed with Flag-specific polyclonal and MYC-specific HRP-conjugated antibodies. (D) HEK 293 cells lysates were incubated with either nonspecific goat or CCT7-specific goat polyclonal antibodies, and immunoblotting was performed using the same CCT7-specific and a β 2 AR-specific rabbit polyclonal antibody. All blots shown are representative of at least three independent experiments. IB, immunoblotting; IP, immunoprecipitation.

    Journal: Molecular Biology of the Cell

    Article Title: Regulation of GPCR expression through an interaction with CCT7, a subunit of the CCT/TRiC complex

    doi: 10.1091/mbc.E16-04-0224

    Figure Lengend Snippet: TPβ and β 2 AR interact with CCT7. (A) A yeast two-hybrid screen was performed using the TPβ C-terminus portion as bait on a human HeLa cell MATCHMAKER cDNA library. The interaction between CCT7 and the TPβ C-terminus was confirmed by the use of the selective yeast medium Trp − , Leu − , His − , and Ade − . (B) Lysates of HEK 293 cells transiently expressing HA-TPβ and CCT7-MYC alone or together were immunoprecipitated with HA-specific monoclonal antibody, and immunoblotting was performed with HA-specific HRP and MYC-specific HRP-conjugated antibodies. (C) Lysates of HEK 293 cells transiently expressing FLAG-β 2 AR and CCT7-MYC alone or together were immunoprecipitated with FLAG-specific monoclonal antibody, and immunoblotting was performed with Flag-specific polyclonal and MYC-specific HRP-conjugated antibodies. (D) HEK 293 cells lysates were incubated with either nonspecific goat or CCT7-specific goat polyclonal antibodies, and immunoblotting was performed using the same CCT7-specific and a β 2 AR-specific rabbit polyclonal antibody. All blots shown are representative of at least three independent experiments. IB, immunoblotting; IP, immunoprecipitation.

    Article Snippet: Transient transfection of HEK 293 cells grown to 50–70% co nfluence was performed using the Trans IT-LT1 Reagent (Mirus, Madison, WI) according to the manufacturer’s instructions.

    Techniques: Two Hybrid Screening, cDNA Library Assay, Expressing, Immunoprecipitation, Incubation

    CCT7 coimmunoprecipitates with other GPCRs. (A) Lysates of HEK 293 cells transiently expressing HA-MOR (HA-tagged rat μ-opioid receptor) alone or with CCT7-MYC were immunoprecipitated with an HA-specific monoclonal antibody and analyzed by immunoblotting with MYC- and HA-specific HRP-conjugated antibodies. Lysates of HEK 293 cells transiently expressing FLAG-DOR (FLAG-tagged rat δ-opioid receptor; B) or FLAG-DP (FLAG-tagged prostaglandin D 2 receptor; C) alone or with CCT7-MYC were immunoprecipitated with a FLAG-specific monoclonal antibody and analyzed by immunoblotting with FLAG-specific polyclonal and HA-specific HRP-conjugated antibodies. The blots shown are representative of three separate experiments. IB, immunoblotting; IP, immunoprecipitation.

    Journal: Molecular Biology of the Cell

    Article Title: Regulation of GPCR expression through an interaction with CCT7, a subunit of the CCT/TRiC complex

    doi: 10.1091/mbc.E16-04-0224

    Figure Lengend Snippet: CCT7 coimmunoprecipitates with other GPCRs. (A) Lysates of HEK 293 cells transiently expressing HA-MOR (HA-tagged rat μ-opioid receptor) alone or with CCT7-MYC were immunoprecipitated with an HA-specific monoclonal antibody and analyzed by immunoblotting with MYC- and HA-specific HRP-conjugated antibodies. Lysates of HEK 293 cells transiently expressing FLAG-DOR (FLAG-tagged rat δ-opioid receptor; B) or FLAG-DP (FLAG-tagged prostaglandin D 2 receptor; C) alone or with CCT7-MYC were immunoprecipitated with a FLAG-specific monoclonal antibody and analyzed by immunoblotting with FLAG-specific polyclonal and HA-specific HRP-conjugated antibodies. The blots shown are representative of three separate experiments. IB, immunoblotting; IP, immunoprecipitation.

    Article Snippet: Transient transfection of HEK 293 cells grown to 50–70% co nfluence was performed using the Trans IT-LT1 Reagent (Mirus, Madison, WI) according to the manufacturer’s instructions.

    Techniques: Expressing, Immunoprecipitation

    CCT7 colocalizes with TPβ and β 2 AR and regulates their intracellular distribution. Confocal microscopy was performed on HEK 293 cells stably expressing HA-TPβ (A) or HA-β 2 AR (B) treated with control or CCT7 DsiRNAs. Cells were fixed, permeabilized, and labeled with goat nonspecific IgG as a control, goat anti-CCT7 IgG, and mouse anti-HA IgG. Secondary antibodies used were Alexa Fluor 488–conjugated anti-mouse IgG and Texas red–conjugated anti-goat IgG. Cells were then stained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize nuclei (a, e, and i). The fourth panel on the right represents a merged image of the blue, green, and red signals where the areas with high degree of colocalization between green-labeled receptors (c, g, and k) and red-labeled CCT7 (f and j) appear yellow (h). Approximately 88% of the 250 CCT7-depleted cells observed showed juxtanuclear accumulation of TPβ compared with 7% of the 250 control cells. Images shown are single confocal slices representative of at least four independent experiments and more than 250 observed cells. Scale bars: 10 μm.

    Journal: Molecular Biology of the Cell

    Article Title: Regulation of GPCR expression through an interaction with CCT7, a subunit of the CCT/TRiC complex

    doi: 10.1091/mbc.E16-04-0224

    Figure Lengend Snippet: CCT7 colocalizes with TPβ and β 2 AR and regulates their intracellular distribution. Confocal microscopy was performed on HEK 293 cells stably expressing HA-TPβ (A) or HA-β 2 AR (B) treated with control or CCT7 DsiRNAs. Cells were fixed, permeabilized, and labeled with goat nonspecific IgG as a control, goat anti-CCT7 IgG, and mouse anti-HA IgG. Secondary antibodies used were Alexa Fluor 488–conjugated anti-mouse IgG and Texas red–conjugated anti-goat IgG. Cells were then stained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize nuclei (a, e, and i). The fourth panel on the right represents a merged image of the blue, green, and red signals where the areas with high degree of colocalization between green-labeled receptors (c, g, and k) and red-labeled CCT7 (f and j) appear yellow (h). Approximately 88% of the 250 CCT7-depleted cells observed showed juxtanuclear accumulation of TPβ compared with 7% of the 250 control cells. Images shown are single confocal slices representative of at least four independent experiments and more than 250 observed cells. Scale bars: 10 μm.

    Article Snippet: Transient transfection of HEK 293 cells grown to 50–70% co nfluence was performed using the Trans IT-LT1 Reagent (Mirus, Madison, WI) according to the manufacturer’s instructions.

    Techniques: Confocal Microscopy, Stable Transfection, Expressing, Labeling, Staining

    Characterization of the TPβ Trp 334 mutant. (A) Confocal microscopy was performed on HEK 293 cells transiently expressing HA-TPβ (a–d) or TPβ W334Q (e–h). Cells were fixed, permeabilized, and labeled with goat anti-CCT7 IgG and mouse anti-HA IgG antibodies. Secondary antibodies used were Alexa Fluor 488–conjugated anti-mouse IgG and Texas red–conjugated anti-goat IgG. Cells were then stained with DAPI to visualize nuclei (a and e). The fourth panel on the right represents a merged image of the blue, green and red signals where the areas with high degree of colocalization between green-labeled receptors (c and g) and red-labeled CCT7 (b and f) appear yellow (d and h). Images shown are single confocal slices representative of at least four independent experiments and more than 100 observed cells. Scale bars: 10 µm. (B) Quantification of the membrane on total receptor fluorescence ratio was calculated using ImageJ software and was performed on at least 100 cells per condition. (C) Mander’s colocalization coefficients represent the ratio of the green signal of the receptors overlapping the red signal of the aggresome and were calculated from at least 100 cells per condition. Results are presented as mean ± SEM.

    Journal: Molecular Biology of the Cell

    Article Title: Regulation of GPCR expression through an interaction with CCT7, a subunit of the CCT/TRiC complex

    doi: 10.1091/mbc.E16-04-0224

    Figure Lengend Snippet: Characterization of the TPβ Trp 334 mutant. (A) Confocal microscopy was performed on HEK 293 cells transiently expressing HA-TPβ (a–d) or TPβ W334Q (e–h). Cells were fixed, permeabilized, and labeled with goat anti-CCT7 IgG and mouse anti-HA IgG antibodies. Secondary antibodies used were Alexa Fluor 488–conjugated anti-mouse IgG and Texas red–conjugated anti-goat IgG. Cells were then stained with DAPI to visualize nuclei (a and e). The fourth panel on the right represents a merged image of the blue, green and red signals where the areas with high degree of colocalization between green-labeled receptors (c and g) and red-labeled CCT7 (b and f) appear yellow (d and h). Images shown are single confocal slices representative of at least four independent experiments and more than 100 observed cells. Scale bars: 10 µm. (B) Quantification of the membrane on total receptor fluorescence ratio was calculated using ImageJ software and was performed on at least 100 cells per condition. (C) Mander’s colocalization coefficients represent the ratio of the green signal of the receptors overlapping the red signal of the aggresome and were calculated from at least 100 cells per condition. Results are presented as mean ± SEM.

    Article Snippet: Transient transfection of HEK 293 cells grown to 50–70% co nfluence was performed using the Trans IT-LT1 Reagent (Mirus, Madison, WI) according to the manufacturer’s instructions.

    Techniques: Mutagenesis, Confocal Microscopy, Expressing, Labeling, Staining, Fluorescence, Software

    CCT7 depletion impairs TPβ and β 2 AR total and cell-surface expression. HEK 293 cells stably expressing HA-TPβ (A) or HA-β 2 AR (B) were transfected with control DsiRNA (DsiCtrl) or CCT7 DsiRNA (DsiCCT7), and lysates were immunoblotted with HA-specific HRP-conjugated, CCT7-specific and GAPDH-specific antibodies. Densitometry was performed on the Western blots to quantify relative expression of HA-TPβ (C) and HA-β 2 AR (D) in cells treated with CCT7 DsiRNA compared with control DsiRNA-transfected cells (100%) and normalized to GAPDH expression. Densitometry was performed using ImageJ software, and the results are presented as mean ± SD of at least four independent experiments. Cell-surface receptor expression was measured in HEK 293 cells expressing HA-TPβ (E), HA-TPα (F), or HA-β 2 AR (G) transfected with control or CCT7 DsiRNAs by ELISA using a monoclonal HA-specific antibody as described in Materials and Methods . Results are shown as a percentage of cell-surface receptor expression when cells were transfected with CCT7 DsiRNA compared with control DsiRNA condition (100%). (H) Lysates of HEK 293 cells transiently expressing FLAG-TPβ or FLAG-β 2 AR and HA-Hsp90 alone or together were immunoprecipitated with FLAG-specific monoclonal antibody, and immunoblotting was performed with FLAG-specific polyclonal and HA-specific HRP-conjugated antibodies. Densitometry on Western blots of five independent experiments are reported in the graphic and expressed as a ratio of HSP90 co-IP on receptors IP. Results are presented as mean ± SEM of at least four independent experiments. IB, immunoblotting; IP, immunoprecipitation.

    Journal: Molecular Biology of the Cell

    Article Title: Regulation of GPCR expression through an interaction with CCT7, a subunit of the CCT/TRiC complex

    doi: 10.1091/mbc.E16-04-0224

    Figure Lengend Snippet: CCT7 depletion impairs TPβ and β 2 AR total and cell-surface expression. HEK 293 cells stably expressing HA-TPβ (A) or HA-β 2 AR (B) were transfected with control DsiRNA (DsiCtrl) or CCT7 DsiRNA (DsiCCT7), and lysates were immunoblotted with HA-specific HRP-conjugated, CCT7-specific and GAPDH-specific antibodies. Densitometry was performed on the Western blots to quantify relative expression of HA-TPβ (C) and HA-β 2 AR (D) in cells treated with CCT7 DsiRNA compared with control DsiRNA-transfected cells (100%) and normalized to GAPDH expression. Densitometry was performed using ImageJ software, and the results are presented as mean ± SD of at least four independent experiments. Cell-surface receptor expression was measured in HEK 293 cells expressing HA-TPβ (E), HA-TPα (F), or HA-β 2 AR (G) transfected with control or CCT7 DsiRNAs by ELISA using a monoclonal HA-specific antibody as described in Materials and Methods . Results are shown as a percentage of cell-surface receptor expression when cells were transfected with CCT7 DsiRNA compared with control DsiRNA condition (100%). (H) Lysates of HEK 293 cells transiently expressing FLAG-TPβ or FLAG-β 2 AR and HA-Hsp90 alone or together were immunoprecipitated with FLAG-specific monoclonal antibody, and immunoblotting was performed with FLAG-specific polyclonal and HA-specific HRP-conjugated antibodies. Densitometry on Western blots of five independent experiments are reported in the graphic and expressed as a ratio of HSP90 co-IP on receptors IP. Results are presented as mean ± SEM of at least four independent experiments. IB, immunoblotting; IP, immunoprecipitation.

    Article Snippet: Transient transfection of HEK 293 cells grown to 50–70% co nfluence was performed using the Trans IT-LT1 Reagent (Mirus, Madison, WI) according to the manufacturer’s instructions.

    Techniques: Expressing, Stable Transfection, Transfection, Western Blot, Software, Cell Surface Receptor Assay, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Co-Immunoprecipitation Assay

    Targeting of the TPβ Trp 334 mutant to the aggresome is diminished compared with wild-type TPβ in CCT7-depleted cells. (A) HEK 293 cells transiently expressing HA-TPβ W334Q were treated with control or CCT7 DsiRNAs. The cells were fixed, permeabilized, labeled with mouse anti-HA IgG, and stained with PROTEOSTAT aggresome dye. Alexa Fluor 633–conjugated anti-mouse IgG was used as the secondary antibody. The third images represent a merged image (c and f) of the green and red signals where the areas with high degree of colocalization between the green signal of the receptors (a and d) and red signal of the aggresome (b and e) appear yellow. Scale bars: 10 μm. Images shown are single confocal slices representative of at least four independent experiments and more than 250 observed cells. (B) Mander’s colocalization coefficients represent the ratio of the green signal of the receptor overlapping the red signal of aggresomes and were calculated from at least 100 cells per condition. Results are presented as mean ± SEM.

    Journal: Molecular Biology of the Cell

    Article Title: Regulation of GPCR expression through an interaction with CCT7, a subunit of the CCT/TRiC complex

    doi: 10.1091/mbc.E16-04-0224

    Figure Lengend Snippet: Targeting of the TPβ Trp 334 mutant to the aggresome is diminished compared with wild-type TPβ in CCT7-depleted cells. (A) HEK 293 cells transiently expressing HA-TPβ W334Q were treated with control or CCT7 DsiRNAs. The cells were fixed, permeabilized, labeled with mouse anti-HA IgG, and stained with PROTEOSTAT aggresome dye. Alexa Fluor 633–conjugated anti-mouse IgG was used as the secondary antibody. The third images represent a merged image (c and f) of the green and red signals where the areas with high degree of colocalization between the green signal of the receptors (a and d) and red signal of the aggresome (b and e) appear yellow. Scale bars: 10 μm. Images shown are single confocal slices representative of at least four independent experiments and more than 250 observed cells. (B) Mander’s colocalization coefficients represent the ratio of the green signal of the receptor overlapping the red signal of aggresomes and were calculated from at least 100 cells per condition. Results are presented as mean ± SEM.

    Article Snippet: Transient transfection of HEK 293 cells grown to 50–70% co nfluence was performed using the Trans IT-LT1 Reagent (Mirus, Madison, WI) according to the manufacturer’s instructions.

    Techniques: Mutagenesis, Expressing, Labeling, Staining

    Trp 334 is involved in TPβ maturation and cell-surface expression. (A) Lysates of HEK 293 cells transiently expressing HA-TPβ or HA-TPβ W334Q were analyzed by immunoblotting with HA-specific HRP-conjugated and GAPDH antibodies. (B) Lysates of HEK 293 cells transiently expressing HA-TPα or HA-TPα Q333W were analyzed by immunoblotting with HA-specific HRP-conjugated and GAPDH-specific antibodies. (C) Lysates from HEK 293 cells transiently expressing HA-TPβ or HA-TPα were subject to deglycosylation with Endo Hf as described in Materials and Methods . Immunoblotting was performed using HA-specific HRP-conjugated and GAPDH-specific antibodies. The blots shown are representative of three separate experiments. IB, immunoblotting. (D) Cell-surface receptor expression was measured in HEK 293 cells transiently expressing HA-TPβ, HA-TPβ W334Q, HA-TPα, or HA-TPα Q333W by ELISA using a monoclonal HA-specific antibody as described in Materials and Methods . The results are shown as the percentage of cell-surface receptor expression compared with HA-TPβ (TPβ, 100%). Results represent mean ± SEM of at least four independent experiments.

