hek293t cells Search Results


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  • 99
    ATCC hek293t cells hek293t cells
    E488Q mutation introduced into DD increases editing efficiency. ( A ) Schematic of the E488Q mutation in λN-DD or 4λN-DD. ( B ) Confocal images of <t>HEK293T</t> cells transfected with various components of SDRE. Scale bars = 12 μm. ( C ) Quantification of correction in transfections by both fluorescence (dark gray) and direct sequencing (light gray). All values for fluorescence correction are significantly different by ANOVA and Tukey's test. n = 200–800 cells, mean ± SEM. Technical duplicates for all fluorescence measurements yielded similar results (11.5 ± 0.1, 32 ± 0.2, 58.9 ± 2.1 and 65 ± 0.02 for λN-DD 1boxB, λN-DD E488Q 1boxB, 4λN-DD 2boxB and 4λN-DD E488Q 2boxB, respectively). Technical duplicates for RNA correction were as follows: 7, 27, 69 and 70 % for direct sequencing measurements of λN-DD 1boxB, λN-DD E488Q 1boxB, 4λN-DD 2boxB and 4λN-DD E488Q 2boxB, respectively.
    Hek293t Cells Hek293t Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 601 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher geneblazer β lactamase bla hek293t cell lines
    Concentration response curves of BPA on (A) <t>ERα-bla</t> and (B) AR-bla assays. <t>GeneBLAzer</t> <t>β-lactamase</t> HEK293 cell lines that co-express Gal4-reporter system with Gal4DBD-linked ERα-LBD or AR-LBD were tested in 12-24 concentrations
    Geneblazer β Lactamase Bla Hek293t Cell Lines, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC hek293t cell culture hek293t cells
    Kv2.1 P404W increases Cav1.2 single channel activity. ( A, B ) Maximum z -projections of TIRF images of Cav1.2-mediated Ca 2+ sparklets in a representative <t>HEK293T</t> cell transfected with Cav1.2 and auxiliary subunits and loaded with Fluo-5F via the patch pipette, before ( A ) and after ( B ) treatment with 500 nM Bay K8644 (scale bar: 5 µm). ( C ) Maximum z -projection of TIRF images of DsRed-Kv2.1 in a representative HEK293T cell cotransfected with Cav1.2 and auxiliary subunits (scale bar: 5 µm). ( D, F ) Maximum z -projections of TIRF images of sparklets in a representative HEK293T cell transfected with DsRed-Kv2.1, Cav1.2, and auxiliary subunits and loaded with Fluo-5F via the patch pipette, before ( D ) and after ( F ) treatment with 500 nM Bay K8644. ( E, G ) Merged images of panels C and D ( E ), or panels C and F ( G ). ( H ) Fluorescence intensity profiles of representative sparklets recorded in 20 mM external Ca 2+ in a control cell (upper panel, ROI depicted in A) or in a cell additionally expressing Kv2.1 (lower panel, ROI depicted in D). ( I ) Fluorescence intensity profiles of representative sparklets recorded in 20 mM external Ca 2+ and treated with Bay K8644 in a control cell (upper panel, ROI depicted in B) or in a cell additionally expressing Kv2.1 (lower panel, ROI depicted in F). ( J ) Summary data of sparklet site n P s measured from n = 6 cells expressing Cav1.2 alone and n = 7 cells coexpressing Cav1.2 and Kv2.1. Each point represents a single sparklet site (vehicle: *p=0.0367; Bay K: p=0.9224; two-tailed Mann-Whitney test). ( K ) Summary data of sparklet site nearest neighbor distance (NND) measured from n = 6 cells expressing Cav1.2 alone and n = 7 cells coexpressing Cav1.2 and Kv2.1. Each point represents a single sparklet site (vehicle: *p=0.0214; Bay K: p
    Hek293t Cell Culture Hek293t Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 125 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC cell culture hek293t cells
    FIN mediates the COX2/PGE 2 pathway by increasing the activity of COX2 promoter. The plasmid harboring a COX2 promoter fragment and a firefly luciferase (FL) gene (5ng/well separately) was transfected into <t>HEK293T</t> cells in a 24-well-plate and a CMV-Renilla luciferase (RL) plasmid (10ng/well) served as a transfection control. FIN (50μM) was treated for 6 h before harvesting the cells for dual-luciferase assay. Transfected cells treated with DMSO were set as negative control. Results are shown in mean ± SEM and representative of 3 independent experiments. **p
    Cell Culture Hek293t Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 205 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam hek293t cells
    Stimulation of poly-ubiquitination of c-Myc by Rabring7. A and B . <t>HEK293T</t> cells were co-transfected with FLAG-wild-type-c-Myc together with two doses of HA-wild-type Rabring7 (A) or HA-C229S-Rabring7 (B). Forty-four hrs after transfection, 25 μM MG132 was added to the culture medium, and the cells were cultured for an additional 4 hrs. Proteins in cells were then immunoprecipitated with an anti-FLAG antibody, and the precipitates were analyzed by Western blotting with an anti-multi-ubiquitin antibody. C . HEK293T cells were co-transfected with FLAG-T58A-c-Myc together with two doses of HA-wild-type Rabring7 and subjected to ubiquitination assays as described in the legends of Figures 6A and 6B . D. HEK293T cells were co-transfected with FLAG-MM-1α, T7-Rabring7 and HA-ubiquitin. Forty-eight hrs after transfection, total cell lysates were prepared and the cytoplasm and nucleus were fractionated as described in Experimental procedures. Proteins extracted from them were immunoprecipitated with an anti-FLAG antibody and analyzed by Western blotting with anti-HA, anti-FLAG and anti-T7 antibodies. Fractions of the cytoplasm and nucleus were also blotted with anti-HSP60 (SC-6216, Santa Cruz biotechnology, Santa Cruz, CA) and anti-Lamin B (SC-13115, Santa Cruz biotechnology) antibodies. E. HEK293T cells were co-transfected with EGFP-c-Myc, FLAG-MM-1α, T7-Rabring7 and HA-ubiquitin. Proteins were analyzed as described in the legend of Figure 6D .
    Hek293t Cells, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 136 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa hek293t cells
    Mitochondrial t 6 A37 formation is sensitive to intracellular bicarbonate concentration. a Kinetic analyses of mitochondrial t 6 A37 formation mediated by YRDC and OSGEPL1. Initial velocities of t 6 A37 formation were measured against variable concentrations of mt-tRNA Thr , L -Thr, ATP, and bicarbonate. Km values for each substrate are indicated. b Hypomodification of t 6 A37 in mt-tRNAs in <t>HEK293T</t> cells cultured in non-bicarbonate medium. XICs generated by integration of multiply-charged negative ions of A37-containing fragments of mt-tRNA Ser(AGY) (top panels), mt-tRNA Asn (second panels), mt-tRNA Thr (third panels), mt-tRNA Lys with s 2 U34 (fourth panels) and ct-tRNA Ile (bottom panels) bearing A37 (blue) and t 6 A37 (black) (Supplementary Table 1 ) isolated from HEK293T cells cultured with normal DMEM medium (44 mM NaHCO 3 ) in 5% CO 2 (left panels) and non-bicarbonate medium in air (right panels). mt-tRNA Ser(AGY) and other tRNAs were isolated from the cells cultured for 6 and 3 days, respectively. t 6 A frequencies (%) are described as mean values ± s.d. of technical triplicate
    Hek293t Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1672 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher geneblazer cckar nfat bla hek 293t cells
    Mitochondrial t 6 A37 formation is sensitive to intracellular bicarbonate concentration. a Kinetic analyses of mitochondrial t 6 A37 formation mediated by YRDC and OSGEPL1. Initial velocities of t 6 A37 formation were measured against variable concentrations of mt-tRNA Thr , L -Thr, ATP, and bicarbonate. Km values for each substrate are indicated. b Hypomodification of t 6 A37 in mt-tRNAs in <t>HEK293T</t> cells cultured in non-bicarbonate medium. XICs generated by integration of multiply-charged negative ions of A37-containing fragments of mt-tRNA Ser(AGY) (top panels), mt-tRNA Asn (second panels), mt-tRNA Thr (third panels), mt-tRNA Lys with s 2 U34 (fourth panels) and ct-tRNA Ile (bottom panels) bearing A37 (blue) and t 6 A37 (black) (Supplementary Table 1 ) isolated from HEK293T cells cultured with normal DMEM medium (44 mM NaHCO 3 ) in 5% CO 2 (left panels) and non-bicarbonate medium in air (right panels). mt-tRNA Ser(AGY) and other tRNAs were isolated from the cells cultured for 6 and 3 days, respectively. t 6 A frequencies (%) are described as mean values ± s.d. of technical triplicate
    Geneblazer Cckar Nfat Bla Hek 293t Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hek293t cells
    Association of C4orf14 with the mitochondrial small ribosomal subunit. ( A ) Affinity purified C4orf14.FLAG.StrepII protein was isolated from mitochondria fractions of transgenic <t>HEK293T</t> cells and the concentrated eluted fractions resolved by SDS–PAGE. Proteins identified by MS are indicated on the left and right of the gel. They included 22 polypeptides of the 28S subunit (MRPS; see Supplementary Table S1 ); ( B ) sucrose-gradient purified mitochondria from HEK293T cells were lysed and fractionated on sucrose gradients. Antibodies to MRPS2 and MRPS18, and MRPL3 and MRPL11 were used as markers of the 28S and 39S subunits, respectively. ( C ) 143B cells were transfected with dsRNA (c3 or c6) targeting C4orf14 mRNA and the effects on mitochondrial protein synthesis (i); selected proteins in mitochondria (ii) and mitochondrial ribosomal RNAs (panel D and Supplementary Figure S2 ) were examined 72 h later. GAPDH: glyceraldehyde-3-phosphate dehydrogenase, the outer mitochondrial membrane protein TOM20, a putative mitochondrial RNA helicase DHX30 and components of the 55S ribosome (MRPS2, MRPS29, MRPL3 and MRPL11).
    Hek293t Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 28498 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    DSMZ hek293t cells
    p38 promotes mTORC1 activation and stress granule formation when PI3K is inactive. (A) p38 mediates mTORC1 activation when PI3K is inactive. MCF-7 cells were serum-starved and treated with arsenite in the presence of carrier (DMSO) or wortmannin (100 nM, PI3K inhibitor). In addition, the cells were treated with carrier (DMSO) versus LY2228820 (1 μM, p38 inhibitor). MK2-pT334, Akt-pT308, Akt-pS473, p70-S6K-pT389, and eIF2α-pS51 were monitored by immunoblot. Data represent five biological replicates. (B) Quantification of data shown in (A) when PI3K is active. Akt-pS473 and p70-S6K-pT389 were compared between carrier (DMSO) and LY2228820-treated cells using a two-way ANOVA followed by a Bonferroni multiple comparison test across five biological replicates. Data represent the mean ± SEM. The P -values for the Bonferroni multiple comparison tests are shown. *** P ≤ 0.001. (C) Quantification of data shown in (A) when PI3K is inactive. Akt-pS473 and p70-S6K-pT389 were compared between wortmannin- and wortmannin + LY2228820–treated cells using a two-way ANOVA followed by a Bonferroni multiple comparison test across five biological replicates. Data represent the mean ± SEM. The P -values for the Bonferroni multiple comparison tests are shown. * P ≤ 0.05; *** P ≤ 0.001. (D) p38 drives mTORC1 activity when PI3K is inactive. Quantification of data shown in Fig S14A . 4E-BP1-pT37/46 relative intensity was normalized separately for conditions without or with wortmannin. Significance of 4E-BP1-pT37/46 inhibition by LY2228820 was tested using a two-tailed t test across five biological replicates. Data represent the mean ± SEM. * P ≤ 0.05. (E) Prediction on the extent of mTORC1 inhibition upon LY2228820 treatment when PI3K is active or inactive. Prediction was performed with model V. The red lines depict the time points measured experimentally ( Fig 5A–C ). (F) When PI3K activity declines, p38 drives mTORC1 activity. Quantification of data shown in Fig S13 L. MCF-7 cells were serum-starved and treated with arsenite for 60 min in the presence of different concentrations of wortmannin (as indicated, PI3K inhibitor) in carrier (DMSO) versus LY2228820 (1 μM, p38 inhibitor)-treated cells. p70-S6K-pT389 relative intensity was normalized separately for each wortmannin concentration. Significance of p70-S6K-pT389 inhibition by LY2228820 was tested using a two-tailed t test across five biological replicates. Data represent the mean ± SEM. * P ≤ 0.05. (G) p38 drives mTORC1 activity in several cell lines, as PI3K activity declines. Quantification of data shown in Fig S14D–G . MCF-7, CAL51, LN18, <t>HEK293T,</t> and HeLa cells were serum-starved and exposed to arsenite for 60 min in combination with wortmannin (100 nM, PI3K inhibitor) and/or LY2228820 (1 mM, p38 inhibitor). Data represent 3–4 biological replicates (see Fig S14D–G ). 4E-BP1-pT37/46 relative intensity was normalized separately for conditions without or with wortmannin. Significance of 4E-BP1-pT37/46 inhibition by LY2228820 was tested using a two-tailed t test across three biological replicates. Data represent the mean ± SEM. * P ≤ 0.05; ** P ≤ 0.01. (H) Stress granule numbers upon PI3K and p38 inhibition. MCF-7 cells were serum-starved and treated with arsenite for 30 min in the presence of carrier (DMSO), wortmannin (100 nM, PI3K inhibitor), LY2228820 (1 μM, p38 inhibitor), or wortmannin + LY2228820. Stress granules were visualized by immunofluorescence staining of G3BP1. Nuclei were visualized with Hoechst 33342. Data represent four biological replicates. White square indicates region of insert and blue arrow highlights stress granules; scale bar 10 μm. (I) Quantification of data shown in (H). The number of stress granules (SGs) per cell (normalized to the arsenite condition) across four biological replicates. Stress granule formation between carrier and LY2228820 as well as wortmannin- and wortmannin + LY2228820–treated cells was compared using a two-tailed t test across four biological replicates. Data represent the mean ± SEM. * P ≤ 0.01. ns, not significant.
    Hek293t Cells, supplied by DSMZ, used in various techniques. Bioz Stars score: 96/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    E488Q mutation introduced into DD increases editing efficiency. ( A ) Schematic of the E488Q mutation in λN-DD or 4λN-DD. ( B ) Confocal images of HEK293T cells transfected with various components of SDRE. Scale bars = 12 μm. ( C ) Quantification of correction in transfections by both fluorescence (dark gray) and direct sequencing (light gray). All values for fluorescence correction are significantly different by ANOVA and Tukey's test. n = 200–800 cells, mean ± SEM. Technical duplicates for all fluorescence measurements yielded similar results (11.5 ± 0.1, 32 ± 0.2, 58.9 ± 2.1 and 65 ± 0.02 for λN-DD 1boxB, λN-DD E488Q 1boxB, 4λN-DD 2boxB and 4λN-DD E488Q 2boxB, respectively). Technical duplicates for RNA correction were as follows: 7, 27, 69 and 70 % for direct sequencing measurements of λN-DD 1boxB, λN-DD E488Q 1boxB, 4λN-DD 2boxB and 4λN-DD E488Q 2boxB, respectively.