    Journal: Molecular Biology of the Cell

    Article Title: Regulation of GPCR expression through an interaction with CCT7, a subunit of the CCT/TRiC complex

    doi: 10.1091/mbc.E16-04-0224

    Figure Lengend Snippet: Trp 334 is involved in TPβ maturation and cell-surface expression. (A) Lysates of HEK 293 cells transiently expressing HA-TPβ or HA-TPβ W334Q were analyzed by immunoblotting with HA-specific HRP-conjugated and GAPDH antibodies. (B) Lysates of HEK 293 cells transiently expressing HA-TPα or HA-TPα Q333W were analyzed by immunoblotting with HA-specific HRP-conjugated and GAPDH-specific antibodies. (C) Lysates from HEK 293 cells transiently expressing HA-TPβ or HA-TPα were subject to deglycosylation with Endo Hf as described in Materials and Methods . Immunoblotting was performed using HA-specific HRP-conjugated and GAPDH-specific antibodies. The blots shown are representative of three separate experiments. IB, immunoblotting. (D) Cell-surface receptor expression was measured in HEK 293 cells transiently expressing HA-TPβ, HA-TPβ W334Q, HA-TPα, or HA-TPα Q333W by ELISA using a monoclonal HA-specific antibody as described in Materials and Methods . The results are shown as the percentage of cell-surface receptor expression compared with HA-TPβ (TPβ, 100%). Results represent mean ± SEM of at least four independent experiments.

    Article Snippet: Transient transfection of HEK 293 cells grown to 50–70% co nfluence was performed using the Trans IT-LT1 Reagent (Mirus, Madison, WI) according to the manufacturer’s instructions.

    Techniques: Expressing, Cell Surface Receptor Assay, Enzyme-linked Immunosorbent Assay

    Deletion of the first cysteine triplet of ASP reduces multimer formation and autophagosome signals. (A) Schematic representation of the different domains of ASP and the deletion mutant of the first cysteine triplet ( 10 CCC 12 ). (B to D) COS-7 cells were transfected with expression vectors for wild-type or cysteine triplet-deleted ASP versus pcDNA3.1 (empty vector). After 48 h of transfection, using anti-Myc antibodies, cells were analyzed by flow cytometry (B), Western blotting (C), and confocal microscopy (60× objective with numerical aperture of 1.4) (D). In panel C, high-molecular-weight (including 200-kDa multimers) versus monomeric 20-kDa ASP signals are indicated. Cells analyzed by confocal microscopy were also stained for their nuclei with Hoechst (D). (E) 293T cells were transfected with expression vectors for optimized (opt) NL4.3 ASP WT, ASPΔN1–15, ASPΔN1–30, and ρCCC versus pcDNA3.1 (mock) and analyzed by Western blotting using anti-ASP antibodies.

    Journal: Journal of Virology

    Article Title: HIV-1 Antisense Protein of Different Clades Induces Autophagy and Associates with the Autophagy Factor p62

    doi: 10.1128/JVI.01757-18

    Figure Lengend Snippet: Deletion of the first cysteine triplet of ASP reduces multimer formation and autophagosome signals. (A) Schematic representation of the different domains of ASP and the deletion mutant of the first cysteine triplet ( 10 CCC 12 ). (B to D) COS-7 cells were transfected with expression vectors for wild-type or cysteine triplet-deleted ASP versus pcDNA3.1 (empty vector). After 48 h of transfection, using anti-Myc antibodies, cells were analyzed by flow cytometry (B), Western blotting (C), and confocal microscopy (60× objective with numerical aperture of 1.4) (D). In panel C, high-molecular-weight (including 200-kDa multimers) versus monomeric 20-kDa ASP signals are indicated. Cells analyzed by confocal microscopy were also stained for their nuclei with Hoechst (D). (E) 293T cells were transfected with expression vectors for optimized (opt) NL4.3 ASP WT, ASPΔN1–15, ASPΔN1–30, and ρCCC versus pcDNA3.1 (mock) and analyzed by Western blotting using anti-ASP antibodies.

    Article Snippet: Pseudotyped lentiviruses were produced by cotransfecting 293T cells with lentiCRISPRv2 or lentiCRISPRv2-sgRNA (specific to ATG5, ATG7, or p62), the HIV-1 backbone-containing psPAX2, and the vesicular stomatitis virus (VSV) envelope-encoding-pVSVg vector (8454; Addgene) using the PEI agent.

    Techniques: Mutagenesis, Countercurrent Chromatography, Transfection, Expressing, Plasmid Preparation, Flow Cytometry, Cytometry, Western Blot, Confocal Microscopy, Molecular Weight, Staining

    The interaction of p62 with ASP requires its homodimerization domain despite ASP ubiquitination. (A) The various domains of the p62 protein are depicted and include the ubiquitination-binding domain UBA, the LC3-binding domain LIR, the regions of the nuclear localization signal (NLS) and nuclear export signal (NES), and the homodimerization domain PB1. (B and C) 293T cells were cotransfected with expression vectors of GST-tagged wild type and deletion mutants of p62 and Myc-tagged (B) or His-tagged (C) NL4.3 ASP. At 48 h posttransfection, immunoprecipitation was performed using the anti-GST antibody, and Western blot analyses of immunoprecipitated samples and total extracts were conducted using anti-GST, anti-Myc/anti-His, and anti-tubulin antibodies. (D and E) 293T cells were cotransfected with expression vectors for His-tagged NL4.3 (clade B) or 94CY032 (clade A) ASP (panel D only) and for the HA-tagged ubiquitin (Ub)-KOR mutant (restricted to monoubiquitination). In panel E, expression vectors for HA-tagged ubiquitin WT or the restrictive ubiquitin-K48 and ubiquitin-K63 were also included. At 48 h posttransfection, immunoprecipitation was performed with anti-His antibodies, and Western blot analyses of immunoprecipitated samples and total extracts were conducted using anti-His, anti-HA, and anti-tubulin antibodies. Arrows on the right side of the right panels indicate the positions and expected molecular weights (25 to 30 kDa) of the ubiquitinated ASPs from the different tested proviral DNAs.

    Journal: Journal of Virology

    Article Title: HIV-1 Antisense Protein of Different Clades Induces Autophagy and Associates with the Autophagy Factor p62

    doi: 10.1128/JVI.01757-18

    Figure Lengend Snippet: The interaction of p62 with ASP requires its homodimerization domain despite ASP ubiquitination. (A) The various domains of the p62 protein are depicted and include the ubiquitination-binding domain UBA, the LC3-binding domain LIR, the regions of the nuclear localization signal (NLS) and nuclear export signal (NES), and the homodimerization domain PB1. (B and C) 293T cells were cotransfected with expression vectors of GST-tagged wild type and deletion mutants of p62 and Myc-tagged (B) or His-tagged (C) NL4.3 ASP. At 48 h posttransfection, immunoprecipitation was performed using the anti-GST antibody, and Western blot analyses of immunoprecipitated samples and total extracts were conducted using anti-GST, anti-Myc/anti-His, and anti-tubulin antibodies. (D and E) 293T cells were cotransfected with expression vectors for His-tagged NL4.3 (clade B) or 94CY032 (clade A) ASP (panel D only) and for the HA-tagged ubiquitin (Ub)-KOR mutant (restricted to monoubiquitination). In panel E, expression vectors for HA-tagged ubiquitin WT or the restrictive ubiquitin-K48 and ubiquitin-K63 were also included. At 48 h posttransfection, immunoprecipitation was performed with anti-His antibodies, and Western blot analyses of immunoprecipitated samples and total extracts were conducted using anti-His, anti-HA, and anti-tubulin antibodies. Arrows on the right side of the right panels indicate the positions and expected molecular weights (25 to 30 kDa) of the ubiquitinated ASPs from the different tested proviral DNAs.

    Article Snippet: Pseudotyped lentiviruses were produced by cotransfecting 293T cells with lentiCRISPRv2 or lentiCRISPRv2-sgRNA (specific to ATG5, ATG7, or p62), the HIV-1 backbone-containing psPAX2, and the vesicular stomatitis virus (VSV) envelope-encoding-pVSVg vector (8454; Addgene) using the PEI agent.

    Techniques: Binding Assay, Expressing, Immunoprecipitation, Western Blot, Mutagenesis

    Suppressed expression of ATG5 and ATG7 leads to increased ASP expression. Two different 293T cell clones knocked out for either ATG5 (sgATG5-1 and sgATG5-2) or ATG7 (sgATG7-1 and sgATG7-2), as indicated, and control stably transfected clones were transfected with a His-tagged NL4.3 ASP (clade B) expression vector. Cell lysates were prepared and analyzed by Western blotting using anti-His, anti-ATG5, anti-ATG7, and anti-tubulin antibodies. Monomeric and multimeric ASP signals are depicted. The signal for ATG5 is represented by the typical covalently linked ATG5-ATG12 complex.

    Journal: Journal of Virology

    Article Title: HIV-1 Antisense Protein of Different Clades Induces Autophagy and Associates with the Autophagy Factor p62

    doi: 10.1128/JVI.01757-18

    Figure Lengend Snippet: Suppressed expression of ATG5 and ATG7 leads to increased ASP expression. Two different 293T cell clones knocked out for either ATG5 (sgATG5-1 and sgATG5-2) or ATG7 (sgATG7-1 and sgATG7-2), as indicated, and control stably transfected clones were transfected with a His-tagged NL4.3 ASP (clade B) expression vector. Cell lysates were prepared and analyzed by Western blotting using anti-His, anti-ATG5, anti-ATG7, and anti-tubulin antibodies. Monomeric and multimeric ASP signals are depicted. The signal for ATG5 is represented by the typical covalently linked ATG5-ATG12 complex.

    Article Snippet: Pseudotyped lentiviruses were produced by cotransfecting 293T cells with lentiCRISPRv2 or lentiCRISPRv2-sgRNA (specific to ATG5, ATG7, or p62), the HIV-1 backbone-containing psPAX2, and the vesicular stomatitis virus (VSV) envelope-encoding-pVSVg vector (8454; Addgene) using the PEI agent.

    Techniques: Expressing, Clone Assay, Stable Transfection, Transfection, Plasmid Preparation, Western Blot

    ASPs from different clade representatives form detectable multimers. (A) Sequence alignment of the predicted amino acid sequence of ASPs from HIV-1 isolates representing different clades. The peptide region used to generate anti-ASP monoclonal antibodies is highlighted above the sequence for amino acids 47 to 61. (B and C) Expression vectors of His-tagged ASPs from various HIV clades were transfected in 293T cells, and cell lysates were subsequently analyzed by Western blotting for ASP (anti-ASP and anti-His). Indicated on the right side of the panels are multimers and high-molecular-weight (MW) and monomeric (expected) 20-kDa ASP signals. The clade type for each tested ASP is indicated at the top of each lane. Symbols underneath the sequence comparison in panel A represent complete conservation (*), residues with conservation between groups of strongly similar properties (:), and residues with conservation between groups of weakly similar properties (.) (sequence alignment using the Clustal Omega tool).

    Journal: Journal of Virology

    Article Title: HIV-1 Antisense Protein of Different Clades Induces Autophagy and Associates with the Autophagy Factor p62

    doi: 10.1128/JVI.01757-18

    Figure Lengend Snippet: ASPs from different clade representatives form detectable multimers. (A) Sequence alignment of the predicted amino acid sequence of ASPs from HIV-1 isolates representing different clades. The peptide region used to generate anti-ASP monoclonal antibodies is highlighted above the sequence for amino acids 47 to 61. (B and C) Expression vectors of His-tagged ASPs from various HIV clades were transfected in 293T cells, and cell lysates were subsequently analyzed by Western blotting for ASP (anti-ASP and anti-His). Indicated on the right side of the panels are multimers and high-molecular-weight (MW) and monomeric (expected) 20-kDa ASP signals. The clade type for each tested ASP is indicated at the top of each lane. Symbols underneath the sequence comparison in panel A represent complete conservation (*), residues with conservation between groups of strongly similar properties (:), and residues with conservation between groups of weakly similar properties (.) (sequence alignment using the Clustal Omega tool).

    Article Snippet: Pseudotyped lentiviruses were produced by cotransfecting 293T cells with lentiCRISPRv2 or lentiCRISPRv2-sgRNA (specific to ATG5, ATG7, or p62), the HIV-1 backbone-containing psPAX2, and the vesicular stomatitis virus (VSV) envelope-encoding-pVSVg vector (8454; Addgene) using the PEI agent.

    Techniques: Sequencing, Expressing, Transfection, Western Blot, Molecular Weight

    Multimerization of ASP involves disulfide bonds. (A and B) 293T (A) and COS-7 (B) cells were transfected with expression vectors for GFP-optimized NL4.3 ASP, Myc-tagged-optimized NL4.3 ASP, and/or the empty vector pcDNA3.1. At 48 h posttransfection, immunoprecipitation (IP) was performed using anti-Myc antibody, and Western blot analyses were conducted using anti-GFP and anti-Myc antibodies. Total cellular extracts were similarly analyzed in parallel. (C and D) Extracts from COS-7 cells transfected with the Myc-tagged optimized NL4.3 ASP expression vector (versus pcDNA3.1) were treated with β-mercaptoethanol and/or DTT (at different concentrations in panel D) prior to Western blot analyses with anti-Myc and anti-GAPDH antibodies. Both stacking and resolving gels are depicted, and high-molecular-weight (HMW) (including 200-kDa multimers) versus monomeric 20-kDa ASP signals are indicated.

    Journal: Journal of Virology

    Article Title: HIV-1 Antisense Protein of Different Clades Induces Autophagy and Associates with the Autophagy Factor p62

    doi: 10.1128/JVI.01757-18

    Figure Lengend Snippet: Multimerization of ASP involves disulfide bonds. (A and B) 293T (A) and COS-7 (B) cells were transfected with expression vectors for GFP-optimized NL4.3 ASP, Myc-tagged-optimized NL4.3 ASP, and/or the empty vector pcDNA3.1. At 48 h posttransfection, immunoprecipitation (IP) was performed using anti-Myc antibody, and Western blot analyses were conducted using anti-GFP and anti-Myc antibodies. Total cellular extracts were similarly analyzed in parallel. (C and D) Extracts from COS-7 cells transfected with the Myc-tagged optimized NL4.3 ASP expression vector (versus pcDNA3.1) were treated with β-mercaptoethanol and/or DTT (at different concentrations in panel D) prior to Western blot analyses with anti-Myc and anti-GAPDH antibodies. Both stacking and resolving gels are depicted, and high-molecular-weight (HMW) (including 200-kDa multimers) versus monomeric 20-kDa ASP signals are indicated.

    Article Snippet: Pseudotyped lentiviruses were produced by cotransfecting 293T cells with lentiCRISPRv2 or lentiCRISPRv2-sgRNA (specific to ATG5, ATG7, or p62), the HIV-1 backbone-containing psPAX2, and the vesicular stomatitis virus (VSV) envelope-encoding-pVSVg vector (8454; Addgene) using the PEI agent.

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

    Association between ASP and p62 and effect of p62 knockout on ASP expression levels. (A) 293T cells were transfected with expression vectors for optimized NL4.3 ASP (clade B) or the empty vector pcDNA3.1. At 48 h posttransfection, coimmunoprecipitation (IP) was performed using the anti-ASP antibody, and Western blot analyses were conducted using anti-ASP and anti-LC3 antibodies. Mouse TrueBlot Ultra (anti-mouse Ig HRP) (for ASP) was used as the secondary antibody for Western blot analyses. The LC3-II signal is shown by an arrow. (B) Expression vectors for optimized NL4.3 ASP and GFP-LC3 were cotransfected in COS-7 cells. After fixation and nuclear staining with DAPI, ASP was detected with anti-ASP antibodies followed by a goat anti-mouse IgG antibody coupled to Alexa Fluor 568. (C) The NL4.3-ASP expression vector was transfected in COS-7 and HeLa cells, as indicated. Nuclei were stained with DAPI, and ASP was detected with anti-ASP antibodies followed by goat anti-mouse IgG antibodies coupled to Alexa Fluor 568 (COS-7) or Alexa Fluor 488 (HeLa), while p62 was detected with anti-p62 antibodies followed by goat anti-rabbit IgG antibodies coupled to Alexa Fluor 488 (COS-7) or Alexa Fluor 568 (HeLa). (D) Expression vectors for NL4.3 (clade B) and 94CY032 (clade A) ASPs were transfected in COS-7 cells. ASP and p62 were detected as described in panel C. Cells were next quantified for the number of punctate structures doubly positive for ASP and p62. (E and F) Expression vectors of His-tagged ASP of various HIV clades were transfected in 293T cells, and after 48 h, cellular extracts were used for coimmunoprecipitation with the anti-His (E) or anti-p62 antibodies (F). Immunoprecipitated samples and total extracts were analyzed by Western blotting using anti-p62, anti-His, and anti-tubulin antibodies. (G) The expression vector for His-tagged NL4.3 ASP (versus empty vector) was transfected in 293T cells, and, at 48 h posttransfection, cellular extracts were used for coimmunoprecipitation with anti-His antibodies. Immunoprecipitated samples and total extracts were analyzed by Western blotting using anti-His, anti-ATG5, anti-ATG7, anti-p62, and anti-tubulin antibodies. The ATG5-ATG12 signal is shown by an arrow. (H) A 293T cell clone knocked out for the p62 gene (sgp62) and a control stably transfected clone (Ctrl) were transfected with His-tagged NL4.3 ASP expression vector. Cell lysates were prepared and analyzed by Western blotting using anti-His, anti-p62, and anti-tubulin antibodies. Monomeric and multimeric signals (including high-molecular-weight) are identified on the right side of the panels. ***, P ≤ 0.001.

    Journal: Journal of Virology

    Article Title: HIV-1 Antisense Protein of Different Clades Induces Autophagy and Associates with the Autophagy Factor p62

    doi: 10.1128/JVI.01757-18

    Figure Lengend Snippet: Association between ASP and p62 and effect of p62 knockout on ASP expression levels. (A) 293T cells were transfected with expression vectors for optimized NL4.3 ASP (clade B) or the empty vector pcDNA3.1. At 48 h posttransfection, coimmunoprecipitation (IP) was performed using the anti-ASP antibody, and Western blot analyses were conducted using anti-ASP and anti-LC3 antibodies. Mouse TrueBlot Ultra (anti-mouse Ig HRP) (for ASP) was used as the secondary antibody for Western blot analyses. The LC3-II signal is shown by an arrow. (B) Expression vectors for optimized NL4.3 ASP and GFP-LC3 were cotransfected in COS-7 cells. After fixation and nuclear staining with DAPI, ASP was detected with anti-ASP antibodies followed by a goat anti-mouse IgG antibody coupled to Alexa Fluor 568. (C) The NL4.3-ASP expression vector was transfected in COS-7 and HeLa cells, as indicated. Nuclei were stained with DAPI, and ASP was detected with anti-ASP antibodies followed by goat anti-mouse IgG antibodies coupled to Alexa Fluor 568 (COS-7) or Alexa Fluor 488 (HeLa), while p62 was detected with anti-p62 antibodies followed by goat anti-rabbit IgG antibodies coupled to Alexa Fluor 488 (COS-7) or Alexa Fluor 568 (HeLa). (D) Expression vectors for NL4.3 (clade B) and 94CY032 (clade A) ASPs were transfected in COS-7 cells. ASP and p62 were detected as described in panel C. Cells were next quantified for the number of punctate structures doubly positive for ASP and p62. (E and F) Expression vectors of His-tagged ASP of various HIV clades were transfected in 293T cells, and after 48 h, cellular extracts were used for coimmunoprecipitation with the anti-His (E) or anti-p62 antibodies (F). Immunoprecipitated samples and total extracts were analyzed by Western blotting using anti-p62, anti-His, and anti-tubulin antibodies. (G) The expression vector for His-tagged NL4.3 ASP (versus empty vector) was transfected in 293T cells, and, at 48 h posttransfection, cellular extracts were used for coimmunoprecipitation with anti-His antibodies. Immunoprecipitated samples and total extracts were analyzed by Western blotting using anti-His, anti-ATG5, anti-ATG7, anti-p62, and anti-tubulin antibodies. The ATG5-ATG12 signal is shown by an arrow. (H) A 293T cell clone knocked out for the p62 gene (sgp62) and a control stably transfected clone (Ctrl) were transfected with His-tagged NL4.3 ASP expression vector. Cell lysates were prepared and analyzed by Western blotting using anti-His, anti-p62, and anti-tubulin antibodies. Monomeric and multimeric signals (including high-molecular-weight) are identified on the right side of the panels. ***, P ≤ 0.001.

    Article Snippet: Pseudotyped lentiviruses were produced by cotransfecting 293T cells with lentiCRISPRv2 or lentiCRISPRv2-sgRNA (specific to ATG5, ATG7, or p62), the HIV-1 backbone-containing psPAX2, and the vesicular stomatitis virus (VSV) envelope-encoding-pVSVg vector (8454; Addgene) using the PEI agent.

    Techniques: Knock-Out, Expressing, Transfection, Plasmid Preparation, Western Blot, Staining, Immunoprecipitation, Stable Transfection, Molecular Weight

    ASPs from different clade representatives induce autophagy. (A and B) Expression vectors of His-tagged ASPs from various HIV clades and the empty vector were transfected in 293T cells, and cell lysates were analyzed by Western blotting for the detection of ASP (anti-His), LC3-II levels (anti-LC3), and tubulin. Signals for LC3-I, LC3-II, and ASP are indicated on the left of each panel. In panel B, after transfection, cells were treated with dimethyl sulfoxide (−) or with (+) 100 nM bafilomycin A1 (Baf A1) for 6 h. LC3-II/LC3-I ratios are indicated for each lane and are representative of fold value over the calculated ratio for untreated cells transfected with the empty vector. (C) An increasing quantity of clade 89.6 ASP expression vector was transfected in 293T cells, and cell lysates were analyzed by Western blotting for ASP, p62, and tubulin. (D) Expression vectors for His-tagged ASPs from NL4.3, 94CY032, IndieC1, and Mal proviral DNAs (versus empty vector) were transfected in 293T cells, and cell lysates were analyzed by Western blotting for ASP (anti-His), p62, and tubulin. (E and F) COS-7 cells were transfected with expression vectors of Myc-tagged ASPs from various clades. After fixation, cells were labeled with anti-Myc antibodies followed by goat anti-mouse IgG coupled to Alexa Fluor 488, stained with DAPI, and observed by confocal microscopy. Cells transfected with the expression vectors for NL4.3 ASP (clade B) and 94CY032 ASP (clade A) were next quantified for the number of ASP-positive punctate structures (F). ***, P ≤ 0.001.

    Journal: Journal of Virology

    Article Title: HIV-1 Antisense Protein of Different Clades Induces Autophagy and Associates with the Autophagy Factor p62

    doi: 10.1128/JVI.01757-18

    Figure Lengend Snippet: ASPs from different clade representatives induce autophagy. (A and B) Expression vectors of His-tagged ASPs from various HIV clades and the empty vector were transfected in 293T cells, and cell lysates were analyzed by Western blotting for the detection of ASP (anti-His), LC3-II levels (anti-LC3), and tubulin. Signals for LC3-I, LC3-II, and ASP are indicated on the left of each panel. In panel B, after transfection, cells were treated with dimethyl sulfoxide (−) or with (+) 100 nM bafilomycin A1 (Baf A1) for 6 h. LC3-II/LC3-I ratios are indicated for each lane and are representative of fold value over the calculated ratio for untreated cells transfected with the empty vector. (C) An increasing quantity of clade 89.6 ASP expression vector was transfected in 293T cells, and cell lysates were analyzed by Western blotting for ASP, p62, and tubulin. (D) Expression vectors for His-tagged ASPs from NL4.3, 94CY032, IndieC1, and Mal proviral DNAs (versus empty vector) were transfected in 293T cells, and cell lysates were analyzed by Western blotting for ASP (anti-His), p62, and tubulin. (E and F) COS-7 cells were transfected with expression vectors of Myc-tagged ASPs from various clades. After fixation, cells were labeled with anti-Myc antibodies followed by goat anti-mouse IgG coupled to Alexa Fluor 488, stained with DAPI, and observed by confocal microscopy. Cells transfected with the expression vectors for NL4.3 ASP (clade B) and 94CY032 ASP (clade A) were next quantified for the number of ASP-positive punctate structures (F). ***, P ≤ 0.001.

    Article Snippet: Pseudotyped lentiviruses were produced by cotransfecting 293T cells with lentiCRISPRv2 or lentiCRISPRv2-sgRNA (specific to ATG5, ATG7, or p62), the HIV-1 backbone-containing psPAX2, and the vesicular stomatitis virus (VSV) envelope-encoding-pVSVg vector (8454; Addgene) using the PEI agent.

    Techniques: Expressing, Plasmid Preparation, Transfection, Western Blot, Labeling, Staining, Confocal Microscopy

    VPg-primed RNA synthesis in the presence of NoV RdRp. (A) Western blot analysis of VPg immunoprecipitated from HEK 293T cells expressing the WT (NoV Rp) or mutant RdRp (Rp GAA ) and WT VPg (VPg) proteins. A plus symbol denotes addition of the corresponding plasmid. The samples were resolved on a 4-to-12% denaturing PAGE gel prior to Western blotting with anti-HA antibodies. The relevant masses from the standards are shown to the left of the Western blot image. The identities of the molecules are shown to the right of the Western blot image. VPg-RNA, VPg linked to RNA; VPg, free VPg. (B) Western blotting results for the total RNAs purified from HEK 293T cells that expressed either the WT or mutant RdRp and VPg proteins. The RNAs were electrophoresed in a 4-to-12% denaturing PAGE gel. A plus symbol in the rows labeled RNase A or RQ1 DNase denotes a 30-min treatment with a 10-μg final concentration or 5 units, respectively, of the appropriate enzyme prior to loading the sample for SDS-PAGE. VPg-RNA, VPg linked to RNA; VPg, free VPg. Lane 2 contains free VPg generated from cells transfected to produce only VPg and was loaded to serve as a molecular mass marker.

    Journal: Journal of Virology

    Article Title: VPg-Primed RNA Synthesis of Norovirus RNA-Dependent RNA Polymerases by Using a Novel Cell-Based Assay ▿

    doi: 10.1128/JVI.06191-11

    Figure Lengend Snippet: VPg-primed RNA synthesis in the presence of NoV RdRp. (A) Western blot analysis of VPg immunoprecipitated from HEK 293T cells expressing the WT (NoV Rp) or mutant RdRp (Rp GAA ) and WT VPg (VPg) proteins. A plus symbol denotes addition of the corresponding plasmid. The samples were resolved on a 4-to-12% denaturing PAGE gel prior to Western blotting with anti-HA antibodies. The relevant masses from the standards are shown to the left of the Western blot image. The identities of the molecules are shown to the right of the Western blot image. VPg-RNA, VPg linked to RNA; VPg, free VPg. (B) Western blotting results for the total RNAs purified from HEK 293T cells that expressed either the WT or mutant RdRp and VPg proteins. The RNAs were electrophoresed in a 4-to-12% denaturing PAGE gel. A plus symbol in the rows labeled RNase A or RQ1 DNase denotes a 30-min treatment with a 10-μg final concentration or 5 units, respectively, of the appropriate enzyme prior to loading the sample for SDS-PAGE. VPg-RNA, VPg linked to RNA; VPg, free VPg. Lane 2 contains free VPg generated from cells transfected to produce only VPg and was loaded to serve as a molecular mass marker.

    Article Snippet: To examine the VPg-linked RNAs, 293T cells (1.0 × 106 ) were seeded in each well of 6-well cell culture plates (BD Falcon), and 1 μg of recombinant plasmid expressing FLAG-tagged RdRp was cotransfected with 100 ng of recombinant plasmid expressing HA-tagged VPg.

    Techniques: Western Blot, Immunoprecipitation, Expressing, Mutagenesis, Plasmid Preparation, Polyacrylamide Gel Electrophoresis, Purification, Labeling, Concentration Assay, SDS Page, Generated, Transfection, Marker

    Effects of coexpressed VPg on NoV GII.4 RdRp activity. (A) Western blot showing that the NoV VPg WT and the Y27A mutant proteins are expressed to comparable levels. (B) Effects of NoV and MNV VPgs on homologous and heterologous combinations of the NoV or MNV RdRps. W, WT VPg; M, mutant VPg. A plus symbol below the x axis shows the presence of the respective plasmids used in the transfection of the HEK 293T cells. The data are the means from three replicates and are representative of three independent experiments, and the standard errors are shown above the bars. Statistical analysis was performed by using the pairwise Student t test, and the P values are shown above the samples that were compared. (C) The NoV VPg does not enhance the activities of the 1b HCV RdRp or the BMV 2a RdRp in transiently transfected HEK 293T cells. The data are the means from three replicates and are representative of two independent experiments, and the standard errors are shown above the bars. Statistical analysis was performed by using the pairwise Student t test. The difference between the NoV Rp and NoV Rp+VPg was statistically significant ( P

    Journal: Journal of Virology

    Article Title: VPg-Primed RNA Synthesis of Norovirus RNA-Dependent RNA Polymerases by Using a Novel Cell-Based Assay ▿

    doi: 10.1128/JVI.06191-11

    Figure Lengend Snippet: Effects of coexpressed VPg on NoV GII.4 RdRp activity. (A) Western blot showing that the NoV VPg WT and the Y27A mutant proteins are expressed to comparable levels. (B) Effects of NoV and MNV VPgs on homologous and heterologous combinations of the NoV or MNV RdRps. W, WT VPg; M, mutant VPg. A plus symbol below the x axis shows the presence of the respective plasmids used in the transfection of the HEK 293T cells. The data are the means from three replicates and are representative of three independent experiments, and the standard errors are shown above the bars. Statistical analysis was performed by using the pairwise Student t test, and the P values are shown above the samples that were compared. (C) The NoV VPg does not enhance the activities of the 1b HCV RdRp or the BMV 2a RdRp in transiently transfected HEK 293T cells. The data are the means from three replicates and are representative of two independent experiments, and the standard errors are shown above the bars. Statistical analysis was performed by using the pairwise Student t test. The difference between the NoV Rp and NoV Rp+VPg was statistically significant ( P

    Article Snippet: To examine the VPg-linked RNAs, 293T cells (1.0 × 106 ) were seeded in each well of 6-well cell culture plates (BD Falcon), and 1 μg of recombinant plasmid expressing FLAG-tagged RdRp was cotransfected with 100 ng of recombinant plasmid expressing HA-tagged VPg.

    Techniques: Activity Assay, Western Blot, Mutagenesis, Transfection

    MDA5, but not TLR3, can replace RIG-I in the NoV-5BR assay. (A) The NoV RdRp can signal through MDA5 in 293T cells. 1b5B is the RdRp from HCV. Rp GAA is an active site mutant of the NoV RdRp. (B) TLR3 signaling was not activated by the products of the NoV RdRp. A TLR3 mutant defective in ligand binding, H539E, served as a control to illustrate signaling that was dependent on the WT TLR3. pI:C denotes poly(I:C), a TLR3 agonist, which was added to the medium of cells to a final concentration of 500 ng/ml.

    Journal: Journal of Virology

    Article Title: VPg-Primed RNA Synthesis of Norovirus RNA-Dependent RNA Polymerases by Using a Novel Cell-Based Assay ▿

    doi: 10.1128/JVI.06191-11

    Figure Lengend Snippet: MDA5, but not TLR3, can replace RIG-I in the NoV-5BR assay. (A) The NoV RdRp can signal through MDA5 in 293T cells. 1b5B is the RdRp from HCV. Rp GAA is an active site mutant of the NoV RdRp. (B) TLR3 signaling was not activated by the products of the NoV RdRp. A TLR3 mutant defective in ligand binding, H539E, served as a control to illustrate signaling that was dependent on the WT TLR3. pI:C denotes poly(I:C), a TLR3 agonist, which was added to the medium of cells to a final concentration of 500 ng/ml.

    Article Snippet: To examine the VPg-linked RNAs, 293T cells (1.0 × 106 ) were seeded in each well of 6-well cell culture plates (BD Falcon), and 1 μg of recombinant plasmid expressing FLAG-tagged RdRp was cotransfected with 100 ng of recombinant plasmid expressing HA-tagged VPg.

    Techniques: Mutagenesis, Ligand Binding Assay, Concentration Assay

    Identification of a template for VPg-primed RNA synthesis in HEK 293T cells. (A) Alignment of the sequences of the cDNAs derived from the VPg-primed RNAs. The sequence from the cellular TGOLN2 mRNA is shown on the top line in the 5′-to-3′ direction, and the sequences of VPg-linked RNAs are complementary to and shown below the TGOLN2 mRNA in the 3′-to-5′ direction. Each sequence was from an independent cDNA clone, and the numbers in parentheses represent the number of independent sequences obtained. Dashes denote that the sequence was not present. (B) Northern blot of the VPg-RNA complexes immunoprecipitated from HEK 293T cells expressing the WT or mutant RdRp and VPg, performed with a radiolabeled oligodeoxyribonucleotide corresponding to the TGOLN2 mRNA sequence. Where RNase A or RQ1 DNase treatment was used, the immunoprecipitated material was incubated with the enzyme for 30 min prior to electrophoresis on a denaturing PAGE gel.