    Journal: Nucleic Acids Research

    Article Title: An efficient system for selectively altering genetic information within mRNAs

    doi: 10.1093/nar/gkw738

    Figure Lengend Snippet: E488Q mutation introduced into DD increases editing efficiency. ( A ) Schematic of the E488Q mutation in λN-DD or 4λN-DD. ( B ) Confocal images of HEK293T cells transfected with various components of SDRE. Scale bars = 12 μm. ( C ) Quantification of correction in transfections by both fluorescence (dark gray) and direct sequencing (light gray). All values for fluorescence correction are significantly different by ANOVA and Tukey's test. n = 200–800 cells, mean ± SEM. Technical duplicates for all fluorescence measurements yielded similar results (11.5 ± 0.1, 32 ± 0.2, 58.9 ± 2.1 and 65 ± 0.02 for λN-DD 1boxB, λN-DD E488Q 1boxB, 4λN-DD 2boxB and 4λN-DD E488Q 2boxB, respectively). Technical duplicates for RNA correction were as follows: 7, 27, 69 and 70 % for direct sequencing measurements of λN-DD 1boxB, λN-DD E488Q 1boxB, 4λN-DD 2boxB and 4λN-DD E488Q 2boxB, respectively.

    Article Snippet: Editing assays in HEK293T cells HEK293T cells (CRL-11268 ATCC, Manassas, VA) were maintained in Dulbecco's Modified Eagle's Medium supplemented with 10 % (v/v) fetal bovine serum, 1% penicillin–streptomycin solution, 1 mM sodium pyruvate, and 2 mM glutamine.

    Techniques: Mutagenesis, Transfection, Fluorescence, Sequencing

    Editing efficiencies of optimized SDRE system on A's in different neighboring contexts. ( A ) A's in different neighboring contexts were introduced into mCherry-eGFP and different 2boxB guide RNAs were designed to target them. These RNA guides were tested in HEK293T cells with either 4λN -DD or 4λN-DD E488Q. Results were tested only by direct sequencing of RT-PCR products. Experiments were done in duplicate. ( B ) Editing percentages of individual A's for the UAA N context with 4λN -DD and 4λN-DD E488Q. Rep 1 = repetition 1 and rep2 = repetition 2.

    Journal: Nucleic Acids Research

    Article Title: An efficient system for selectively altering genetic information within mRNAs

    doi: 10.1093/nar/gkw738

    Figure Lengend Snippet: Editing efficiencies of optimized SDRE system on A's in different neighboring contexts. ( A ) A's in different neighboring contexts were introduced into mCherry-eGFP and different 2boxB guide RNAs were designed to target them. These RNA guides were tested in HEK293T cells with either 4λN -DD or 4λN-DD E488Q. Results were tested only by direct sequencing of RT-PCR products. Experiments were done in duplicate. ( B ) Editing percentages of individual A's for the UAA N context with 4λN -DD and 4λN-DD E488Q. Rep 1 = repetition 1 and rep2 = repetition 2.