    Journal: Journal of Virology

    Article Title: VPg-Primed RNA Synthesis of Norovirus RNA-Dependent RNA Polymerases by Using a Novel Cell-Based Assay ▿

    doi: 10.1128/JVI.06191-11

    Figure Lengend Snippet: Identification of a template for VPg-primed RNA synthesis in HEK 293T cells. (A) Alignment of the sequences of the cDNAs derived from the VPg-primed RNAs. The sequence from the cellular TGOLN2 mRNA is shown on the top line in the 5′-to-3′ direction, and the sequences of VPg-linked RNAs are complementary to and shown below the TGOLN2 mRNA in the 3′-to-5′ direction. Each sequence was from an independent cDNA clone, and the numbers in parentheses represent the number of independent sequences obtained. Dashes denote that the sequence was not present. (B) Northern blot of the VPg-RNA complexes immunoprecipitated from HEK 293T cells expressing the WT or mutant RdRp and VPg, performed with a radiolabeled oligodeoxyribonucleotide corresponding to the TGOLN2 mRNA sequence. Where RNase A or RQ1 DNase treatment was used, the immunoprecipitated material was incubated with the enzyme for 30 min prior to electrophoresis on a denaturing PAGE gel.

    Article Snippet: To examine the VPg-linked RNAs, 293T cells (1.0 × 106 ) were seeded in each well of 6-well cell culture plates (BD Falcon), and 1 μg of recombinant plasmid expressing FLAG-tagged RdRp was cotransfected with 100 ng of recombinant plasmid expressing HA-tagged VPg.

    Techniques: Derivative Assay, Sequencing, Northern Blot, Immunoprecipitation, Expressing, Mutagenesis, Incubation, Electrophoresis, Polyacrylamide Gel Electrophoresis

    Effects of other NoV proteins on RdRp activity. (A) Western blotting of the NoV proteins showed their expression in HEK 293T cells. All of the proteins were HA tagged at their C termini and detected with the goat anti-HA antibody and an HRP-conjugated mouse anti-goat antibody. (B) Summary of the effects of coexpressed NoV GII.4 structural and nonstructural proteins on RIG-I signaling or NoV RdRp activity. Plasmid carrying NoV RdRp was transfected at 50 ng/well, and plasmid carrying each other protein was transfected at 10 ng/well. All data are presented as the means and standard errors of three replicates from three independent assays. The results are normalized to the vector-only control to better allow comparisons. Results statistically different from that of the vector-alone sample are shown with P values in parentheses. (C) Effects of the NoV proteins on the RdRps from NoV, MNV, HCV (1b5b), and BMV (BMV 2a). Plasmids encoding all RdRps were transfected into HEK 293T cells at 50 ng, and the plasmids that express the other NoV GII.4 proteins were transfected at 10 ng. Each bar represents the means and standard errors of three replicates from two independent assays. Statistical analysis was performed by using the pairwise Student t test, and the P values are indicated above the samples that were compared.

    Journal: Journal of Virology

    Article Title: VPg-Primed RNA Synthesis of Norovirus RNA-Dependent RNA Polymerases by Using a Novel Cell-Based Assay ▿

    doi: 10.1128/JVI.06191-11

    Figure Lengend Snippet: Effects of other NoV proteins on RdRp activity. (A) Western blotting of the NoV proteins showed their expression in HEK 293T cells. All of the proteins were HA tagged at their C termini and detected with the goat anti-HA antibody and an HRP-conjugated mouse anti-goat antibody. (B) Summary of the effects of coexpressed NoV GII.4 structural and nonstructural proteins on RIG-I signaling or NoV RdRp activity. Plasmid carrying NoV RdRp was transfected at 50 ng/well, and plasmid carrying each other protein was transfected at 10 ng/well. All data are presented as the means and standard errors of three replicates from three independent assays. The results are normalized to the vector-only control to better allow comparisons. Results statistically different from that of the vector-alone sample are shown with P values in parentheses. (C) Effects of the NoV proteins on the RdRps from NoV, MNV, HCV (1b5b), and BMV (BMV 2a). Plasmids encoding all RdRps were transfected into HEK 293T cells at 50 ng, and the plasmids that express the other NoV GII.4 proteins were transfected at 10 ng. Each bar represents the means and standard errors of three replicates from two independent assays. Statistical analysis was performed by using the pairwise Student t test, and the P values are indicated above the samples that were compared.

    Article Snippet: To examine the VPg-linked RNAs, 293T cells (1.0 × 106 ) were seeded in each well of 6-well cell culture plates (BD Falcon), and 1 μg of recombinant plasmid expressing FLAG-tagged RdRp was cotransfected with 100 ng of recombinant plasmid expressing HA-tagged VPg.

    Techniques: Activity Assay, Western Blot, Expressing, Plasmid Preparation, Transfection

    RNA synthesis-competent NoV RdRp can activate innate immune receptor-mediated signaling. (A) Typical genome organization of human NoV. (B) Western blot of the expression of wild-type or an active site mutant NoV RdRp (norovirus Hu/GII.4/MD-2004/2004/US). Cells transfected to express the FLAG-tagged WT (NoV Rp) or the active site mutant (Rp GAA ) of the NoV GII.4 RdRp were lysed in sample buffer, resolved with a 4-to-12% bis-Tris gel, and subjected to Western blotting with mouse monoclonal antibody to FLAG tag (Sigma). HRP-coupled rabbit anti-mouse secondary antibody was used to detect the primary antibody, and the signal was developed with the ECL Plus Western blot detection system (Amersham, United Kingdom). The sizes of the relevant molecular weight markers are shown to the left of the Western blot image. (C) Results from the NoV-5BR assay for the NoV RdRp in HEK 293T cells. Plasmids that expressed the proteins are denoted on the x axis. 1b5B is the RdRp of HCV genotype 1b. Ratios of firefly to Renilla luciferase activities are shown on the y axis. The data are the means from three replicates and are representative of three independent experiments, and the standard errors are shown above the bars. (D) Results from a NoV-5BR assay performed in cultured human hepatocytes (Huh-7). The data are the means from three replicates, and the standard errors are shown above the bars. (E) Effects of Mn 2+ or Mg 2+ on NoV RdRp activity. The divalent metals were added directly to the medium of the cultured HEK 293T cells. The control reaction monitored the effect of Mn 2+ on cells that expressed the RIG-I receptor and the two luciferases but not the GII.4 polymerase.

    Journal: Journal of Virology

    Article Title: VPg-Primed RNA Synthesis of Norovirus RNA-Dependent RNA Polymerases by Using a Novel Cell-Based Assay ▿

    doi: 10.1128/JVI.06191-11

    Figure Lengend Snippet: RNA synthesis-competent NoV RdRp can activate innate immune receptor-mediated signaling. (A) Typical genome organization of human NoV. (B) Western blot of the expression of wild-type or an active site mutant NoV RdRp (norovirus Hu/GII.4/MD-2004/2004/US). Cells transfected to express the FLAG-tagged WT (NoV Rp) or the active site mutant (Rp GAA ) of the NoV GII.4 RdRp were lysed in sample buffer, resolved with a 4-to-12% bis-Tris gel, and subjected to Western blotting with mouse monoclonal antibody to FLAG tag (Sigma). HRP-coupled rabbit anti-mouse secondary antibody was used to detect the primary antibody, and the signal was developed with the ECL Plus Western blot detection system (Amersham, United Kingdom). The sizes of the relevant molecular weight markers are shown to the left of the Western blot image. (C) Results from the NoV-5BR assay for the NoV RdRp in HEK 293T cells. Plasmids that expressed the proteins are denoted on the x axis. 1b5B is the RdRp of HCV genotype 1b. Ratios of firefly to Renilla luciferase activities are shown on the y axis. The data are the means from three replicates and are representative of three independent experiments, and the standard errors are shown above the bars. (D) Results from a NoV-5BR assay performed in cultured human hepatocytes (Huh-7). The data are the means from three replicates, and the standard errors are shown above the bars. (E) Effects of Mn 2+ or Mg 2+ on NoV RdRp activity. The divalent metals were added directly to the medium of the cultured HEK 293T cells. The control reaction monitored the effect of Mn 2+ on cells that expressed the RIG-I receptor and the two luciferases but not the GII.4 polymerase.

    Article Snippet: To examine the VPg-linked RNAs, 293T cells (1.0 × 106 ) were seeded in each well of 6-well cell culture plates (BD Falcon), and 1 μg of recombinant plasmid expressing FLAG-tagged RdRp was cotransfected with 100 ng of recombinant plasmid expressing HA-tagged VPg.

    Techniques: Western Blot, Expressing, Mutagenesis, Transfection, FLAG-tag, Molecular Weight, Luciferase, Cell Culture, Activity Assay

    RNA synthesis-competent MNV RdRp can activate innate immune receptor-mediated signaling. (A) Western blot of the FLAG-tagged MNV proteins produced in HEK 293T cells. A monoclonal antibody recognizing the FLAG epitope was used to detect the MNV RdRp and VPg proteins. HRP-coupled rabbit anti-mouse secondary antibody was used to detect the primary antibody, and the signal was developed with the ECL Plus Western blot detection system (Amersham, United Kingdom). (B) WT MNV RdRp can induce RIG-I signaling in HEK 293T cells. Rp GAA is the active site mutant of MNV RdRp. (C) Sequence alignment of VPg proteins from representative members of the Caliciviridae . The predicted site of RNA linkage in the calicivirus VPg is highlighted in bold. Tyr26 is the site of nucleotidylylation in MNV VPg, while the site in NoV corresponds to Tyr27. Tyr117 of the MNV VPg protein, reported to be important for nucleotidylylation in vitro , is also highlighted in bold. The conserved signature motif of the calicivirus VPg proteins is shown (E/DE Y DEΩ). (D) Effects of coexpressed wild-type or mutant VPgs on the activity of the MNV polymerase. The presence of a plasmid used in the transfection of HEK 293T cells is shown by a plus symbol below the x axis. A plus symbol in the row titled “Vec” denotes that an empty vector was transfected into the cells. The data are the means from three replicates and are representative of three independent experiments, and the standard errors are shown above the bars. P values less than 0.05 were considered statistically significant.

    Journal: Journal of Virology

    Article Title: VPg-Primed RNA Synthesis of Norovirus RNA-Dependent RNA Polymerases by Using a Novel Cell-Based Assay ▿

    doi: 10.1128/JVI.06191-11

    Figure Lengend Snippet: RNA synthesis-competent MNV RdRp can activate innate immune receptor-mediated signaling. (A) Western blot of the FLAG-tagged MNV proteins produced in HEK 293T cells. A monoclonal antibody recognizing the FLAG epitope was used to detect the MNV RdRp and VPg proteins. HRP-coupled rabbit anti-mouse secondary antibody was used to detect the primary antibody, and the signal was developed with the ECL Plus Western blot detection system (Amersham, United Kingdom). (B) WT MNV RdRp can induce RIG-I signaling in HEK 293T cells. Rp GAA is the active site mutant of MNV RdRp. (C) Sequence alignment of VPg proteins from representative members of the Caliciviridae . The predicted site of RNA linkage in the calicivirus VPg is highlighted in bold. Tyr26 is the site of nucleotidylylation in MNV VPg, while the site in NoV corresponds to Tyr27. Tyr117 of the MNV VPg protein, reported to be important for nucleotidylylation in vitro , is also highlighted in bold. The conserved signature motif of the calicivirus VPg proteins is shown (E/DE Y DEΩ). (D) Effects of coexpressed wild-type or mutant VPgs on the activity of the MNV polymerase. The presence of a plasmid used in the transfection of HEK 293T cells is shown by a plus symbol below the x axis. A plus symbol in the row titled “Vec” denotes that an empty vector was transfected into the cells. The data are the means from three replicates and are representative of three independent experiments, and the standard errors are shown above the bars. P values less than 0.05 were considered statistically significant.

    Article Snippet: To examine the VPg-linked RNAs, 293T cells (1.0 × 106 ) were seeded in each well of 6-well cell culture plates (BD Falcon), and 1 μg of recombinant plasmid expressing FLAG-tagged RdRp was cotransfected with 100 ng of recombinant plasmid expressing HA-tagged VPg.

    Techniques: Western Blot, Produced, FLAG-tag, Mutagenesis, Sequencing, In Vitro, Activity Assay, Plasmid Preparation, Transfection

    RNase L participates in the processing of the VPg-primed RNA products synthesized by the NoV RdRp in the NoV-5BR assay. (A) siRNA knockdown of RNase L decreased the levels of RNase L protein in HEK 293T cells. The cells were treated for 48 h with the concentrations of the siRNAs shown above the Western blot image. Twenty microliters of the cell lysate was then subjected to electrophoresis through a 4-to-12% denaturing PAGE gel. The blot was probed with antibodies to RNase L or β-actin, as shown to the right of the Western blot image. (B) Effects of RNase L knockdown on RIG-I signaling with exogenously supplied ligands or ligands produced by the NoV RdRp. The exogenously provided ligands were shR9 and S4dsRNA, produced by in vitro transcription using the T7 RNA polymerase and transfected into the cells using Lipofectamine. The NoV RdRp and VPg were transiently expressed from transfected plasmids. NS siRNA, a control, nonspecific siRNA (Invitrogen). The data are the means from three replicates and are representative of two independent experiments, and the standard errors are shown above the bars. Statistical analysis was performed by using the pairwise Student t test, and the P values are above the samples that were compared. (C) Knockdown of RNase L increased the abundance of the VPg-RNA complex made by the NoV RdRp. The Western blotting results are from immunoprecipitation reactions of cell lysates that expressed the NoV VPg without or with the NoV RdRp probed to detect the VPg protein. The lane VPg alone served as a molecular mass control for free VPg. (D) Confirmation of the effect of RNase L on the abundance of the VPg-RNA complex. Samples shown were used in the Western blot assay containing total RNAs purified from cells that were transfected to expressed the NoV RdRp and VPg after RNase L knockdown. The lane labeled VPg alone served as a molecular mass control for VPg that was not covalently linked to RNA. This species of VPg would not be present in the affinity purification of RNA.

    Journal: Journal of Virology

    Article Title: VPg-Primed RNA Synthesis of Norovirus RNA-Dependent RNA Polymerases by Using a Novel Cell-Based Assay ▿

    doi: 10.1128/JVI.06191-11

    Figure Lengend Snippet: RNase L participates in the processing of the VPg-primed RNA products synthesized by the NoV RdRp in the NoV-5BR assay. (A) siRNA knockdown of RNase L decreased the levels of RNase L protein in HEK 293T cells. The cells were treated for 48 h with the concentrations of the siRNAs shown above the Western blot image. Twenty microliters of the cell lysate was then subjected to electrophoresis through a 4-to-12% denaturing PAGE gel. The blot was probed with antibodies to RNase L or β-actin, as shown to the right of the Western blot image. (B) Effects of RNase L knockdown on RIG-I signaling with exogenously supplied ligands or ligands produced by the NoV RdRp. The exogenously provided ligands were shR9 and S4dsRNA, produced by in vitro transcription using the T7 RNA polymerase and transfected into the cells using Lipofectamine. The NoV RdRp and VPg were transiently expressed from transfected plasmids. NS siRNA, a control, nonspecific siRNA (Invitrogen). The data are the means from three replicates and are representative of two independent experiments, and the standard errors are shown above the bars. Statistical analysis was performed by using the pairwise Student t test, and the P values are above the samples that were compared. (C) Knockdown of RNase L increased the abundance of the VPg-RNA complex made by the NoV RdRp. The Western blotting results are from immunoprecipitation reactions of cell lysates that expressed the NoV VPg without or with the NoV RdRp probed to detect the VPg protein. The lane VPg alone served as a molecular mass control for free VPg. (D) Confirmation of the effect of RNase L on the abundance of the VPg-RNA complex. Samples shown were used in the Western blot assay containing total RNAs purified from cells that were transfected to expressed the NoV RdRp and VPg after RNase L knockdown. The lane labeled VPg alone served as a molecular mass control for VPg that was not covalently linked to RNA. This species of VPg would not be present in the affinity purification of RNA.