    Article Snippet: Editing assays in HEK293T cells HEK293T cells (CRL-11268 ATCC, Manassas, VA) were maintained in Dulbecco's Modified Eagle's Medium supplemented with 10 % (v/v) fetal bovine serum, 1% penicillin–streptomycin solution, 1 mM sodium pyruvate, and 2 mM glutamine.

    Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction

    Increasing the number of λNs and boxBs improves editing. ( A i) A schematic showing the addition of N-terminal λN's. ( A ii) Reaction kinetics of λN-DD and 2λN-DD, purified from Pichia pastoris , were measured using mCherry-eGFP W58X in vitro using our standard 1boxB RNA guide. Rate constants, estimated from fits (see Materials and Methods section), were k obs = 0.1 ± 0.02 and 0.3 ± 0.05 min −1 for λN-DD and 2λN-DD, respectively. Experiments were performed at 35°C. Constructs with 1–5λNs connected to DD were generated and tested in HEK293T cells using the fluorescent reporter assay (dark gray) and by RT-PCR (light gray; A iii). Technical duplicates for all fluorescence measurements yielded similar results (11.6 ± 0.5, 19.2 ± 0.9, 32.1 ± 1.3, 38.5 ± 0.7 and 35.7 ± 1.3 % for 1–5λN-DD, respectively). Technical duplicates for RNA correction were as follows: 8, 27, 32, 48 and 41 % for direct sequencing measurements of 1–5λN-DD, respectively. ( B i) 1boxB and 2boxB RNA guides. With the 2boxB guide, the target A (red) is 19 nt 3′ to boxB 1 and 11 nt 5′ to boxB 2. ( B ii) The kinetics of editing using a 1boxB or 2BoxB guide was tested with purified λN-DD and mCherry-eGFP W58X in vitro . Temperature = 35°C. The data for 2boxB was fitted to a double exponential and the rate constants were k 1 obs = 1.54 ± 0.09 and k 2 obs = 0.05 ± 0.001. ( B iii) Editing efficiency of 2boxB guide was tested in HEK293T cells using 1–4λN-DD. As before, correction was estimated by both fluorescence (dark gray) and direct sequencing (light gray). All results were tested with a one-way ANOVA and Tukey's test. An asterisk indicates significance ( P

    Journal: Nucleic Acids Research

    Article Title: An efficient system for selectively altering genetic information within mRNAs

    doi: 10.1093/nar/gkw738

    Figure Lengend Snippet: Increasing the number of λNs and boxBs improves editing. ( A i) A schematic showing the addition of N-terminal λN's. ( A ii) Reaction kinetics of λN-DD and 2λN-DD, purified from Pichia pastoris , were measured using mCherry-eGFP W58X in vitro using our standard 1boxB RNA guide. Rate constants, estimated from fits (see Materials and Methods section), were k obs = 0.1 ± 0.02 and 0.3 ± 0.05 min −1 for λN-DD and 2λN-DD, respectively. Experiments were performed at 35°C. Constructs with 1–5λNs connected to DD were generated and tested in HEK293T cells using the fluorescent reporter assay (dark gray) and by RT-PCR (light gray; A iii). Technical duplicates for all fluorescence measurements yielded similar results (11.6 ± 0.5, 19.2 ± 0.9, 32.1 ± 1.3, 38.5 ± 0.7 and 35.7 ± 1.3 % for 1–5λN-DD, respectively). Technical duplicates for RNA correction were as follows: 8, 27, 32, 48 and 41 % for direct sequencing measurements of 1–5λN-DD, respectively. ( B i) 1boxB and 2boxB RNA guides. With the 2boxB guide, the target A (red) is 19 nt 3′ to boxB 1 and 11 nt 5′ to boxB 2. ( B ii) The kinetics of editing using a 1boxB or 2BoxB guide was tested with purified λN-DD and mCherry-eGFP W58X in vitro . Temperature = 35°C. The data for 2boxB was fitted to a double exponential and the rate constants were k 1 obs = 1.54 ± 0.09 and k 2 obs = 0.05 ± 0.001. ( B iii) Editing efficiency of 2boxB guide was tested in HEK293T cells using 1–4λN-DD. As before, correction was estimated by both fluorescence (dark gray) and direct sequencing (light gray). All results were tested with a one-way ANOVA and Tukey's test. An asterisk indicates significance ( P

    Article Snippet: Editing assays in HEK293T cells HEK293T cells (CRL-11268 ATCC, Manassas, VA) were maintained in Dulbecco's Modified Eagle's Medium supplemented with 10 % (v/v) fetal bovine serum, 1% penicillin–streptomycin solution, 1 mM sodium pyruvate, and 2 mM glutamine.

    Techniques: Purification, In Vitro, Construct, Generated, Reporter Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence, Sequencing

    Fluorescent reporter system for quantifying editing. ( A ) Cartoons of fluorescent protein constructs and plasmids used in this study. Full-length mCherry and eGFP were fused together and separated by a 2A peptide (black) followed by a FLAG epitope tag (grey). In addition, a version with a stop codon (U A G) inserted at position 58 of eGFP was made (W58X). These constructs were cloned into a vector driven by the CMV promoter, as was λN-DD. RNA guides were driven by a U6 promoter. ( B ) Confocal images of HEK293T cells transfected with different combinations of components are shown. Red, green, and DIC images are shown for the same field of cells. Pictures are taken 96 h post-transfection. Scale bars = 12 μm. Fluorescence correction was estimated to be 11 ± 0.05 %. ( C ) Sequences from RT-PCR products of corrected cells and cells transfected with mCherry-eGFP W58X alone. Asterisks indicate the target A. Editing percentage for experimental samples was estimated to be 11 %. ( D ) Estimates of editing efficiencies from fluorescence (dark gray) and direct sequencing (light grey) were compared at various days post-transfection. Fluorescence estimates were based on 200–800 cells per sample (mean ± SEM) and RT-PCR products came from the same samples. Technical duplicates for all fluorescence measurements yielded similar results (2.3 ± 0.3, 8.7 ± 1.5, 8.9 ± 1.5, and 12.9 ± 1.4 % for days 1, 2, 3, and 4, respectively). Technical duplicates for RNA correction based on direct sequencing of were as follows: 4, 6, 9 and 14 %, for days 1, 2, 3 and 4, respectively.

    Journal: Nucleic Acids Research

    Article Title: An efficient system for selectively altering genetic information within mRNAs

    doi: 10.1093/nar/gkw738

    Figure Lengend Snippet: Fluorescent reporter system for quantifying editing. ( A ) Cartoons of fluorescent protein constructs and plasmids used in this study. Full-length mCherry and eGFP were fused together and separated by a 2A peptide (black) followed by a FLAG epitope tag (grey). In addition, a version with a stop codon (U A G) inserted at position 58 of eGFP was made (W58X). These constructs were cloned into a vector driven by the CMV promoter, as was λN-DD. RNA guides were driven by a U6 promoter. ( B ) Confocal images of HEK293T cells transfected with different combinations of components are shown. Red, green, and DIC images are shown for the same field of cells. Pictures are taken 96 h post-transfection. Scale bars = 12 μm. Fluorescence correction was estimated to be 11 ± 0.05 %. ( C ) Sequences from RT-PCR products of corrected cells and cells transfected with mCherry-eGFP W58X alone. Asterisks indicate the target A. Editing percentage for experimental samples was estimated to be 11 %. ( D ) Estimates of editing efficiencies from fluorescence (dark gray) and direct sequencing (light grey) were compared at various days post-transfection. Fluorescence estimates were based on 200–800 cells per sample (mean ± SEM) and RT-PCR products came from the same samples. Technical duplicates for all fluorescence measurements yielded similar results (2.3 ± 0.3, 8.7 ± 1.5, 8.9 ± 1.5, and 12.9 ± 1.4 % for days 1, 2, 3, and 4, respectively). Technical duplicates for RNA correction based on direct sequencing of were as follows: 4, 6, 9 and 14 %, for days 1, 2, 3 and 4, respectively.

    Article Snippet: Editing assays in HEK293T cells HEK293T cells (CRL-11268 ATCC, Manassas, VA) were maintained in Dulbecco's Modified Eagle's Medium supplemented with 10 % (v/v) fetal bovine serum, 1% penicillin–streptomycin solution, 1 mM sodium pyruvate, and 2 mM glutamine.