    Article Snippet: To examine the VPg-linked RNAs, 293T cells (1.0 × 106 ) were seeded in each well of 6-well cell culture plates (BD Falcon), and 1 μg of recombinant plasmid expressing FLAG-tagged RdRp was cotransfected with 100 ng of recombinant plasmid expressing HA-tagged VPg.

    Techniques: Synthesized, Western Blot, Electrophoresis, Polyacrylamide Gel Electrophoresis, Produced, In Vitro, Transfection, Immunoprecipitation, Purification, Labeling, Affinity Purification

    TACI co-localizes with MyD88 and TRAF6 in the late endosomal compartment. (A) mCherry labeled TACI-S, TACI-L or TACI-S194X isoform transfected HEK-293T cells, were stained with rabbit Ab to MyD88 or TRAF6; nuclei were stained with DAPI. Merged images show that each TACI isoform co-stains with MyD88 (A) and TRAF6 (B) , but colocalization is absent for the S194X mutant. (C) HEK-293T cells transfected with mCherry labeled TACI-L and TACI-S were incubated with Alexa Fluor 647-conjugated transferrin (40 μg/ml Tfn-647) at 37°C for 5 min fixed with 4% paraformaldehyde. Merged images show that both TACI-L and TACI-S co-localize with Tfn-647 (white arrows). (D) In other experiments, transfected HEK-293T cells were stained with mAb Rab7 as a marker of late endosomes, also showing co-localization. Samples were examined by Leica SP5 DMI confocal microscopy, acquiring 3 different xy planes with 63×/1.4 NA objective lenses (Carl Zeiss) with optimal z spacing (~0.016 μm). Images were processed using Adobe Photoshop.

    Journal: Frontiers in Immunology

    Article Title: TACI Isoforms Regulate Ligand Binding and Receptor Function

    doi: 10.3389/fimmu.2018.02125

    Figure Lengend Snippet: TACI co-localizes with MyD88 and TRAF6 in the late endosomal compartment. (A) mCherry labeled TACI-S, TACI-L or TACI-S194X isoform transfected HEK-293T cells, were stained with rabbit Ab to MyD88 or TRAF6; nuclei were stained with DAPI. Merged images show that each TACI isoform co-stains with MyD88 (A) and TRAF6 (B) , but colocalization is absent for the S194X mutant. (C) HEK-293T cells transfected with mCherry labeled TACI-L and TACI-S were incubated with Alexa Fluor 647-conjugated transferrin (40 μg/ml Tfn-647) at 37°C for 5 min fixed with 4% paraformaldehyde. Merged images show that both TACI-L and TACI-S co-localize with Tfn-647 (white arrows). (D) In other experiments, transfected HEK-293T cells were stained with mAb Rab7 as a marker of late endosomes, also showing co-localization. Samples were examined by Leica SP5 DMI confocal microscopy, acquiring 3 different xy planes with 63×/1.4 NA objective lenses (Carl Zeiss) with optimal z spacing (~0.016 μm). Images were processed using Adobe Photoshop.

    Article Snippet: To examine the cytoplasmic intersection of TACI isoforms with TLR9 by confocal microscopy, we transfected HEK-293T cells with mCherry labeled TACI-S or TACI-L, or for comparison the S194X mutant, along with TLR9-YFP (pcDNA3-TLR9-YFP was a gift from Doug Golenbock; Addgene plasmid # 13642).

    Techniques: Labeling, Transfection, Staining, Mutagenesis, Incubation, Marker, Confocal Microscopy

    TACI variants with missense mutations bind un-mutated isoforms but lack signaling function. (A) TACI mCherry labeled mutants, C104R, A181E, and S194X were generated in the TACI-L and TACI-S isoforms, and co-transfected into HEK-293T cells with WT TACI-YFP. These were examined in FRET experiments by FACS (LSRII) to judge complex formation using CD40-eYFP as a control. (B) Frequency of FRET positive cells in the double positive YFP and mCherry gate. Data shows average ± SD from 6 independent experiments. In other experiments, ligands, APRIL, or BAFF (0, 5, 20, or 50 ng/ml) were added to judge the effects on FRET signal, showing no alteration in the signal (not shown). (C) For validation of complexes found in FRET, complexes forming with FLAG-TACI were precipitated with anti-FLAG sepharose beads and run on 10% PAGE gels; immunoblots were developed with an anti-HA antibody. Lower panel shows FLAG expression control. (D) To examine NF-kB luciferase induction, TACI-S, TACI-L or mutant C104R, A181E and S194X constructs were transfected into HEK-293T cells, along with NF-kB–luc reporter and control pRL-null plasmids and cultured for 48hrs; TACI expression was confirmed by western blot. (E) These cells were cultured with or without activation for 6h with 100 ng/ml APRIL or BAFF. Reporter gene activity was determined and NF-kB luciferase induction normalized to Renilla luciferase. Values reported are represented as Relative Luciferase Units (RLU) and are the mean ± SD from 5 to 7 independent experiments. * p

    Journal: Frontiers in Immunology

    Article Title: TACI Isoforms Regulate Ligand Binding and Receptor Function

    doi: 10.3389/fimmu.2018.02125

    Figure Lengend Snippet: TACI variants with missense mutations bind un-mutated isoforms but lack signaling function. (A) TACI mCherry labeled mutants, C104R, A181E, and S194X were generated in the TACI-L and TACI-S isoforms, and co-transfected into HEK-293T cells with WT TACI-YFP. These were examined in FRET experiments by FACS (LSRII) to judge complex formation using CD40-eYFP as a control. (B) Frequency of FRET positive cells in the double positive YFP and mCherry gate. Data shows average ± SD from 6 independent experiments. In other experiments, ligands, APRIL, or BAFF (0, 5, 20, or 50 ng/ml) were added to judge the effects on FRET signal, showing no alteration in the signal (not shown). (C) For validation of complexes found in FRET, complexes forming with FLAG-TACI were precipitated with anti-FLAG sepharose beads and run on 10% PAGE gels; immunoblots were developed with an anti-HA antibody. Lower panel shows FLAG expression control. (D) To examine NF-kB luciferase induction, TACI-S, TACI-L or mutant C104R, A181E and S194X constructs were transfected into HEK-293T cells, along with NF-kB–luc reporter and control pRL-null plasmids and cultured for 48hrs; TACI expression was confirmed by western blot. (E) These cells were cultured with or without activation for 6h with 100 ng/ml APRIL or BAFF. Reporter gene activity was determined and NF-kB luciferase induction normalized to Renilla luciferase. Values reported are represented as Relative Luciferase Units (RLU) and are the mean ± SD from 5 to 7 independent experiments. * p

    Article Snippet: To examine the cytoplasmic intersection of TACI isoforms with TLR9 by confocal microscopy, we transfected HEK-293T cells with mCherry labeled TACI-S or TACI-L, or for comparison the S194X mutant, along with TLR9-YFP (pcDNA3-TLR9-YFP was a gift from Doug Golenbock; Addgene plasmid # 13642).

    Techniques: Labeling, Generated, Transfection, FACS, Polyacrylamide Gel Electrophoresis, Western Blot, Expressing, Luciferase, Mutagenesis, Construct, Cell Culture, Activation Assay, Activity Assay

    (A) TACI variants with diverse deleted extracellular domains retain capacity for complex assembly. Upper panel shows HA labeled-TACI deletion mutant products: Full length TACI-L, TACI-S (minus CRD1), exon 1 and CRD1 (ΔE1-CRD1), CRD2 (ΔCDR2), minus the remaining extracellular 59 amino acids (Δaa105-164) and excision of both CRD1 and CRD2 (Δ21-104) were constructed. (B) Constructs were transfected into HEK-293T cells along with TACI-L-FLAG; after cell lysis, precipitates were harvested with anti-FLAG sepharose beads, and complexes in lysates examined after PAGE and immunoblotting using an anti-HA antibody. FLAG expression was tested as control (lower panel). Data are representative of 3 independent experiments.

    Journal: Frontiers in Immunology

    Article Title: TACI Isoforms Regulate Ligand Binding and Receptor Function

    doi: 10.3389/fimmu.2018.02125

    Figure Lengend Snippet: (A) TACI variants with diverse deleted extracellular domains retain capacity for complex assembly. Upper panel shows HA labeled-TACI deletion mutant products: Full length TACI-L, TACI-S (minus CRD1), exon 1 and CRD1 (ΔE1-CRD1), CRD2 (ΔCDR2), minus the remaining extracellular 59 amino acids (Δaa105-164) and excision of both CRD1 and CRD2 (Δ21-104) were constructed. (B) Constructs were transfected into HEK-293T cells along with TACI-L-FLAG; after cell lysis, precipitates were harvested with anti-FLAG sepharose beads, and complexes in lysates examined after PAGE and immunoblotting using an anti-HA antibody. FLAG expression was tested as control (lower panel). Data are representative of 3 independent experiments.

    Article Snippet: To examine the cytoplasmic intersection of TACI isoforms with TLR9 by confocal microscopy, we transfected HEK-293T cells with mCherry labeled TACI-S or TACI-L, or for comparison the S194X mutant, along with TLR9-YFP (pcDNA3-TLR9-YFP was a gift from Doug Golenbock; Addgene plasmid # 13642).

    Techniques: Labeling, Mutagenesis, Construct, Transfection, Lysis, Polyacrylamide Gel Electrophoresis, Expressing

    TACI-S display increased ligand binding. (A) To compare ligand binding capacities of isoforms, varying concentrations of FLAG-APRIL (0–50 ng/ml) were incubated with TACI-L or TACI-S transduced BJAB cells. Ligand binding was determined by FACS (LSRII) on washed cells, after incubation with anti-FLAG-PE. Comparable expression of TACI was determined by FACS (LSRII) using GFP expression. (B,C) To compare ligand binding in TACI-L or TACI-S transfected HEK-293T cells, cells were incubated with increasing amounts (0-800 ng) of either FLAG-BAFF (B) or FLAG-APRIL (C) . Comparable expression of TACI isoforms in each case was determined by FACS (LSRII) using anti-TACI antibody. (D) The binding affinity of TACI-L and TACI-S to ligands BAFF and APRIL was determined by Microscale thermophoresis (MST). The change in the thermophoretic signal produces these K d values (nM). Data represent the mean and SD of three independent thermophoresis measurements. (E) BJAB cells transduced with TACI-S and TACI-L, displayed binding of FLAG-APRIL intermediate between the TACI-S and TACI-L. (F) Diminishing ligand binding was also found for TACI-S HEK-293T cells also transfected with increasing ratios of TACI-L (ratios 1:1, 5:1, or 10:1) and incubated with to 0–500 ng of FLAG-APRIL. (G) HEK-293T cells transfected with both TACI-S and TACI-L, demonstrated intermediate NF-kB luciferase induction as compared to cells with either isoform. Values reported are represented as Mean of Fluorescence Intensity (MFI) (A-C,E,F) and as Relative Luciferase Units (RLU) (G) and are the mean ± SEM from 5 to 7 independent experiments. * p

    Journal: Frontiers in Immunology

    Article Title: TACI Isoforms Regulate Ligand Binding and Receptor Function

    doi: 10.3389/fimmu.2018.02125

    Figure Lengend Snippet: TACI-S display increased ligand binding. (A) To compare ligand binding capacities of isoforms, varying concentrations of FLAG-APRIL (0–50 ng/ml) were incubated with TACI-L or TACI-S transduced BJAB cells. Ligand binding was determined by FACS (LSRII) on washed cells, after incubation with anti-FLAG-PE. Comparable expression of TACI was determined by FACS (LSRII) using GFP expression. (B,C) To compare ligand binding in TACI-L or TACI-S transfected HEK-293T cells, cells were incubated with increasing amounts (0-800 ng) of either FLAG-BAFF (B) or FLAG-APRIL (C) . Comparable expression of TACI isoforms in each case was determined by FACS (LSRII) using anti-TACI antibody. (D) The binding affinity of TACI-L and TACI-S to ligands BAFF and APRIL was determined by Microscale thermophoresis (MST). The change in the thermophoretic signal produces these K d values (nM). Data represent the mean and SD of three independent thermophoresis measurements. (E) BJAB cells transduced with TACI-S and TACI-L, displayed binding of FLAG-APRIL intermediate between the TACI-S and TACI-L. (F) Diminishing ligand binding was also found for TACI-S HEK-293T cells also transfected with increasing ratios of TACI-L (ratios 1:1, 5:1, or 10:1) and incubated with to 0–500 ng of FLAG-APRIL. (G) HEK-293T cells transfected with both TACI-S and TACI-L, demonstrated intermediate NF-kB luciferase induction as compared to cells with either isoform. Values reported are represented as Mean of Fluorescence Intensity (MFI) (A-C,E,F) and as Relative Luciferase Units (RLU) (G) and are the mean ± SEM from 5 to 7 independent experiments. * p

    Article Snippet: To examine the cytoplasmic intersection of TACI isoforms with TLR9 by confocal microscopy, we transfected HEK-293T cells with mCherry labeled TACI-S or TACI-L, or for comparison the S194X mutant, along with TLR9-YFP (pcDNA3-TLR9-YFP was a gift from Doug Golenbock; Addgene plasmid # 13642).

    Techniques: Ligand Binding Assay, Incubation, FACS, Expressing, Transfection, Binding Assay, Microscale Thermophoresis, Transduction, Luciferase, Fluorescence

    TACI isoforms are associated with activated cleaved TLR9. (A) HEK-293T cells were transfected with either mCherry labeled TACI-S or TACI-L or the S194X mutant (red) along with TLR9-YFP (green); cells were examined by confocal microscopy showing co-localization (yellow) in cells expressing TACI-L or –S, but absent in cells with the S194X mutant. (B) HEK-293T cells were transfected with either TACI-S, TACI-L, or the S194X mutant with TLR9-YFP, to determine complex formation using FRET using FACS (LSRFortessa) as described above. Data is representative of 10 experiments. Right panel shows the % of FRET positive cells in the double positive YFP and mCherry gate in these experiments. Scatter-plot graph shows the mean ± SD. *** p

    Journal: Frontiers in Immunology

    Article Title: TACI Isoforms Regulate Ligand Binding and Receptor Function

    doi: 10.3389/fimmu.2018.02125

    Figure Lengend Snippet: TACI isoforms are associated with activated cleaved TLR9. (A) HEK-293T cells were transfected with either mCherry labeled TACI-S or TACI-L or the S194X mutant (red) along with TLR9-YFP (green); cells were examined by confocal microscopy showing co-localization (yellow) in cells expressing TACI-L or –S, but absent in cells with the S194X mutant. (B) HEK-293T cells were transfected with either TACI-S, TACI-L, or the S194X mutant with TLR9-YFP, to determine complex formation using FRET using FACS (LSRFortessa) as described above. Data is representative of 10 experiments. Right panel shows the % of FRET positive cells in the double positive YFP and mCherry gate in these experiments. Scatter-plot graph shows the mean ± SD. *** p

    Article Snippet: To examine the cytoplasmic intersection of TACI isoforms with TLR9 by confocal microscopy, we transfected HEK-293T cells with mCherry labeled TACI-S or TACI-L, or for comparison the S194X mutant, along with TLR9-YFP (pcDNA3-TLR9-YFP was a gift from Doug Golenbock; Addgene plasmid # 13642).

    Techniques: Transfection, Labeling, Mutagenesis, Confocal Microscopy, Expressing, FACS

    (A) TACI isoforms form hybrid complexes detected by FRET. To examine isoform complexes, YFP and mCherry labeled TACI-L, TACI-S, and/or CD40-eYFP as a control, were co-transfected into HEK-293T cells. After 48 h, the molecular association was analyzed by FRET using FACS (LSRII). A minimum of 50,000 positive cells were examined in all experiments. Data are representative of 6 experiments. (B) Frequency of FRET positive cells in the double positive YFP and mCherry gate. Graph shows the mean ± SD. *** p

    Journal: Frontiers in Immunology

    Article Title: TACI Isoforms Regulate Ligand Binding and Receptor Function

    doi: 10.3389/fimmu.2018.02125

    Figure Lengend Snippet: (A) TACI isoforms form hybrid complexes detected by FRET. To examine isoform complexes, YFP and mCherry labeled TACI-L, TACI-S, and/or CD40-eYFP as a control, were co-transfected into HEK-293T cells. After 48 h, the molecular association was analyzed by FRET using FACS (LSRII). A minimum of 50,000 positive cells were examined in all experiments. Data are representative of 6 experiments. (B) Frequency of FRET positive cells in the double positive YFP and mCherry gate. Graph shows the mean ± SD. *** p

    Article Snippet: To examine the cytoplasmic intersection of TACI isoforms with TLR9 by confocal microscopy, we transfected HEK-293T cells with mCherry labeled TACI-S or TACI-L, or for comparison the S194X mutant, along with TLR9-YFP (pcDNA3-TLR9-YFP was a gift from Doug Golenbock; Addgene plasmid # 13642).