    Techniques: Construct, FLAG-tag, Clone Assay, Plasmid Preparation, Transfection, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Sequencing

    TDP‐43 NTD high‐order oligomerization is impaired by S48E Peptides composed of TDP43 (40–53), with and without phosphorylated Ser48, were serially diluted and spotted to nitrocellulose membranes. Polyclonal antibody (α‐TDP‐43 pSer48) specific to the phosphorylated peptide was used in the top panel showing specificity for pS48, and α‐TDP‐43 “pan antibody” recognizing the same peptide irrespective of phosphorylation was used in the bottom panel. The α‐TDP‐43 pSer48 antibody and commercial TDP‐43 antibody used in Western blots of HEK293T cell lysates both show reactivity at ˜43 kDa, consistent with TDP‐43 SDS–PAGE migration. Standard Western blotting was performed on HEK293T cell lysates that had been transferred onto nitrocellulose membranes, except calf intestinal phosphatase (CIP, bottom) or a mock treatment (top) was used to treat the membranes prior to immunoprobing with α‐TDP‐43 (pSer48). Whole HEK293T cell lysates were used in the left panel. In the right panel, TDP‐43 was first immunoprecipitated using commercial α‐TDP‐43 antibody prior to Western blotting. Gel filtration chromatogram of 200 μM wild‐type (black) and S48E (red) TDP‐43 NTD. The shorter retention time and skewed profile of wild‐type NTD is consistent with self‐assembly. The single‐point variant S48E results in a symmetric peak at longer retention time, consistent with predominantly monomer. CG‐MALS derived mass average as a function of increasing TDP‐43 NTD concentration data are fit to an isodesmic self‐association model (bold black line) with K D ˜ 95 μM. Fits for dimer, trimer, tetramer, and pentamer models are poor (dashed lines), shown for comparison. CG‐MALS data for wild‐type are effectively the same at 150 mM (black, repeated from E for clarity) and 300 mM (gray) NaCl. S48E at 150 mM NaCl (red) shows dramatically disrupted assembly with K D ˜ 2,000 μM. The concentration‐dependent chemical shift deviations of 1 H‐ 15 N HSQC are large for wild‐type and small for S48E TDP‐43 NTD, consistent with disrupted binding. The CSDs are measured for 200 μM (cyan), 100 μM (green), 40 μM (yellow), and 20 μM (orange) WT compared to a monomeric control: 5 μM. For S48E, only 200 and 100 μM are shown. The chemical shift deviations (at 100 μM with a cutoff of 0.02 ppm, shown in green) map to two different sides of TDP‐43 NTD (PDB 2N4P), supporting a view that TDP‐43 can assemble into linear chains via multiple interfaces. S48 is highlighted with red spheres.

    Journal: The EMBO Journal

    Article Title: A single N‐terminal phosphomimic disrupts TDP‐43 polymerization, phase separation, and RNA splicing

    doi: 10.15252/embj.201797452

    Figure Lengend Snippet: TDP‐43 NTD high‐order oligomerization is impaired by S48E Peptides composed of TDP43 (40–53), with and without phosphorylated Ser48, were serially diluted and spotted to nitrocellulose membranes. Polyclonal antibody (α‐TDP‐43 pSer48) specific to the phosphorylated peptide was used in the top panel showing specificity for pS48, and α‐TDP‐43 “pan antibody” recognizing the same peptide irrespective of phosphorylation was used in the bottom panel. The α‐TDP‐43 pSer48 antibody and commercial TDP‐43 antibody used in Western blots of HEK293T cell lysates both show reactivity at ˜43 kDa, consistent with TDP‐43 SDS–PAGE migration. Standard Western blotting was performed on HEK293T cell lysates that had been transferred onto nitrocellulose membranes, except calf intestinal phosphatase (CIP, bottom) or a mock treatment (top) was used to treat the membranes prior to immunoprobing with α‐TDP‐43 (pSer48). Whole HEK293T cell lysates were used in the left panel. In the right panel, TDP‐43 was first immunoprecipitated using commercial α‐TDP‐43 antibody prior to Western blotting. Gel filtration chromatogram of 200 μM wild‐type (black) and S48E (red) TDP‐43 NTD. The shorter retention time and skewed profile of wild‐type NTD is consistent with self‐assembly. The single‐point variant S48E results in a symmetric peak at longer retention time, consistent with predominantly monomer. CG‐MALS derived mass average as a function of increasing TDP‐43 NTD concentration data are fit to an isodesmic self‐association model (bold black line) with K D ˜ 95 μM. Fits for dimer, trimer, tetramer, and pentamer models are poor (dashed lines), shown for comparison. CG‐MALS data for wild‐type are effectively the same at 150 mM (black, repeated from E for clarity) and 300 mM (gray) NaCl. S48E at 150 mM NaCl (red) shows dramatically disrupted assembly with K D ˜ 2,000 μM. The concentration‐dependent chemical shift deviations of 1 H‐ 15 N HSQC are large for wild‐type and small for S48E TDP‐43 NTD, consistent with disrupted binding. The CSDs are measured for 200 μM (cyan), 100 μM (green), 40 μM (yellow), and 20 μM (orange) WT compared to a monomeric control: 5 μM. For S48E, only 200 and 100 μM are shown. The chemical shift deviations (at 100 μM with a cutoff of 0.02 ppm, shown in green) map to two different sides of TDP‐43 NTD (PDB 2N4P), supporting a view that TDP‐43 can assemble into linear chains via multiple interfaces. S48 is highlighted with red spheres.

    Article Snippet: HEK293T cells were obtained from ATCC (CRL‐11268) and maintained under standard conditions at 37°C in DMEM‐based media (Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum and 1% P/S/G (penicillin–streptomycin–glutamine).

    Techniques: Western Blot, SDS Page, Migration, Immunoprecipitation, Filtration, Variant Assay, Derivative Assay, Concentration Assay, Binding Assay

    Gaussian fitting of the factor of viscosity for HEK293T cells. The cells were treated with (A) 0 μg/mL, (B) 10 μg/mL, (C) 20 μg/mL, and (D) 40 μg/mL AgNPs for 24 h, respectively.

    Journal: ACS Omega

    Article Title: Nanotoxicity of Silver Nanoparticles on HEK293T Cells: A Combined Study Using Biomechanical and Biological Techniques

    doi: 10.1021/acsomega.8b00608

    Figure Lengend Snippet: Gaussian fitting of the factor of viscosity for HEK293T cells. The cells were treated with (A) 0 μg/mL, (B) 10 μg/mL, (C) 20 μg/mL, and (D) 40 μg/mL AgNPs for 24 h, respectively.

    Article Snippet: 4.2 Cells Culture and Cell Viability Measurement HEK293T cells (American Type Culture Collection, CRL-11268, Shanghai, China) were cultured in Gibco Roswell Park Memorial Institute medium 1640 basic (1×) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Shanghai, China) and 1% penicillin–streptomycin solution (Beyotime, Jiangsu, China) at 37 °C in an incubator humidified with 5% CO2 atmosphere.

    Techniques:

    Images of DNA damages detected by comet assays for HEK293T cells treated with varying AgNPs concentrations for 24 h: (A) 0 μg/mL, (B) 10 μg/mL, (C) 20 μg/mL, and (D) 40 μg/mL.

    Journal: ACS Omega

    Article Title: Nanotoxicity of Silver Nanoparticles on HEK293T Cells: A Combined Study Using Biomechanical and Biological Techniques

    doi: 10.1021/acsomega.8b00608

    Figure Lengend Snippet: Images of DNA damages detected by comet assays for HEK293T cells treated with varying AgNPs concentrations for 24 h: (A) 0 μg/mL, (B) 10 μg/mL, (C) 20 μg/mL, and (D) 40 μg/mL.

    Article Snippet: 4.2 Cells Culture and Cell Viability Measurement HEK293T cells (American Type Culture Collection, CRL-11268, Shanghai, China) were cultured in Gibco Roswell Park Memorial Institute medium 1640 basic (1×) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Shanghai, China) and 1% penicillin–streptomycin solution (Beyotime, Jiangsu, China) at 37 °C in an incubator humidified with 5% CO2 atmosphere.

    Techniques:

    Cell viability of HEK293T cells tested by the MTT assay. HEK293T cells were treated with varying AgNPs concentrations (0, 10, 20, and 40 μg/mL) for 24 h. Following treatment with MTT reagents, viable cells were quantified by measuring the OD 490 of sample wells. * indicates p

    Journal: ACS Omega

    Article Title: Nanotoxicity of Silver Nanoparticles on HEK293T Cells: A Combined Study Using Biomechanical and Biological Techniques

    doi: 10.1021/acsomega.8b00608

    Figure Lengend Snippet: Cell viability of HEK293T cells tested by the MTT assay. HEK293T cells were treated with varying AgNPs concentrations (0, 10, 20, and 40 μg/mL) for 24 h. Following treatment with MTT reagents, viable cells were quantified by measuring the OD 490 of sample wells. * indicates p

    Article Snippet: 4.2 Cells Culture and Cell Viability Measurement HEK293T cells (American Type Culture Collection, CRL-11268, Shanghai, China) were cultured in Gibco Roswell Park Memorial Institute medium 1640 basic (1×) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Shanghai, China) and 1% penicillin–streptomycin solution (Beyotime, Jiangsu, China) at 37 °C in an incubator humidified with 5% CO2 atmosphere.