    Techniques: Labeling, Transfection, FACS

    Expression of MetYPCP of HEV ORF1 decreased signal transducer and activator of transcription protein (STAT)1 nuclear translocation upon IFN-β treatment. ( a ) Here, 293T cells were transfected with a pCINeo-3xFLAG empty vector or a plasmid coding for MetYPCP, PCP or MV-V fused to a 3xFLAG tag. Twenty-four hours post-transfection, cells were stimulated or not for 30 min with 1000 IU/mL of IFN-β. Cells were then washed, fixed and stained with primary antibodies raised against STAT1 and FLAG, followed by fluorescent dye-conjugated secondary antibodies. Intracellular localization of 4,6-diamidine-2-phenylindole dihydrochloride (DAPI)-stained nuclei (blue), FLAG (green) and STAT1 (red) was visualized by microscopy (magnification, 630×). Scale bars: 10 μm. Cells showing diffuse cytoplasmic/nuclear localisation of STAT1 upon IFN-β treatment are indicated by arrows. ( b ) STAT1 localization was visualized after immunostaining as described in ( a ) in 293T cells transfected with a pCINeo-3xFLAG empty vector or a plasmid coding for MetYPCP, PCP or MV-V fused to a FLAG tag. For each condition, STAT1 localisation (predominant nuclear localisation or diffuse localisation within the cytoplasm and nucleus) was determined in 70 to 172 cells expressing the corresponding FLAG-tagged protein (except for the EV control, for which 356 to 384 cells were randomly assessed). The mean percentage (± standard deviation) of cells showing a predominant nuclear localization of STAT1 from three independent experiments is shown: ** p

    Journal: Viruses

    Article Title: The Amino-Terminal Region of Hepatitis E Virus ORF1 Containing a Methyltransferase (Met) and a Papain-Like Cysteine Protease (PCP) Domain Counteracts Type I Interferon Response

    doi: 10.3390/v10120726

    Figure Lengend Snippet: Expression of MetYPCP of HEV ORF1 decreased signal transducer and activator of transcription protein (STAT)1 nuclear translocation upon IFN-β treatment. ( a ) Here, 293T cells were transfected with a pCINeo-3xFLAG empty vector or a plasmid coding for MetYPCP, PCP or MV-V fused to a 3xFLAG tag. Twenty-four hours post-transfection, cells were stimulated or not for 30 min with 1000 IU/mL of IFN-β. Cells were then washed, fixed and stained with primary antibodies raised against STAT1 and FLAG, followed by fluorescent dye-conjugated secondary antibodies. Intracellular localization of 4,6-diamidine-2-phenylindole dihydrochloride (DAPI)-stained nuclei (blue), FLAG (green) and STAT1 (red) was visualized by microscopy (magnification, 630×). Scale bars: 10 μm. Cells showing diffuse cytoplasmic/nuclear localisation of STAT1 upon IFN-β treatment are indicated by arrows. ( b ) STAT1 localization was visualized after immunostaining as described in ( a ) in 293T cells transfected with a pCINeo-3xFLAG empty vector or a plasmid coding for MetYPCP, PCP or MV-V fused to a FLAG tag. For each condition, STAT1 localisation (predominant nuclear localisation or diffuse localisation within the cytoplasm and nucleus) was determined in 70 to 172 cells expressing the corresponding FLAG-tagged protein (except for the EV control, for which 356 to 384 cells were randomly assessed). The mean percentage (± standard deviation) of cells showing a predominant nuclear localization of STAT1 from three independent experiments is shown: ** p

    Article Snippet: Transfections For this, 293T cells were transfected with plasmid DNA using JetPRIME (Polyplus transfection, Strasbourg, France) according to the manufacturer’s instructions.

    Techniques: Expressing, Translocation Assay, Transfection, Plasmid Preparation, Staining, Microscopy, Immunostaining, FLAG-tag, Standard Deviation

    Expression of MetYPCP of HEV ORF1 inhibited weakly STAT1 translocation but not STAT1 phosphorylation in response to IFN-II. ( a ) Here, 293T cells were transfected with a pCINeo-3xFLAG empty vector or a plasmid coding for MetYPCP or MV-V fused to a 3xFLAG tag. Twenty-four hours post-transfection, cells were stimulated for 30 min with 1000 IU/mL of IFN-β or 250 ng/mL of IFN-γ. Cells were then washed, fixed and stained with primary antibodies raised against STAT1 and FLAG, followed by fluorescent dye-conjugated secondary antibodies. STAT1 localization was determined in 64 to 102 cells expressing the corresponding FLAG-tagged protein (except for the EV control, for which 299 to 328 cells were randomly assessed). The mean percentage (± standard deviation) of cells showing a predominant nuclear localization of STAT1 from three independent experiments is shown: * p

    Journal: Viruses

    Article Title: The Amino-Terminal Region of Hepatitis E Virus ORF1 Containing a Methyltransferase (Met) and a Papain-Like Cysteine Protease (PCP) Domain Counteracts Type I Interferon Response

    doi: 10.3390/v10120726

    Figure Lengend Snippet: Expression of MetYPCP of HEV ORF1 inhibited weakly STAT1 translocation but not STAT1 phosphorylation in response to IFN-II. ( a ) Here, 293T cells were transfected with a pCINeo-3xFLAG empty vector or a plasmid coding for MetYPCP or MV-V fused to a 3xFLAG tag. Twenty-four hours post-transfection, cells were stimulated for 30 min with 1000 IU/mL of IFN-β or 250 ng/mL of IFN-γ. Cells were then washed, fixed and stained with primary antibodies raised against STAT1 and FLAG, followed by fluorescent dye-conjugated secondary antibodies. STAT1 localization was determined in 64 to 102 cells expressing the corresponding FLAG-tagged protein (except for the EV control, for which 299 to 328 cells were randomly assessed). The mean percentage (± standard deviation) of cells showing a predominant nuclear localization of STAT1 from three independent experiments is shown: * p

    Article Snippet: Transfections For this, 293T cells were transfected with plasmid DNA using JetPRIME (Polyplus transfection, Strasbourg, France) according to the manufacturer’s instructions.

    Techniques: Expressing, Translocation Assay, Transfection, Plasmid Preparation, Staining, Standard Deviation

    Expression of MetYPCP of HEV ORF1 downregulated mRNA levels of several ISGs following IFN-β treatment. ( a – c ) Here, 293T cells were transfected with a pCINeo-3xFLAG empty vector or a plasmid coding for MetYPCP, PCP or MV-V fused to a 3xFLAG tag. Forty hours post-transfection, cells were stimulated or not (NT) with 500 IU/mL of IFN-β for 6 h. Total RNA was extracted, and expression of the mRNA coding for ( a ) ISG56, ( b ) melanoma differentiation-associated protein (MDA)5 and ( c ) 2′,5′-oligoadenylate synthetase (OAS)1 were measured by RT-qPCR. Glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) was used as a reference gene. Relative mRNA expression was calculated for each condition and is presented as percentages of the treated EV control (± standard deviations). Results shown represent the mean of three independent experiments performed in triplicate: *, p

    Journal: Viruses

    Article Title: The Amino-Terminal Region of Hepatitis E Virus ORF1 Containing a Methyltransferase (Met) and a Papain-Like Cysteine Protease (PCP) Domain Counteracts Type I Interferon Response

    doi: 10.3390/v10120726

    Figure Lengend Snippet: Expression of MetYPCP of HEV ORF1 downregulated mRNA levels of several ISGs following IFN-β treatment. ( a – c ) Here, 293T cells were transfected with a pCINeo-3xFLAG empty vector or a plasmid coding for MetYPCP, PCP or MV-V fused to a 3xFLAG tag. Forty hours post-transfection, cells were stimulated or not (NT) with 500 IU/mL of IFN-β for 6 h. Total RNA was extracted, and expression of the mRNA coding for ( a ) ISG56, ( b ) melanoma differentiation-associated protein (MDA)5 and ( c ) 2′,5′-oligoadenylate synthetase (OAS)1 were measured by RT-qPCR. Glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) was used as a reference gene. Relative mRNA expression was calculated for each condition and is presented as percentages of the treated EV control (± standard deviations). Results shown represent the mean of three independent experiments performed in triplicate: *, p

    Article Snippet: Transfections For this, 293T cells were transfected with plasmid DNA using JetPRIME (Polyplus transfection, Strasbourg, France) according to the manufacturer’s instructions.

    Techniques: Expressing, Transfection, Plasmid Preparation, Multiple Displacement Amplification, Quantitative RT-PCR

    Comparison of the effect of MetYPCP from HEV-1 and HEV-3 on the Janus kinase (JAK)/STAT pathway. ( a ) Expression of FLAG-tagged MetYPCP and PCP from a strain of HEV-1 (MetYPCP-G1 and PCP-G1) and HEV-3 (MetYPCP-G3 and PCP-G3) in 293T cells detected by immunoblotting using an anti-FLAG antibody. Actin served as a loading control. Cells were lysed 24 h post-transfection. ( b ) Effect of MetYPCP and PCP from HEV-1 and HEV-3 on ISRE promoter activation: 293T cells were transfected with pISRE-Luc, pCMV-Luc and a pCINeo-3xFLAG empty vector or a plasmid coding for MV-V, MetYPCP-G1, MetYPCP-G3, PCP-G1 or PCP-G3. Forty h later, cells were treated or not (NT) with IFN-β for 7 h and lysed to determine firefly and Renilla luciferase activities. Mean ratios between firefly and Renilla luciferase activities were calculated and are presented as percentages of the treated EV control (± standard deviations). Results shown represent the mean of five independent experiments performed in triplicate: * p

    Journal: Viruses

    Article Title: The Amino-Terminal Region of Hepatitis E Virus ORF1 Containing a Methyltransferase (Met) and a Papain-Like Cysteine Protease (PCP) Domain Counteracts Type I Interferon Response

    doi: 10.3390/v10120726

    Figure Lengend Snippet: Comparison of the effect of MetYPCP from HEV-1 and HEV-3 on the Janus kinase (JAK)/STAT pathway. ( a ) Expression of FLAG-tagged MetYPCP and PCP from a strain of HEV-1 (MetYPCP-G1 and PCP-G1) and HEV-3 (MetYPCP-G3 and PCP-G3) in 293T cells detected by immunoblotting using an anti-FLAG antibody. Actin served as a loading control. Cells were lysed 24 h post-transfection. ( b ) Effect of MetYPCP and PCP from HEV-1 and HEV-3 on ISRE promoter activation: 293T cells were transfected with pISRE-Luc, pCMV-Luc and a pCINeo-3xFLAG empty vector or a plasmid coding for MV-V, MetYPCP-G1, MetYPCP-G3, PCP-G1 or PCP-G3. Forty h later, cells were treated or not (NT) with IFN-β for 7 h and lysed to determine firefly and Renilla luciferase activities. Mean ratios between firefly and Renilla luciferase activities were calculated and are presented as percentages of the treated EV control (± standard deviations). Results shown represent the mean of five independent experiments performed in triplicate: * p

    Article Snippet: Transfections For this, 293T cells were transfected with plasmid DNA using JetPRIME (Polyplus transfection, Strasbourg, France) according to the manufacturer’s instructions.

    Techniques: Expressing, Transfection, Activation Assay, Plasmid Preparation, Luciferase

    Effect of the expression of full-length HEV ORF1 and several of its domains on IFN-stimulated response element (ISRE) promoter activation. ( a ) Schematic representation of the different domains of HEV ORF1. Met: Methyltransferase domain; Y: Y domain; PCP: Papain-like cysteine protease; HVR: Hypervariable region; X: Macrodomain; Hel: Helicase domain; RdRp: RNA-dependent RNA polymerase. The position of the different putative functional domains present in the ORF1 amino acid sequence of the HEV-3 strain used in this study is indicated. The different fragments of ORF1 that were cloned and expressed in 293T cells are represented by arrows. ( b ) Expression of FLAG-tagged full-length and domains of ORF1 in 293T cells detected by immunoblotting using an anti-FLAG antibody. Bands corresponding to PCP (arrow) and PCP products of higher molecular weight (asterisks) are indicated. Actin served as a loading control. Cells were lysed 18 h post-transfection. ( c ) Effect of full-length ORF1, MetYPCP, Y, PCP, macrodomain (X), Met, MetY and YPCP on ISRE promoter activation: 293T cells were transfected with pISRE-Luc, pCMV-Luc and a pCINeo-3xFLAG empty vector (EV) or a plasmid coding for ORF1, MetYPCP, Y, PCP, X, Met, MetY, YPCP or MV-V. Forty hours later, cells were treated or not (NT) with IFN-β for 7 h and lysed to determine firefly and Renilla luciferase activities. Mean ratios between firefly and Renilla luciferase activities were calculated and are presented as percentages of the treated EV control (± standard deviations). Results shown represent the mean of four independent experiments performed in triplicate. Here, * p

    Journal: Viruses

    Article Title: The Amino-Terminal Region of Hepatitis E Virus ORF1 Containing a Methyltransferase (Met) and a Papain-Like Cysteine Protease (PCP) Domain Counteracts Type I Interferon Response

    doi: 10.3390/v10120726

    Figure Lengend Snippet: Effect of the expression of full-length HEV ORF1 and several of its domains on IFN-stimulated response element (ISRE) promoter activation. ( a ) Schematic representation of the different domains of HEV ORF1. Met: Methyltransferase domain; Y: Y domain; PCP: Papain-like cysteine protease; HVR: Hypervariable region; X: Macrodomain; Hel: Helicase domain; RdRp: RNA-dependent RNA polymerase. The position of the different putative functional domains present in the ORF1 amino acid sequence of the HEV-3 strain used in this study is indicated. The different fragments of ORF1 that were cloned and expressed in 293T cells are represented by arrows. ( b ) Expression of FLAG-tagged full-length and domains of ORF1 in 293T cells detected by immunoblotting using an anti-FLAG antibody. Bands corresponding to PCP (arrow) and PCP products of higher molecular weight (asterisks) are indicated. Actin served as a loading control. Cells were lysed 18 h post-transfection. ( c ) Effect of full-length ORF1, MetYPCP, Y, PCP, macrodomain (X), Met, MetY and YPCP on ISRE promoter activation: 293T cells were transfected with pISRE-Luc, pCMV-Luc and a pCINeo-3xFLAG empty vector (EV) or a plasmid coding for ORF1, MetYPCP, Y, PCP, X, Met, MetY, YPCP or MV-V. Forty hours later, cells were treated or not (NT) with IFN-β for 7 h and lysed to determine firefly and Renilla luciferase activities. Mean ratios between firefly and Renilla luciferase activities were calculated and are presented as percentages of the treated EV control (± standard deviations). Results shown represent the mean of four independent experiments performed in triplicate. Here, * p

    Article Snippet: Transfections For this, 293T cells were transfected with plasmid DNA using JetPRIME (Polyplus transfection, Strasbourg, France) according to the manufacturer’s instructions.