    Techniques: MTT Assay

    Gene expression levels for HEK293T cells after exposure to varying AgNPs concentrations (0, 10, 20, and 40 μg/mL) for 24 h: (A) Bcl2-t , (B) Bclw , and (C) Bid . * indicates p

    Journal: ACS Omega

    Article Title: Nanotoxicity of Silver Nanoparticles on HEK293T Cells: A Combined Study Using Biomechanical and Biological Techniques

    doi: 10.1021/acsomega.8b00608

    Figure Lengend Snippet: Gene expression levels for HEK293T cells after exposure to varying AgNPs concentrations (0, 10, 20, and 40 μg/mL) for 24 h: (A) Bcl2-t , (B) Bclw , and (C) Bid . * indicates p

    Article Snippet: 4.2 Cells Culture and Cell Viability Measurement HEK293T cells (American Type Culture Collection, CRL-11268, Shanghai, China) were cultured in Gibco Roswell Park Memorial Institute medium 1640 basic (1×) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Shanghai, China) and 1% penicillin–streptomycin solution (Beyotime, Jiangsu, China) at 37 °C in an incubator humidified with 5% CO2 atmosphere.

    Techniques: Expressing

    Biomechanical analysis of HEK293T cells in phosphate buffer solution (PBS) buffer. A sample force versus distance curve obtained on an untreated HEK293T cell shows the tip approach (red) and withdrawal (back). The energy involved in the indentation process includes two parts: elastic energy (green) and viscous energy (yellow).

    Journal: ACS Omega

    Article Title: Nanotoxicity of Silver Nanoparticles on HEK293T Cells: A Combined Study Using Biomechanical and Biological Techniques

    doi: 10.1021/acsomega.8b00608

    Figure Lengend Snippet: Biomechanical analysis of HEK293T cells in phosphate buffer solution (PBS) buffer. A sample force versus distance curve obtained on an untreated HEK293T cell shows the tip approach (red) and withdrawal (back). The energy involved in the indentation process includes two parts: elastic energy (green) and viscous energy (yellow).

    Article Snippet: 4.2 Cells Culture and Cell Viability Measurement HEK293T cells (American Type Culture Collection, CRL-11268, Shanghai, China) were cultured in Gibco Roswell Park Memorial Institute medium 1640 basic (1×) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Shanghai, China) and 1% penicillin–streptomycin solution (Beyotime, Jiangsu, China) at 37 °C in an incubator humidified with 5% CO2 atmosphere.

    Techniques:

    Representative images of HEK293T cells following exposure to varying AgNPs concentrations for 24 h, observed using an inverted optical microscope: (A) 0 μg/mL, (B) 10 μg/mL, (C) 20 μg/mL, and (D) 40 μg/mL.

    Journal: ACS Omega

    Article Title: Nanotoxicity of Silver Nanoparticles on HEK293T Cells: A Combined Study Using Biomechanical and Biological Techniques

    doi: 10.1021/acsomega.8b00608

    Figure Lengend Snippet: Representative images of HEK293T cells following exposure to varying AgNPs concentrations for 24 h, observed using an inverted optical microscope: (A) 0 μg/mL, (B) 10 μg/mL, (C) 20 μg/mL, and (D) 40 μg/mL.

    Article Snippet: 4.2 Cells Culture and Cell Viability Measurement HEK293T cells (American Type Culture Collection, CRL-11268, Shanghai, China) were cultured in Gibco Roswell Park Memorial Institute medium 1640 basic (1×) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Shanghai, China) and 1% penicillin–streptomycin solution (Beyotime, Jiangsu, China) at 37 °C in an incubator humidified with 5% CO2 atmosphere.

    Techniques: Microscopy

    Dose–response relationship for DNA damage in HEK293T cells after exposure to AgNPs measured using the comet assay. DNA damage in terms of (A) tail DNA% and (B) tail moment obtained at different AgNPs concentrations (0, 10, 20, and 40 μg/mL), fitted each with a linear regression line.

    Journal: ACS Omega

    Article Title: Nanotoxicity of Silver Nanoparticles on HEK293T Cells: A Combined Study Using Biomechanical and Biological Techniques

    doi: 10.1021/acsomega.8b00608

    Figure Lengend Snippet: Dose–response relationship for DNA damage in HEK293T cells after exposure to AgNPs measured using the comet assay. DNA damage in terms of (A) tail DNA% and (B) tail moment obtained at different AgNPs concentrations (0, 10, 20, and 40 μg/mL), fitted each with a linear regression line.

    Article Snippet: 4.2 Cells Culture and Cell Viability Measurement HEK293T cells (American Type Culture Collection, CRL-11268, Shanghai, China) were cultured in Gibco Roswell Park Memorial Institute medium 1640 basic (1×) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Shanghai, China) and 1% penicillin–streptomycin solution (Beyotime, Jiangsu, China) at 37 °C in an incubator humidified with 5% CO2 atmosphere.

    Techniques: Single Cell Gel Electrophoresis

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M113.529339

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

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

    Techniques: Transfection, SDS Page

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M113.529339

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

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

    Techniques: Transfection, Luciferase, Activity Assay

    CstF64 Modulates Alternative 3′ End Processing of MRJ (A) Diagram showing the MRJ minigene spanning exons 7 to 9 of the human MRJ gene with truncated introns. Arrows depict the primers used for RT-PCR of minigene MRJ-L or MRJ-S transcripts ( Table S1 ), 5′ splice site (5′SS) alteration, and SNP-derived mutations that were downstream of the proximal PAS or within the polypyrimidine tract (PYT). (B) HEK293T cells were co-transfected with the MRJ minigene and the empty or HA-tagged CstF64-expression vector. RT-PCR was performed using the indicated primers (arrows) followed by Southern blotting (see Materials and Methods ). Bar graph indicates the fold increase of minigene MRJ-S to total MRJ (T) in the CstF64 transfection. Immunoblotting (IB) was performed by using anti-HA and anti-GAPDH antibodies. (C) HEK293T cells were cotransfected with the MRJ minigene and the empty or expression vector encoding HA-tagged SRSF1, SRSF2, or SRSF3. Relative fold changes of minigene MRJ-S to total MRJ (T) are indicated below the gel. Immunoblotting was performed with anti-HA and anti-actin (alternative name ACTB). (D) HEK293T cells were transfected with the wild-type or mutant MRJ minigene vectors. Bar graph is as described in (B). For (B) and (D), mean values were obtained from three independent experiments. *p ⩽ 0.05; **p ⩽ 0.01; ***p ⩽ 0.001.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Interference of DNAJB6/MRJ Isoform Switch by Morpholino Inhibits Replication of HIV-1 and RSV

    doi: 10.1016/j.omtn.2018.12.001

    Figure Lengend Snippet: CstF64 Modulates Alternative 3′ End Processing of MRJ (A) Diagram showing the MRJ minigene spanning exons 7 to 9 of the human MRJ gene with truncated introns. Arrows depict the primers used for RT-PCR of minigene MRJ-L or MRJ-S transcripts ( Table S1 ), 5′ splice site (5′SS) alteration, and SNP-derived mutations that were downstream of the proximal PAS or within the polypyrimidine tract (PYT). (B) HEK293T cells were co-transfected with the MRJ minigene and the empty or HA-tagged CstF64-expression vector. RT-PCR was performed using the indicated primers (arrows) followed by Southern blotting (see Materials and Methods ). Bar graph indicates the fold increase of minigene MRJ-S to total MRJ (T) in the CstF64 transfection. Immunoblotting (IB) was performed by using anti-HA and anti-GAPDH antibodies. (C) HEK293T cells were cotransfected with the MRJ minigene and the empty or expression vector encoding HA-tagged SRSF1, SRSF2, or SRSF3. Relative fold changes of minigene MRJ-S to total MRJ (T) are indicated below the gel. Immunoblotting was performed with anti-HA and anti-actin (alternative name ACTB). (D) HEK293T cells were transfected with the wild-type or mutant MRJ minigene vectors. Bar graph is as described in (B). For (B) and (D), mean values were obtained from three independent experiments. *p ⩽ 0.05; **p ⩽ 0.01; ***p ⩽ 0.001.

    Article Snippet: Cell Cultures and Chemicals HEK293T cells (ATCC, CRL-11268) and human lung epithelial A549 cells (A549; ATCC, CCL-185) were maintained in DMEM (HyClone) containing 10% fetal bovine serum (FBS; Biological Industries).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Transfection, Expressing, Plasmid Preparation, Southern Blot, Mutagenesis

    MoMRJ Suppresses MRJ-L Expression (A) Diagram showing the MRJ pre-mRNA substrate containing exons 8 and 9 and an internally truncated intron 8. The antisense morpholino was complementary to the 5′ splice site of MRJ intron 8. In vitro splicing of 32 P-labeled MRJ pre-mRNA was performed in HeLa cell nuclear extract. MoMRJ or the negative control morpholino (MoC) was added into the reactions (mock, without morpholino). Pre-mRNA and splicing intermediates and products are depicted at the right of the gel. (B) HEK293T cells were treated with different amounts of MoMRJ or control MoC in serum-free medium for 24 hr. RT-PCR and immunoblotting were performed to determine the RNA and protein levels, respectively, of MRJ isoforms. Bar graphs indicate relative ratios of MRJ-L to total MRJ (T); data were obtained from three independent experiments. *p ⩽ 0.05; **p ⩽ 0.01; ***p ⩽ 0.001.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Interference of DNAJB6/MRJ Isoform Switch by Morpholino Inhibits Replication of HIV-1 and RSV

    doi: 10.1016/j.omtn.2018.12.001

    Figure Lengend Snippet: MoMRJ Suppresses MRJ-L Expression (A) Diagram showing the MRJ pre-mRNA substrate containing exons 8 and 9 and an internally truncated intron 8. The antisense morpholino was complementary to the 5′ splice site of MRJ intron 8. In vitro splicing of 32 P-labeled MRJ pre-mRNA was performed in HeLa cell nuclear extract. MoMRJ or the negative control morpholino (MoC) was added into the reactions (mock, without morpholino). Pre-mRNA and splicing intermediates and products are depicted at the right of the gel. (B) HEK293T cells were treated with different amounts of MoMRJ or control MoC in serum-free medium for 24 hr. RT-PCR and immunoblotting were performed to determine the RNA and protein levels, respectively, of MRJ isoforms. Bar graphs indicate relative ratios of MRJ-L to total MRJ (T); data were obtained from three independent experiments. *p ⩽ 0.05; **p ⩽ 0.01; ***p ⩽ 0.001.

    Article Snippet: Cell Cultures and Chemicals HEK293T cells (ATCC, CRL-11268) and human lung epithelial A549 cells (A549; ATCC, CCL-185) were maintained in DMEM (HyClone) containing 10% fetal bovine serum (FBS; Biological Industries).

    Techniques: Expressing, In Vitro, Labeling, Negative Control, Reverse Transcription Polymerase Chain Reaction

    Expression of CstF64 and MRJ Isoforms in Monocytes and Macrophages (A) Schematic diagram showing the MRJ ( DNAJB6 ) gene structure and two transcript isoforms generated by alternative splicing and polyadenylation. Arrows depict the primers used for RT-PCR of MRJ-L or MRJ-S ( Table S1 ). (B) Monocytes (Mo) collected from healthy donors were differentiated to macrophages (Mψ). Total cellular proteins were subjected to immunoblotting using antibodies against CstF64, MRJ, and GAPDH. Bar graphs indicate the levels of CstF64, MRJ-L, and MRJ-S in Mψ relative to that of Mo (set to 1); their levels were normalized to GAPDH. (C) Immunoblotting of THP-1 monocytes that were mock treated (Mo) or treated with PMA for 24 or 48 hr to differentiate into macrophage-like cells (Mψ). Antibodies used and bar graphs for relative CstF64 and MRJ levels are as described in (B). (D) THP-1 monocytes were transduced with shRNA (Luc or CstF64)-expressing lentiviruses. Immunoblotting analysis was performed using antibodies against CstF64, MRJ, and GAPDH (left panel). HEK293T cells were transduced with indicated shRNA (Luc or CstF64) and mock transformed or transformed with the CstF64 expression vector (+), followed by immunoblotting (right panel). Bar graphs indicate the ratio of MRJ-L to total MRJ (T, i.e., L+S); the data were obtained from two independent experiments. ***p ⩽ 0.001. (E) Schematic diagram showing that the level of CstF64 influences alternative 3′ end processing of the MRJ pre-mRNA. Downregulation of CstF64 promoted MRJ-L expression.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Interference of DNAJB6/MRJ Isoform Switch by Morpholino Inhibits Replication of HIV-1 and RSV

    doi: 10.1016/j.omtn.2018.12.001

    Figure Lengend Snippet: Expression of CstF64 and MRJ Isoforms in Monocytes and Macrophages (A) Schematic diagram showing the MRJ ( DNAJB6 ) gene structure and two transcript isoforms generated by alternative splicing and polyadenylation. Arrows depict the primers used for RT-PCR of MRJ-L or MRJ-S ( Table S1 ). (B) Monocytes (Mo) collected from healthy donors were differentiated to macrophages (Mψ). Total cellular proteins were subjected to immunoblotting using antibodies against CstF64, MRJ, and GAPDH. Bar graphs indicate the levels of CstF64, MRJ-L, and MRJ-S in Mψ relative to that of Mo (set to 1); their levels were normalized to GAPDH. (C) Immunoblotting of THP-1 monocytes that were mock treated (Mo) or treated with PMA for 24 or 48 hr to differentiate into macrophage-like cells (Mψ). Antibodies used and bar graphs for relative CstF64 and MRJ levels are as described in (B). (D) THP-1 monocytes were transduced with shRNA (Luc or CstF64)-expressing lentiviruses. Immunoblotting analysis was performed using antibodies against CstF64, MRJ, and GAPDH (left panel). HEK293T cells were transduced with indicated shRNA (Luc or CstF64) and mock transformed or transformed with the CstF64 expression vector (+), followed by immunoblotting (right panel). Bar graphs indicate the ratio of MRJ-L to total MRJ (T, i.e., L+S); the data were obtained from two independent experiments. ***p ⩽ 0.001. (E) Schematic diagram showing that the level of CstF64 influences alternative 3′ end processing of the MRJ pre-mRNA. Downregulation of CstF64 promoted MRJ-L expression.

    Article Snippet: Cell Cultures and Chemicals HEK293T cells (ATCC, CRL-11268) and human lung epithelial A549 cells (A549; ATCC, CCL-185) were maintained in DMEM (HyClone) containing 10% fetal bovine serum (FBS; Biological Industries).

    Techniques: Expressing, Generated, Reverse Transcription Polymerase Chain Reaction, Transduction, shRNA, Transformation Assay, Plasmid Preparation

    Evaluation of the effect of copy number on riboswitch dynamics. (A) Schematic representation of constructs generated through insertion of 1, 2, 3 or 4 copies of each riboswitch into the 3′-UTR and depiction of the mechanism of an ‘ON-type’ riboswitch in the presence or absence of activating ligand. hPGK; human phosphoglycerate kinase promoter, p(A); polyadenylation signal, SV40; Simian vacuolating virus 40 promoter, RS; riboswitch. Dual luciferase assay in HEK293T cells with (grey bars) or without (blue bars) the activating ligand at saturating levels for cells transfected with 1,2,3 or 4 copies of (B) K19, (C) L2Bulge9, (D) L2Bulge18tc, (E) GuaM8HDV or (F) TC45; n = 3 for all groups, green arrow represents the optimal copy number. Values are normalized to unmodified Firefly luciferase activity with standard error shown.

    Journal: Scientific Reports

    Article Title: Development of an inducible anti-VEGF rAAV gene therapy strategy for the treatment of wet AMD

    doi: 10.1038/s41598-018-29726-7

    Figure Lengend Snippet: Evaluation of the effect of copy number on riboswitch dynamics. (A) Schematic representation of constructs generated through insertion of 1, 2, 3 or 4 copies of each riboswitch into the 3′-UTR and depiction of the mechanism of an ‘ON-type’ riboswitch in the presence or absence of activating ligand. hPGK; human phosphoglycerate kinase promoter, p(A); polyadenylation signal, SV40; Simian vacuolating virus 40 promoter, RS; riboswitch. Dual luciferase assay in HEK293T cells with (grey bars) or without (blue bars) the activating ligand at saturating levels for cells transfected with 1,2,3 or 4 copies of (B) K19, (C) L2Bulge9, (D) L2Bulge18tc, (E) GuaM8HDV or (F) TC45; n = 3 for all groups, green arrow represents the optimal copy number. Values are normalized to unmodified Firefly luciferase activity with standard error shown.

    Article Snippet: Cell culture conditions HEK293T cells were obtained from ATCC (#CRL-11268, Manassas, VA) and cultured in Dulbecco’s Modified Eagle Medium with high glucose and GlutaMAX (Thermo Fisher Technologies, Carlsbad, CA).

    Techniques: Construct, Generated, Luciferase, Transfection, Activity Assay

    Assessment of riboswitch dosing kinetics in HEK293T cells. Normalized GFP fluorescence and representative GFP (inducible) and mCherry (non-inducible) images at varying concentrations of activating ligand for (A) K19, (B) L2Bulge9, (C) L2Bulge18tc, (D) GuaM8HDV and (E) TC45. Dynamic range is represented as fold-change. n = 3 , ** p > 0.01, *** p > 0.001, **** p > 0.001, One-Way ANOVA with Tukey’s post-hoc test. For each group, respective fluorescent channels were recorded with constant exposure and gain; scale bars = 50 μM.

    Journal: Scientific Reports

    Article Title: Development of an inducible anti-VEGF rAAV gene therapy strategy for the treatment of wet AMD

    doi: 10.1038/s41598-018-29726-7

    Figure Lengend Snippet: Assessment of riboswitch dosing kinetics in HEK293T cells. Normalized GFP fluorescence and representative GFP (inducible) and mCherry (non-inducible) images at varying concentrations of activating ligand for (A) K19, (B) L2Bulge9, (C) L2Bulge18tc, (D) GuaM8HDV and (E) TC45. Dynamic range is represented as fold-change. n = 3 , ** p > 0.01, *** p > 0.001, **** p > 0.001, One-Way ANOVA with Tukey’s post-hoc test. For each group, respective fluorescent channels were recorded with constant exposure and gain; scale bars = 50 μM.

    Article Snippet: Cell culture conditions HEK293T cells were obtained from ATCC (#CRL-11268, Manassas, VA) and cultured in Dulbecco’s Modified Eagle Medium with high glucose and GlutaMAX (Thermo Fisher Technologies, Carlsbad, CA).