    Techniques: Expressing, Activation Assay, Functional Assay, Sequencing, Clone Assay, Molecular Weight, Transfection, Plasmid Preparation, Luciferase

    Expression of MetYPCP of HEV ORF1 inhibited STAT1 but not STAT2 phosphorylation upon IFN-β treatment. ( a ) Here, 293T cells were transfected with a pCINeo-3xFLAG empty vector or a plasmid coding for MetYPCP, PCP or MV-V fused to a 3xFLAG tag. Twenty-four hours post-transfection, cells were stimulated for 30 min with 500 IU/mL of IFN-β. Cell lysates were extracted and used for the detection of FLAG-tagged proteins, total STAT1, phosphorylated STAT1 (p-STAT1), total STAT2 and phosphorylated STAT2 (p-STAT2) by immunoblotting. Actin served as an internal control. ( b ) Here, 293T cells were transfected with an empty vector or a plasmid coding for MetYPCP, PCP or MV-V and treated as described in ( a ). Cell lysates were extracted and used for the detection of total STAT1, p-STAT1 and actin by immunoblotting. Band intensities were quantified using ImageJ software, and relative levels of STAT1, p-STAT1 and actin were determined for each treated sample. Ratios between p-STAT1 and actin, STAT1 and actin, and p-STAT1 and STAT1 were calculated and expressed as a relative percentage in comparison to the EV control. ( c ) Here, 293T cells were transfected with an empty vector or a plasmid coding for MetYPCP or MV-V and treated as described in ( a ). Cell lysates were extracted and used for the detection of total STAT2, p-STAT2 and p-STAT1 by immunoblotting. Band intensities were quantified using ImageJ software, and relative levels of STAT2, p-STAT2, p-STAT1 and actin were determined for each treated sample. Ratios between p-STAT2 and actin and STAT2 and actin were calculated and expressed as a relative percentage in comparison to the EV control. The ratio between p-STAT1 and actin was also determined to ensure significant inhibition of the p-STAT1 level by MetYPCP in this set of experiments. In ( b – c ), the mean percentage (± standard deviation) of four independent experiments is presented for each panel: * p

    Journal: Viruses

    Article Title: The Amino-Terminal Region of Hepatitis E Virus ORF1 Containing a Methyltransferase (Met) and a Papain-Like Cysteine Protease (PCP) Domain Counteracts Type I Interferon Response

    doi: 10.3390/v10120726

    Figure Lengend Snippet: Expression of MetYPCP of HEV ORF1 inhibited STAT1 but not STAT2 phosphorylation upon IFN-β treatment. ( a ) Here, 293T cells were transfected with a pCINeo-3xFLAG empty vector or a plasmid coding for MetYPCP, PCP or MV-V fused to a 3xFLAG tag. Twenty-four hours post-transfection, cells were stimulated for 30 min with 500 IU/mL of IFN-β. Cell lysates were extracted and used for the detection of FLAG-tagged proteins, total STAT1, phosphorylated STAT1 (p-STAT1), total STAT2 and phosphorylated STAT2 (p-STAT2) by immunoblotting. Actin served as an internal control. ( b ) Here, 293T cells were transfected with an empty vector or a plasmid coding for MetYPCP, PCP or MV-V and treated as described in ( a ). Cell lysates were extracted and used for the detection of total STAT1, p-STAT1 and actin by immunoblotting. Band intensities were quantified using ImageJ software, and relative levels of STAT1, p-STAT1 and actin were determined for each treated sample. Ratios between p-STAT1 and actin, STAT1 and actin, and p-STAT1 and STAT1 were calculated and expressed as a relative percentage in comparison to the EV control. ( c ) Here, 293T cells were transfected with an empty vector or a plasmid coding for MetYPCP or MV-V and treated as described in ( a ). Cell lysates were extracted and used for the detection of total STAT2, p-STAT2 and p-STAT1 by immunoblotting. Band intensities were quantified using ImageJ software, and relative levels of STAT2, p-STAT2, p-STAT1 and actin were determined for each treated sample. Ratios between p-STAT2 and actin and STAT2 and actin were calculated and expressed as a relative percentage in comparison to the EV control. The ratio between p-STAT1 and actin was also determined to ensure significant inhibition of the p-STAT1 level by MetYPCP in this set of experiments. In ( b – c ), the mean percentage (± standard deviation) of four independent experiments is presented for each panel: * p

    Article Snippet: Transfections For this, 293T cells were transfected with plasmid DNA using JetPRIME (Polyplus transfection, Strasbourg, France) according to the manufacturer’s instructions.

    Techniques: Expressing, Transfection, Plasmid Preparation, Software, Inhibition, Standard Deviation

    Cell Surface Shed and Secreted, Soluble HLA-G Is Endocytosed into KIR2DL4-Containing Vesicles (A) Endocytosis of soluble HLA-G in resting NK cells. The 221 cells and 221 cells transfected with HLA-Cw3 (221-Cw3) were fixed, permeabilized, and stained with mAb F4/326. Resting NK cells were incubated at 37 °C for 120 min with soluble, refolded HLA-C or HLA-G. Cells were then fixed, permeabilized, and stained with reagents to detect HLA-C (F4/326) or HLA-G (G233) as indicated. (B) The NK cell line YTS-2DL4-gfp was loaded at 37 °C for 120 min with refolded HLA-G. Cells were fixed, permeabilized, and stained with mAb G233 to detect co-localization of soluble HLA-G with gfp-tagged KIR2DL4. (C) Recombinant soluble molecules of HLA-G but not HLA-C are endocytosed into 293T-2DL4-gfp cells. Refolded HLA-G and HLA-C were incubated with 293T-2DL4-gfp cells for 2 h. Cells were then fixed, permeabilized, and stained with either mAb G233 (to detect endocytosed HLA-G; upper) or mAb F4/326 (to detect endocytosed HLA-C; middle). (D) The 293T-2DL4-gfp cells were co-cultured with an equal number of 221 cells, 221 cells expressing transmembrane HLA-G (221-G), and 221 cells expressing a soluble isoform of HLA-G (221-sG) for 48 h. Adherent 293T-2DL4-gfp cells were fixed, permeabilized, and stained with mAb G233 followed by Alexa-568–conjugated secondary antibodies prior to acquisition of confocal images. Two 221-G cells are visible in the middle panel.

    Journal: PLoS Biology

    Article Title: Activation of NK Cells by an Endocytosed Receptor for Soluble HLA-GInside a Killer: Immune Signals May Promote Vascular Growth

    doi: 10.1371/journal.pbio.0040009

    Figure Lengend Snippet: Cell Surface Shed and Secreted, Soluble HLA-G Is Endocytosed into KIR2DL4-Containing Vesicles (A) Endocytosis of soluble HLA-G in resting NK cells. The 221 cells and 221 cells transfected with HLA-Cw3 (221-Cw3) were fixed, permeabilized, and stained with mAb F4/326. Resting NK cells were incubated at 37 °C for 120 min with soluble, refolded HLA-C or HLA-G. Cells were then fixed, permeabilized, and stained with reagents to detect HLA-C (F4/326) or HLA-G (G233) as indicated. (B) The NK cell line YTS-2DL4-gfp was loaded at 37 °C for 120 min with refolded HLA-G. Cells were fixed, permeabilized, and stained with mAb G233 to detect co-localization of soluble HLA-G with gfp-tagged KIR2DL4. (C) Recombinant soluble molecules of HLA-G but not HLA-C are endocytosed into 293T-2DL4-gfp cells. Refolded HLA-G and HLA-C were incubated with 293T-2DL4-gfp cells for 2 h. Cells were then fixed, permeabilized, and stained with either mAb G233 (to detect endocytosed HLA-G; upper) or mAb F4/326 (to detect endocytosed HLA-C; middle). (D) The 293T-2DL4-gfp cells were co-cultured with an equal number of 221 cells, 221 cells expressing transmembrane HLA-G (221-G), and 221 cells expressing a soluble isoform of HLA-G (221-sG) for 48 h. Adherent 293T-2DL4-gfp cells were fixed, permeabilized, and stained with mAb G233 followed by Alexa-568–conjugated secondary antibodies prior to acquisition of confocal images. Two 221-G cells are visible in the middle panel.

    Article Snippet: Expression constructs for transfection into 293T cells consisted of cDNAs for 2B4 [ ], mouse gp49B [ ], KIR2DL4, and KIR2DL4 variants cloned into the pDisplay vector (Stratagene, La Jolla, California, United States) that introduces an HA-tag at the amino-terminus.

    Techniques: Transfection, Staining, Incubation, Recombinant, Cell Culture, Expressing

    Endocytosis of Soluble HLA-G into 29 3T-2DL4-gfp Cells Is Blocked by Anti-KIR2DL4 mAb and by Soluble KIR2DL4 (A) Recombinant, soluble, sHLA-G at 50 μg/ml or mAb 33 (50 μg/ml) was incubated with 293T-2DL4-gfp cells. sHLA-G was also incubated together with 50 μg/ml mAb 33, 50 μg/ml KIR2DL1-Ig, or 50 μg/ml KIR2DL4-Ig, as indicated on the left. Cells were fixed, permeabilized, and stained with either Alexa-568–conjugated secondary antibodies to detect mAb 33 or anti-HLA-G mAb G233, as indicated. Individual confocal sections are shown. (B) Uptake of sHLA-G into 293T-2DL4-gfp cells correlates with level of KIR2DL4 expression. Red fluorescence intensity of G233 staining and green fluorescence intensity of gfp were quantified in 38 individual cells and plotted on a log scale. A best-fit line was generated by linear regression analysis using EXCEL data analysis software. (C) Ratio of red to green fluorescence was quantified for each loading condition as indicated. Average of 10 cells is shown, and standard deviation is shown as bars.

    Journal: PLoS Biology

    Article Title: Activation of NK Cells by an Endocytosed Receptor for Soluble HLA-GInside a Killer: Immune Signals May Promote Vascular Growth

    doi: 10.1371/journal.pbio.0040009

    Figure Lengend Snippet: Endocytosis of Soluble HLA-G into 29 3T-2DL4-gfp Cells Is Blocked by Anti-KIR2DL4 mAb and by Soluble KIR2DL4 (A) Recombinant, soluble, sHLA-G at 50 μg/ml or mAb 33 (50 μg/ml) was incubated with 293T-2DL4-gfp cells. sHLA-G was also incubated together with 50 μg/ml mAb 33, 50 μg/ml KIR2DL1-Ig, or 50 μg/ml KIR2DL4-Ig, as indicated on the left. Cells were fixed, permeabilized, and stained with either Alexa-568–conjugated secondary antibodies to detect mAb 33 or anti-HLA-G mAb G233, as indicated. Individual confocal sections are shown. (B) Uptake of sHLA-G into 293T-2DL4-gfp cells correlates with level of KIR2DL4 expression. Red fluorescence intensity of G233 staining and green fluorescence intensity of gfp were quantified in 38 individual cells and plotted on a log scale. A best-fit line was generated by linear regression analysis using EXCEL data analysis software. (C) Ratio of red to green fluorescence was quantified for each loading condition as indicated. Average of 10 cells is shown, and standard deviation is shown as bars.

    Article Snippet: Expression constructs for transfection into 293T cells consisted of cDNAs for 2B4 [ ], mouse gp49B [ ], KIR2DL4, and KIR2DL4 variants cloned into the pDisplay vector (Stratagene, La Jolla, California, United States) that introduces an HA-tag at the amino-terminus.

    Techniques: Recombinant, Incubation, Staining, Expressing, Fluorescence, Generated, Software, Standard Deviation

    KIR2DL4 Is Endocytosed into Intracellular Vesicles (A) The B cell line 721.221 (221), resting NK cells, and activated NK cells were fixed, permeabilized, and stained with Cy3-conjugated mAb 33. In the image on the right, resting NK cells were fixed, permeabilized, and stained with a polyclonal antibody specific for the tail of KIR2DL1, followed by Alexa-568–conjugated secondary antibodies. (B) Resting NK cells were incubated at 37 °C for 30 min or 120 min with KIR2DL4-specific mAb 33-Cy3, with a Fab of control mAb 2A8 or with a Fab of anti-KIR2DL4 mAb 33, as indicated. Cells were then fixed and analyzed by confocal microscopy. (C) The 293T cells stably transfected with KIR2DL4-gfp (293T-2DL4-gfp) were fixed, permeabilized, and stained with anti-KIR2DL4 mAb 33 followed by Alexa-568–conjugated secondary antibodies to show co-localization with KIR2DL4-gfp. Single confocal sections are shown. (D) The 293T-2DL4-gfp cells were incubated at 37 °C for 120 min with anti-KIR2DL4 mAb 33, either intact (33 whole Ab) or as a Fab (33-Fab). Cells were fixed, stained with Alexa-568–conjugated secondary antibodies, and analyzed by confocal microscopy. Single confocal sections are shown.

    Journal: PLoS Biology

    Article Title: Activation of NK Cells by an Endocytosed Receptor for Soluble HLA-GInside a Killer: Immune Signals May Promote Vascular Growth

    doi: 10.1371/journal.pbio.0040009

    Figure Lengend Snippet: KIR2DL4 Is Endocytosed into Intracellular Vesicles (A) The B cell line 721.221 (221), resting NK cells, and activated NK cells were fixed, permeabilized, and stained with Cy3-conjugated mAb 33. In the image on the right, resting NK cells were fixed, permeabilized, and stained with a polyclonal antibody specific for the tail of KIR2DL1, followed by Alexa-568–conjugated secondary antibodies. (B) Resting NK cells were incubated at 37 °C for 30 min or 120 min with KIR2DL4-specific mAb 33-Cy3, with a Fab of control mAb 2A8 or with a Fab of anti-KIR2DL4 mAb 33, as indicated. Cells were then fixed and analyzed by confocal microscopy. (C) The 293T cells stably transfected with KIR2DL4-gfp (293T-2DL4-gfp) were fixed, permeabilized, and stained with anti-KIR2DL4 mAb 33 followed by Alexa-568–conjugated secondary antibodies to show co-localization with KIR2DL4-gfp. Single confocal sections are shown. (D) The 293T-2DL4-gfp cells were incubated at 37 °C for 120 min with anti-KIR2DL4 mAb 33, either intact (33 whole Ab) or as a Fab (33-Fab). Cells were fixed, stained with Alexa-568–conjugated secondary antibodies, and analyzed by confocal microscopy. Single confocal sections are shown.

    Article Snippet: Expression constructs for transfection into 293T cells consisted of cDNAs for 2B4 [ ], mouse gp49B [ ], KIR2DL4, and KIR2DL4 variants cloned into the pDisplay vector (Stratagene, La Jolla, California, United States) that introduces an HA-tag at the amino-terminus.

    Techniques: Staining, Incubation, Confocal Microscopy, Stable Transfection, Transfection

    IL-8 Secretion Induced by KIR2DL4 Is Independent of the Transmembrane Arginine Residue and Requires Trafficking to Intracellular Vesicles (A) Expression of KIR2DL4 in 293T cells induces IL-8 secretion. The 293T cells were transfected with HA-tagged 2B4, gp49B, and KIR2DL4. After 48 h, culture supernatants were tested for IL-8 by ELISA. (B) Schematic representation of KIR2DL4 variants used in this study. Receptor localization was determined by confocal analysis of immunofluorescence staining of 293T cells transfected with the indicated constructs for 48 h. (C) IL-8 secretion induced by KIR2DL4 mutants in 293T cells. The 293T cells were transfected with HA-tagged KIR2DL4, KIR2DL4(RY-GT), and KIR2DL4-TR. After 48 h, culture supernatants were tested for IL-8 by ELISA. Transfection efficiency was verified by monitoring HA-positive cells by confocal microscopy. (D) Cell surface–expressed gp49B/2DL4 chimera does not induce IL-8 secretion. The 293T cells were transfected with HA-tagged 2B4, KIR2DL4, and gp49B/2DL4 chimera, as indicated. After 48 h, beads coated with control IgG (solid bars) or anti-HA mAb (hatched bars) were added at four beads per cell. Twelve hours later, culture supernatants were tested for IL-8 secretion. The percentage of 293T cells expressing receptor at the cell surface was monitored by HA staining and flow cytometry, and was as follows: vector control, 2%; 2B4, 39%; KIR2DL4, 9%; gp49B, 31%; gp49B/2DL4, 26%.

    Journal: PLoS Biology

    Article Title: Activation of NK Cells by an Endocytosed Receptor for Soluble HLA-GInside a Killer: Immune Signals May Promote Vascular Growth

    doi: 10.1371/journal.pbio.0040009

    Figure Lengend Snippet: IL-8 Secretion Induced by KIR2DL4 Is Independent of the Transmembrane Arginine Residue and Requires Trafficking to Intracellular Vesicles (A) Expression of KIR2DL4 in 293T cells induces IL-8 secretion. The 293T cells were transfected with HA-tagged 2B4, gp49B, and KIR2DL4. After 48 h, culture supernatants were tested for IL-8 by ELISA. (B) Schematic representation of KIR2DL4 variants used in this study. Receptor localization was determined by confocal analysis of immunofluorescence staining of 293T cells transfected with the indicated constructs for 48 h. (C) IL-8 secretion induced by KIR2DL4 mutants in 293T cells. The 293T cells were transfected with HA-tagged KIR2DL4, KIR2DL4(RY-GT), and KIR2DL4-TR. After 48 h, culture supernatants were tested for IL-8 by ELISA. Transfection efficiency was verified by monitoring HA-positive cells by confocal microscopy. (D) Cell surface–expressed gp49B/2DL4 chimera does not induce IL-8 secretion. The 293T cells were transfected with HA-tagged 2B4, KIR2DL4, and gp49B/2DL4 chimera, as indicated. After 48 h, beads coated with control IgG (solid bars) or anti-HA mAb (hatched bars) were added at four beads per cell. Twelve hours later, culture supernatants were tested for IL-8 secretion. The percentage of 293T cells expressing receptor at the cell surface was monitored by HA staining and flow cytometry, and was as follows: vector control, 2%; 2B4, 39%; KIR2DL4, 9%; gp49B, 31%; gp49B/2DL4, 26%.

    Article Snippet: Expression constructs for transfection into 293T cells consisted of cDNAs for 2B4 [ ], mouse gp49B [ ], KIR2DL4, and KIR2DL4 variants cloned into the pDisplay vector (Stratagene, La Jolla, California, United States) that introduces an HA-tag at the amino-terminus.