    Techniques: Fluorescence

    Measurements of different cell populations in spleen cells by flow cytometry using appropriate surface markers conjugated with different fluorochromes. Fluorescence signal of T-cell surface marker CD3e conjugated with PE-Vio770 (P1 = T cells) and B-cell surface marker CD19 conjugated with APC-Vio770 (P2 = B cells) on the cells from mice receiving (a) vehicle control or (b) lipopolysaccharide (LPS). The percentage of each cell population in mice (c) 24 h after LPS treatment (three doses, n = 4) or (d) 3 weeks after treatment with extracellular vesicles (10 doses, n = 10). Bars and error bars denote the mean and standard deviation, respectively, of experimental groups. PBS, phosphate-buffered saline; WT, wild-type HEK293T cells. * p

    Journal: Journal of Extracellular Vesicles

    Article Title: Comprehensive toxicity and immunogenicity studies reveal minimal effects in mice following sustained dosing of extracellular vesicles derived from HEK293T cells

    doi: 10.1080/20013078.2017.1324730

    Figure Lengend Snippet: Measurements of different cell populations in spleen cells by flow cytometry using appropriate surface markers conjugated with different fluorochromes. Fluorescence signal of T-cell surface marker CD3e conjugated with PE-Vio770 (P1 = T cells) and B-cell surface marker CD19 conjugated with APC-Vio770 (P2 = B cells) on the cells from mice receiving (a) vehicle control or (b) lipopolysaccharide (LPS). The percentage of each cell population in mice (c) 24 h after LPS treatment (three doses, n = 4) or (d) 3 weeks after treatment with extracellular vesicles (10 doses, n = 10). Bars and error bars denote the mean and standard deviation, respectively, of experimental groups. PBS, phosphate-buffered saline; WT, wild-type HEK293T cells. * p

    Article Snippet: Cell lines Wild-type HEK293T cells (WT, ATCC CRL-11268) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Carlsbad, CA, USA) supplemented with 5% foetal bovine serum (Sigma, St Louis, MO, USA) that was depleted of EVs by ultracentrifugation (110,000 × g , 18 h).

    Techniques: Flow Cytometry, Cytometry, Fluorescence, Marker, Mouse Assay, Standard Deviation

    Interrogating NADH/NAD + Redox in Live HEK283T Cells. Panel A displays sensor emission of three channels (green, red and ratiometric) of HEK293T cells. Panel B depicts a quantification of the average of five replicate experiments using the ratiometric channel to express the percentage of LipDH-mCherry reduction.

    Journal: Biochemistry

    Article Title: Designing Flavoprotein-GFP fusion Probes for Analyte-specific Ratiometric Fluorescence Imaging

    doi: 10.1021/acs.biochem.7b01132

    Figure Lengend Snippet: Interrogating NADH/NAD + Redox in Live HEK283T Cells. Panel A displays sensor emission of three channels (green, red and ratiometric) of HEK293T cells. Panel B depicts a quantification of the average of five replicate experiments using the ratiometric channel to express the percentage of LipDH-mCherry reduction.

    Article Snippet: Stock HEK293T cells (ATCC CRL-11268) were grown and maintained at 37 °C using 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM) with high glucose supplemented with 10% fetal bovine serum and 100 units/mL penicillin and streptomycin.

    Techniques:

    The Del6 mutant subunit presents increased Kv7.2 total protein. A.- Western blot of increasing sample load from HEK293T cells extracts expressing the same amount of DNA coding WT- or Del6-Kv7.2 Myc-tagged subunits. The optical densities (OD) of the bands were analyzed using ImageJ software. A linear regression fit was obtained from the OD vs load relation. From this regression, the amount required to obtain the same OD’s was found to be 4.7±1.22 fold larger ( n = 5) for WT subunits than for Del6 mutant Kv7.2 subunits. Top, the cartoons are schematic representations of the tetrameric assemblies. Each square represents a subunit of the tetrameric channel, the ovals represent the A-B loops, and the black circles highlights the presence of a Myc tag. B.- The steady-state protein levels are inversely proportional to the number of A–B loops present in the tetrameric Kv7.2 assemblies. Western blot of protein extracts from HEK293T cells expressing Del6- (right panel) or WT- Myc-Kv7.2 subunits (left panel) when co-expressed with a six fold larger amount of plasmid DNA coding the indicated YFP-tagged protein ( n = 4). Myc tagged WT and Del6 Kv7.2 channels were detected using anti-Myc antibody. The Del6 protein levels decreased when co-expressed with WT subunits, whereas increased protein levels of WT Kv7.2 were detected when co-expressed with Del6. A schematic representation of the theoretical assemblies detected with the anti-Myc antibody is represented at the top. The subunits that were overexpressed and that lack a Myc tag, so they are not detected in the Western blot, are indicated at the bottom of each lane. C.- Normalized quantification of Western blot signals as a function of the theoretical number of A–B loops present on the detected channel assemblies. On each Western blot, the OD’s were normalized to the signal obtained for tubulin. The values were subsequently normalized to those obtained with Myc-Del6 or Myc-WT in the same Western blot. Data points for 0 (Del6) and 4 (WT) loops were derived from data obtained as in panel A, data points for 1 and 3 loops were obtained from Western blots equivalent to the left and right columns in panel B, respectively. The points represent the means ± S.E.M. (n ≥4). D.- Pulse-chase analysis of WT-, Del2- and Del6-Kv7.2 subunit stability. Densitometric quantification of the bands normalized to the value at time 0 (no chase). Each data point is the mean ± SEM calculated from three separate experiments. Inset: representative images of autoradiographic films of experiments in HEK293T cells transfected with the indicated plasmids. Metabolic labeling was performed for 1 h, 36 h post-transfection, followed by chase times of 1, 2 and 4 h.

    Journal: PLoS ONE

    Article Title: Surface Expression and Subunit Specific Control of Steady Protein Levels by the Kv7.2 Helix A-B Linker

    doi: 10.1371/journal.pone.0047263

    Figure Lengend Snippet: The Del6 mutant subunit presents increased Kv7.2 total protein. A.- Western blot of increasing sample load from HEK293T cells extracts expressing the same amount of DNA coding WT- or Del6-Kv7.2 Myc-tagged subunits. The optical densities (OD) of the bands were analyzed using ImageJ software. A linear regression fit was obtained from the OD vs load relation. From this regression, the amount required to obtain the same OD’s was found to be 4.7±1.22 fold larger ( n = 5) for WT subunits than for Del6 mutant Kv7.2 subunits. Top, the cartoons are schematic representations of the tetrameric assemblies. Each square represents a subunit of the tetrameric channel, the ovals represent the A-B loops, and the black circles highlights the presence of a Myc tag. B.- The steady-state protein levels are inversely proportional to the number of A–B loops present in the tetrameric Kv7.2 assemblies. Western blot of protein extracts from HEK293T cells expressing Del6- (right panel) or WT- Myc-Kv7.2 subunits (left panel) when co-expressed with a six fold larger amount of plasmid DNA coding the indicated YFP-tagged protein ( n = 4). Myc tagged WT and Del6 Kv7.2 channels were detected using anti-Myc antibody. The Del6 protein levels decreased when co-expressed with WT subunits, whereas increased protein levels of WT Kv7.2 were detected when co-expressed with Del6. A schematic representation of the theoretical assemblies detected with the anti-Myc antibody is represented at the top. The subunits that were overexpressed and that lack a Myc tag, so they are not detected in the Western blot, are indicated at the bottom of each lane. C.- Normalized quantification of Western blot signals as a function of the theoretical number of A–B loops present on the detected channel assemblies. On each Western blot, the OD’s were normalized to the signal obtained for tubulin. The values were subsequently normalized to those obtained with Myc-Del6 or Myc-WT in the same Western blot. Data points for 0 (Del6) and 4 (WT) loops were derived from data obtained as in panel A, data points for 1 and 3 loops were obtained from Western blots equivalent to the left and right columns in panel B, respectively. The points represent the means ± S.E.M. (n ≥4). D.- Pulse-chase analysis of WT-, Del2- and Del6-Kv7.2 subunit stability. Densitometric quantification of the bands normalized to the value at time 0 (no chase). Each data point is the mean ± SEM calculated from three separate experiments. Inset: representative images of autoradiographic films of experiments in HEK293T cells transfected with the indicated plasmids. Metabolic labeling was performed for 1 h, 36 h post-transfection, followed by chase times of 1, 2 and 4 h.

    Article Snippet: Cell Culture and Transfection HEK293T cells (HEK 293T/17, ATCC, CRL-11268) were maintained at 37°C and 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich), supplemented with non-essential amino acids (Sigma) and 10% FBS (Lonza).