    Techniques: Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Construct, Confocal Microscopy, Flow Cytometry, Cytometry, Plasmid Preparation

    KIR2DL4 Resides in Endocytic Compartments Resting NK cells and 293T-2DL4-gfp cells were fixed, permeabilized, and stained with antibodies against Rab5, EEA-1, and M6PR, followed by Alexa-568–conjugated secondary antibodies. NK cells were further stained with mAb 33 coupled to Alexa-488 to detect KIR2DL4.

    Journal: PLoS Biology

    Article Title: Activation of NK Cells by an Endocytosed Receptor for Soluble HLA-GInside a Killer: Immune Signals May Promote Vascular Growth

    doi: 10.1371/journal.pbio.0040009

    Figure Lengend Snippet: KIR2DL4 Resides in Endocytic Compartments Resting NK cells and 293T-2DL4-gfp cells were fixed, permeabilized, and stained with antibodies against Rab5, EEA-1, and M6PR, followed by Alexa-568–conjugated secondary antibodies. NK cells were further stained with mAb 33 coupled to Alexa-488 to detect KIR2DL4.

    Article Snippet: Expression constructs for transfection into 293T cells consisted of cDNAs for 2B4 [ ], mouse gp49B [ ], KIR2DL4, and KIR2DL4 variants cloned into the pDisplay vector (Stratagene, La Jolla, California, United States) that introduces an HA-tag at the amino-terminus.

    Techniques: Staining

    KIR2DL4 Localizes to a Subset of Endosomes Containing Rab5 The 293T cells were transfected with HA-tagged KIR2DL4 and gfp-tagged versions of Rab4, Rab5, Rab7, and Rab11. Forty hours after transfection, cells were fixed and stained with anti-HA mAb, followed by Alexa-568–conjugated secondary antibodies to detect KIR2DL4. Single confocal sections are shown.

    Journal: PLoS Biology

    Article Title: Activation of NK Cells by an Endocytosed Receptor for Soluble HLA-GInside a Killer: Immune Signals May Promote Vascular Growth

    doi: 10.1371/journal.pbio.0040009

    Figure Lengend Snippet: KIR2DL4 Localizes to a Subset of Endosomes Containing Rab5 The 293T cells were transfected with HA-tagged KIR2DL4 and gfp-tagged versions of Rab4, Rab5, Rab7, and Rab11. Forty hours after transfection, cells were fixed and stained with anti-HA mAb, followed by Alexa-568–conjugated secondary antibodies to detect KIR2DL4. Single confocal sections are shown.

    Article Snippet: Expression constructs for transfection into 293T cells consisted of cDNAs for 2B4 [ ], mouse gp49B [ ], KIR2DL4, and KIR2DL4 variants cloned into the pDisplay vector (Stratagene, La Jolla, California, United States) that introduces an HA-tag at the amino-terminus.

    Techniques: Transfection, Staining

    Internalization of KIR2DL4 Is Dynamin Dependent The 293T cells transfected with HA-tagged KIR2DL4 together with either wild-type dynamin-gfp (Dynamin Egfp-WT) or a dominant-negative mutant of dynamin (dynamin Egfp-K44A) were loaded with KIR2DL4-specific Cy3-conjugated mAb 33 for 120 min and fixed. Individual confocal sections are shown.

    Journal: PLoS Biology

    Article Title: Activation of NK Cells by an Endocytosed Receptor for Soluble HLA-GInside a Killer: Immune Signals May Promote Vascular Growth

    doi: 10.1371/journal.pbio.0040009

    Figure Lengend Snippet: Internalization of KIR2DL4 Is Dynamin Dependent The 293T cells transfected with HA-tagged KIR2DL4 together with either wild-type dynamin-gfp (Dynamin Egfp-WT) or a dominant-negative mutant of dynamin (dynamin Egfp-K44A) were loaded with KIR2DL4-specific Cy3-conjugated mAb 33 for 120 min and fixed. Individual confocal sections are shown.

    Article Snippet: Expression constructs for transfection into 293T cells consisted of cDNAs for 2B4 [ ], mouse gp49B [ ], KIR2DL4, and KIR2DL4 variants cloned into the pDisplay vector (Stratagene, La Jolla, California, United States) that introduces an HA-tag at the amino-terminus.

    Techniques: Transfection, Dominant Negative Mutation

    Micrographs of 293T cells transfected with pEGFP-N1 or pEGFP-N1-BDNF after transfection at the various time points by fluorescence microscopy (× 200). (A1, A2, B1, B2, C1, C2, D1 and D2): 293T cells transfected with pEGFP-N1-BDNF 24–96 hours (h) after transfection under fluorescence and light microscopes. A2, B2, C2, D2 are light micrographs of the same visual field as A1, B1, C1, D1, respectively. With increasing time after transfection, EGFP signals (green) increased, suggesting increased expression of EGFP-BDNF protein. (A3, A4, B3, B4, C3, C4, D3 and D4) 293T cells transfected with pEGFP-N1 24–96 h after transfection under fluorescence and light microscopes. A4, B4, C4, D4 are light micrographs of the same visual fields as A3, B3, C3, D3, respectively. With increasing time after transfection, EGFP signals increased, suggesting increased expression of EGFP protein. Scale bars: 250 μm. EGFP: Enhanced green fluorescent protein; BDNF: brain-derived neurotrophic factor.

    Journal: Neural Regeneration Research

    Article Title: Construction of a plasmid for human brain-derived neurotrophic factor and its effect on retinal pigment epithelial cell viability

    doi: 10.4103/1673-5374.197142

    Figure Lengend Snippet: Micrographs of 293T cells transfected with pEGFP-N1 or pEGFP-N1-BDNF after transfection at the various time points by fluorescence microscopy (× 200). (A1, A2, B1, B2, C1, C2, D1 and D2): 293T cells transfected with pEGFP-N1-BDNF 24–96 hours (h) after transfection under fluorescence and light microscopes. A2, B2, C2, D2 are light micrographs of the same visual field as A1, B1, C1, D1, respectively. With increasing time after transfection, EGFP signals (green) increased, suggesting increased expression of EGFP-BDNF protein. (A3, A4, B3, B4, C3, C4, D3 and D4) 293T cells transfected with pEGFP-N1 24–96 h after transfection under fluorescence and light microscopes. A4, B4, C4, D4 are light micrographs of the same visual fields as A3, B3, C3, D3, respectively. With increasing time after transfection, EGFP signals increased, suggesting increased expression of EGFP protein. Scale bars: 250 μm. EGFP: Enhanced green fluorescent protein; BDNF: brain-derived neurotrophic factor.

    Article Snippet: Upon reaching confluence, the cells were harvested and co-cultured with 293T cells in 6-well Transwell bicameral chambers (Corning Costar, Cambridge, MA, USA).

    Techniques: Transfection, Fluorescence, Microscopy, Expressing, Derivative Assay

    Expression of standard purified BDNF (positive control) and BDNF-GFP fusion protein (experimental group) in 293T cells, assessed by western blot assay 48 hours after transfection. (A) Beta-actin control showing a 43 kDa band, except for the positive control group. (B) Positive control group showing the location of monomers and dimers of BDNF at 14 kDa and 28 kDa, respectively. 293T cells transfected with pEGFP-N1 (negative control) and ARPE-19 cells (ARPE-19 control) showed no signal at the corresponding locations. The BDNF group showed a band at 55 kDa, suggesting the production of fusion protein, GFP-BDNF (BDNF: 28 kDa + GFP: 27 kDa = 55 kDa), after transfection with pEGFP-N1-BDNF. BDNF: Brain-derived neurotrophic factor; EGFP: enhanced green fluorescent protein; ARPE-19: acute retinal pigment epithelial-19.

    Journal: Neural Regeneration Research

    Article Title: Construction of a plasmid for human brain-derived neurotrophic factor and its effect on retinal pigment epithelial cell viability

    doi: 10.4103/1673-5374.197142

    Figure Lengend Snippet: Expression of standard purified BDNF (positive control) and BDNF-GFP fusion protein (experimental group) in 293T cells, assessed by western blot assay 48 hours after transfection. (A) Beta-actin control showing a 43 kDa band, except for the positive control group. (B) Positive control group showing the location of monomers and dimers of BDNF at 14 kDa and 28 kDa, respectively. 293T cells transfected with pEGFP-N1 (negative control) and ARPE-19 cells (ARPE-19 control) showed no signal at the corresponding locations. The BDNF group showed a band at 55 kDa, suggesting the production of fusion protein, GFP-BDNF (BDNF: 28 kDa + GFP: 27 kDa = 55 kDa), after transfection with pEGFP-N1-BDNF. BDNF: Brain-derived neurotrophic factor; EGFP: enhanced green fluorescent protein; ARPE-19: acute retinal pigment epithelial-19.

    Article Snippet: Upon reaching confluence, the cells were harvested and co-cultured with 293T cells in 6-well Transwell bicameral chambers (Corning Costar, Cambridge, MA, USA).

    Techniques: Expressing, Purification, Positive Control, Western Blot, Transfection, Negative Control, Derivative Assay

    BDNF-GFP or GFP expression in 293T cells after transfection by confocal laser scanning microscopy. GFP distribution in 293T cells was determined by confocal laser scanning microscopy (× 600). Hoechst 3342 staining was blue when excited by a 405 nm light. GFP was green when excited by a 514 nm light. (A1) Image of cells transfected with pEGFP-N1-BDNF in the early stage (24 hours after transfection). The micrograph shows a few vesicles expressing GFP around the nuclei of cells (marked with white arrows). (A2) Image of cells transfected with pEGFP-N1-BDNF in the middle stage (48 hours after transfection). The micrograph shows more GFP vesicles mainly around the nuclei of cells, with some GFP signals in the cytoplasm. (A3) Image of cells transfected with pEGFP-N1-BDNF in the late stage (72 hours after transfection). GFP is distributed uniformly in the cytoplasm, with some vesicles near the nuclei. (B) Cells transfected with control pEGFP-N1 in the mid stage, showing GFP distributed uniformly in both nuclei and the cytoplasm from the early stage to the end stage without vesicles. Scale bar: 20 μm. BDNF: Brain-derived neurotrophic factor; EGFP: enhanced green fluorescent protein; GFP: green fluorescent protein.

    Journal: Neural Regeneration Research

    Article Title: Construction of a plasmid for human brain-derived neurotrophic factor and its effect on retinal pigment epithelial cell viability

    doi: 10.4103/1673-5374.197142

    Figure Lengend Snippet: BDNF-GFP or GFP expression in 293T cells after transfection by confocal laser scanning microscopy. GFP distribution in 293T cells was determined by confocal laser scanning microscopy (× 600). Hoechst 3342 staining was blue when excited by a 405 nm light. GFP was green when excited by a 514 nm light. (A1) Image of cells transfected with pEGFP-N1-BDNF in the early stage (24 hours after transfection). The micrograph shows a few vesicles expressing GFP around the nuclei of cells (marked with white arrows). (A2) Image of cells transfected with pEGFP-N1-BDNF in the middle stage (48 hours after transfection). The micrograph shows more GFP vesicles mainly around the nuclei of cells, with some GFP signals in the cytoplasm. (A3) Image of cells transfected with pEGFP-N1-BDNF in the late stage (72 hours after transfection). GFP is distributed uniformly in the cytoplasm, with some vesicles near the nuclei. (B) Cells transfected with control pEGFP-N1 in the mid stage, showing GFP distributed uniformly in both nuclei and the cytoplasm from the early stage to the end stage without vesicles. Scale bar: 20 μm. BDNF: Brain-derived neurotrophic factor; EGFP: enhanced green fluorescent protein; GFP: green fluorescent protein.

    Article Snippet: Upon reaching confluence, the cells were harvested and co-cultured with 293T cells in 6-well Transwell bicameral chambers (Corning Costar, Cambridge, MA, USA).

    Techniques: Expressing, Transfection, Confocal Laser Scanning Microscopy, Staining, Derivative Assay

    BDNF release profiles of transfected 293T cells detected by ELISA. pEGFP-N1-BDNF-transfected 293T cells released BDNF persistently, and the concentration was significantly higher than in the negative control group. At 24–72 hours, the secretion of BDNF rose gradually and reached a peak at 72 hours, which was 12-fold more than in the control group (*** P

    Journal: Neural Regeneration Research

    Article Title: Construction of a plasmid for human brain-derived neurotrophic factor and its effect on retinal pigment epithelial cell viability

    doi: 10.4103/1673-5374.197142

    Figure Lengend Snippet: BDNF release profiles of transfected 293T cells detected by ELISA. pEGFP-N1-BDNF-transfected 293T cells released BDNF persistently, and the concentration was significantly higher than in the negative control group. At 24–72 hours, the secretion of BDNF rose gradually and reached a peak at 72 hours, which was 12-fold more than in the control group (*** P

    Article Snippet: Upon reaching confluence, the cells were harvested and co-cultured with 293T cells in 6-well Transwell bicameral chambers (Corning Costar, Cambridge, MA, USA).

    Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Concentration Assay, Negative Control

    Flow chart of transfected 293T cells co-cultured with ARPE-19 cells using the Transwell double chamber system. Untransfected 293T cells and cells transfected with pEGFP-N1-BDNF or pEGFP-N1 were seeded in the upper chamber of a Transwell plate 12 hours before transfection. ARPE-19 cells were seeded in the lower chamber of an adjacent well 7 hours before transfection of 293T cells. The upper chamber was then transferred to the corresponding ARPE-19 well at hour 0 after transfection. Purified standard BDNF (50 ng/mL or the same concentration as BDNF in the medium) was administered to other ARPE-19 wells as a control. ARPE-19 cells were incubated for 24, 48, 72 or 96 hours before harvesting, for the MTT and western blot assays. ARPE-19: Acute retinal pigment epithelial-19; BDNF: brain-derived neurotrophic factor; EGFP: enhanced green fluorescent protein.

    Journal: Neural Regeneration Research

    Article Title: Construction of a plasmid for human brain-derived neurotrophic factor and its effect on retinal pigment epithelial cell viability

    doi: 10.4103/1673-5374.197142

    Figure Lengend Snippet: Flow chart of transfected 293T cells co-cultured with ARPE-19 cells using the Transwell double chamber system. Untransfected 293T cells and cells transfected with pEGFP-N1-BDNF or pEGFP-N1 were seeded in the upper chamber of a Transwell plate 12 hours before transfection. ARPE-19 cells were seeded in the lower chamber of an adjacent well 7 hours before transfection of 293T cells. The upper chamber was then transferred to the corresponding ARPE-19 well at hour 0 after transfection. Purified standard BDNF (50 ng/mL or the same concentration as BDNF in the medium) was administered to other ARPE-19 wells as a control. ARPE-19 cells were incubated for 24, 48, 72 or 96 hours before harvesting, for the MTT and western blot assays. ARPE-19: Acute retinal pigment epithelial-19; BDNF: brain-derived neurotrophic factor; EGFP: enhanced green fluorescent protein.

    Article Snippet: Upon reaching confluence, the cells were harvested and co-cultured with 293T cells in 6-well Transwell bicameral chambers (Corning Costar, Cambridge, MA, USA).

    Techniques: Flow Cytometry, Transfection, Cell Culture, Purification, Concentration Assay, Incubation, MTT Assay, Western Blot, Derivative Assay

    Efficiency of 293T cells transfected with pEGFP-N1-BDNF at different time points after transfection. We calculated efficiency with Image-Pro Plus 6.0 or ImageJ v1.48 software using positive area or positive cell number. A total of 36 samples were separately counted at 24, 48, 72 and 96 hours. The two methods provided the same results; i.e ., a gradual rise in efficiency from the beginning to 96 hours, although the efficiency measured by area was higher than that measured by positive cell number. BDNF: Brain-derived neurotrophic factor; EGFP: enhanced green fluorescent protein.

    Journal: Neural Regeneration Research

    Article Title: Construction of a plasmid for human brain-derived neurotrophic factor and its effect on retinal pigment epithelial cell viability

    doi: 10.4103/1673-5374.197142

    Figure Lengend Snippet: Efficiency of 293T cells transfected with pEGFP-N1-BDNF at different time points after transfection. We calculated efficiency with Image-Pro Plus 6.0 or ImageJ v1.48 software using positive area or positive cell number. A total of 36 samples were separately counted at 24, 48, 72 and 96 hours. The two methods provided the same results; i.e ., a gradual rise in efficiency from the beginning to 96 hours, although the efficiency measured by area was higher than that measured by positive cell number. BDNF: Brain-derived neurotrophic factor; EGFP: enhanced green fluorescent protein.

    Article Snippet: Upon reaching confluence, the cells were harvested and co-cultured with 293T cells in 6-well Transwell bicameral chambers (Corning Costar, Cambridge, MA, USA).

    Techniques: Transfection, Software, Derivative Assay