    Techniques: Mutagenesis, Western Blot, Expressing, Software, Plasmid Preparation, Derivative Assay, Pulse Chase, Transfection, Labeling

    Single-molecule analysis of rhomboid protease and substrate diffusion in living human cells. ( A ) smTIRF image of a HEK293T cell expressing Halo-RHBDL2 labeled with HTL-JF646. White scale bars indicate 5 μm throughout. ( B ) Electrophoretic analysis of whole cell lysates of HTL-JF646-labeled HEK293T cells expressing the indicated RHBDL2 construct. ( C ) Processing of GFP-Ephrin B3-Flag in transfected HEK293T cells co-expressing Halo-tagged versus untagged RHBDL2. Denoted are uncut (asterisks) and cut (arrow) fragments. ( D ) D for protein names/sources.( E ) Parallel comparison of Halo-RHBDL2 versus Halo-Rhodopsin diffusion in HEK293T cells.( F ) smTIRF image of a HEK293T cell with its endogenous RHBDL2 tagged with Halo (labeled with HTL-JF549), and single-molecule tracks of the same cell over 2,000 frames. Tracks are color-coded by D , and the mean±s.d. is shown for the indicated number of cells throughout. ( G ) Tracks of JF549-SNAP-Ephrin B3-Flag diffusion in HEK293T cells. ( H ) Halo-RHBDL2 mobility in living HEK293T cells treated with actin or microtubule destabilizing or stabilizing agents (see Methods). Means are normalized to each untreated sample analysed in parallel. Cohen’s d-values ranged from 0.004-0.21, indicating effect sizes between very small and small.

    Journal: Science (New York, N.Y.)

    Article Title: Rhomboid distorts lipids to break the viscosity-imposed speed limit of membrane diffusion

    doi: 10.1126/science.aao0076

    Figure Lengend Snippet: Single-molecule analysis of rhomboid protease and substrate diffusion in living human cells. ( A ) smTIRF image of a HEK293T cell expressing Halo-RHBDL2 labeled with HTL-JF646. White scale bars indicate 5 μm throughout. ( B ) Electrophoretic analysis of whole cell lysates of HTL-JF646-labeled HEK293T cells expressing the indicated RHBDL2 construct. ( C ) Processing of GFP-Ephrin B3-Flag in transfected HEK293T cells co-expressing Halo-tagged versus untagged RHBDL2. Denoted are uncut (asterisks) and cut (arrow) fragments. ( D ) D for protein names/sources.( E ) Parallel comparison of Halo-RHBDL2 versus Halo-Rhodopsin diffusion in HEK293T cells.( F ) smTIRF image of a HEK293T cell with its endogenous RHBDL2 tagged with Halo (labeled with HTL-JF549), and single-molecule tracks of the same cell over 2,000 frames. Tracks are color-coded by D , and the mean±s.d. is shown for the indicated number of cells throughout. ( G ) Tracks of JF549-SNAP-Ephrin B3-Flag diffusion in HEK293T cells. ( H ) Halo-RHBDL2 mobility in living HEK293T cells treated with actin or microtubule destabilizing or stabilizing agents (see Methods). Means are normalized to each untreated sample analysed in parallel. Cohen’s d-values ranged from 0.004-0.21, indicating effect sizes between very small and small.

    Article Snippet: Human HEK293T cells (CRL-11268, purchased from American Type Culture Collection) were grown in DMEM (Life Technologies) supplemented with 2mM L-glutamine, 10mM Hepes pH 7, 10% fetal bovine serum (F4135, Sigma), and 10 μg/ml gentamicin at 37°C and humidified 5% CO2 .

    Techniques: Diffusion-based Assay, Expressing, Labeling, Construct, Transfection

    Rhomboid proteolysis is diffusion-limited in living cells. ( A ) Calorigrams of DMPC vesicles showing a shift in transition temperature (T m ) and trough broadening exerted by Mg 2+ /Ionomycin relative to methanol vehicle. ( B ) NHalo-RHBDL2 molecule tracks (colored by D ) on the same field of HEK293T cells before and after Mg 2+ /Ionomycin addition. White scale bars indicate 5 μm. ( C ) Diffusion of NHalo-RHBDL2 in living HEK293T cells treated with Mg 2+ /Ionomycin (MgIono) or Thapsigargin (Thap) versus buffer (normalized to the untreated mean). Mglono accelerated diffusion (p

    Journal: Science (New York, N.Y.)

    Article Title: Rhomboid distorts lipids to break the viscosity-imposed speed limit of membrane diffusion

    doi: 10.1126/science.aao0076

    Figure Lengend Snippet: Rhomboid proteolysis is diffusion-limited in living cells. ( A ) Calorigrams of DMPC vesicles showing a shift in transition temperature (T m ) and trough broadening exerted by Mg 2+ /Ionomycin relative to methanol vehicle. ( B ) NHalo-RHBDL2 molecule tracks (colored by D ) on the same field of HEK293T cells before and after Mg 2+ /Ionomycin addition. White scale bars indicate 5 μm. ( C ) Diffusion of NHalo-RHBDL2 in living HEK293T cells treated with Mg 2+ /Ionomycin (MgIono) or Thapsigargin (Thap) versus buffer (normalized to the untreated mean). Mglono accelerated diffusion (p

    Article Snippet: Human HEK293T cells (CRL-11268, purchased from American Type Culture Collection) were grown in DMEM (Life Technologies) supplemented with 2mM L-glutamine, 10mM Hepes pH 7, 10% fetal bovine serum (F4135, Sigma), and 10 μg/ml gentamicin at 37°C and humidified 5% CO2 .

    Techniques: Diffusion-based Assay

    Single-molecule analysis of rhomboid protease and substrate diffusion in living Drosophila cells. ( A ) smTIRF image of DmRho4-HaloC-JF549 molecules in a S2R+ cell (left), and their diffusion tracks (right, recorded for 2,000 frames at 25 Hz). Tracks are color-coded by D , the mean±s.d. is shown for the indicated number of cells, and white scale bars indicate 5 μm throughout. ( B ) Electrophoretic analysis of whole cell lysates of HTL-JF646-labeled S2R+ cells expressing the indicated DmRho4 construct. ( C ) Processing of GFP-Spitz in transfected S2R+ cells co-expressing Halo-tagged versus untagged DmRho4, and in the absence (−) or presence (+) of Ca 2+ stimulation. Uncut (asterisks) and cut (arrow) fragments are indicated. Untransfected cells were analysed in the leftmost lane. ( D ) Tracks of JF549-SNAP-Spitz diffusing in a S2R+ cell. ( E ) D comparisons: DmRho4 diffused faster than RHBDL2 in both S2R+ cells (p=2.0×10 −184 ) and HEK293T cells (p=4.1×10 −244 ), and diffusion of both proteins was faster in S2R+ cells than in HEK293T cells (DmR4, p=1.6×10 −195 ; RHBDL2, p=3.4×10 −233 ). Data is normalized to DmRho4 in S2R+ cells

    Journal: Science (New York, N.Y.)

    Article Title: Rhomboid distorts lipids to break the viscosity-imposed speed limit of membrane diffusion

    doi: 10.1126/science.aao0076

    Figure Lengend Snippet: Single-molecule analysis of rhomboid protease and substrate diffusion in living Drosophila cells. ( A ) smTIRF image of DmRho4-HaloC-JF549 molecules in a S2R+ cell (left), and their diffusion tracks (right, recorded for 2,000 frames at 25 Hz). Tracks are color-coded by D , the mean±s.d. is shown for the indicated number of cells, and white scale bars indicate 5 μm throughout. ( B ) Electrophoretic analysis of whole cell lysates of HTL-JF646-labeled S2R+ cells expressing the indicated DmRho4 construct. ( C ) Processing of GFP-Spitz in transfected S2R+ cells co-expressing Halo-tagged versus untagged DmRho4, and in the absence (−) or presence (+) of Ca 2+ stimulation. Uncut (asterisks) and cut (arrow) fragments are indicated. Untransfected cells were analysed in the leftmost lane. ( D ) Tracks of JF549-SNAP-Spitz diffusing in a S2R+ cell. ( E ) D comparisons: DmRho4 diffused faster than RHBDL2 in both S2R+ cells (p=2.0×10 −184 ) and HEK293T cells (p=4.1×10 −244 ), and diffusion of both proteins was faster in S2R+ cells than in HEK293T cells (DmR4, p=1.6×10 −195 ; RHBDL2, p=3.4×10 −233 ). Data is normalized to DmRho4 in S2R+ cells

    Article Snippet: Human HEK293T cells (CRL-11268, purchased from American Type Culture Collection) were grown in DMEM (Life Technologies) supplemented with 2mM L-glutamine, 10mM Hepes pH 7, 10% fetal bovine serum (F4135, Sigma), and 10 μg/ml gentamicin at 37°C and humidified 5% CO2 .

    Techniques: Diffusion-based Assay, Labeling, Expressing, Construct, Transfection

    Experimental and evolutionary tuning of rhomboid diffusivity. ( A ) Tracks (colored by D ) of RHBDL2-HaloC molecule diffusion in HEK293T cell after being complexed with quantum dots. Mean±s.d. is shown. White scale bar indicates 5 μm. ( B ) Relative protease activity of RHBDL2-HaloC (extracellular tag) versus NHalo-RHBDL2 (cytoplasmic tag) on Ephrin B3 in HEK293T cells treated with extracellular quantum dots relative to buffer. ( C ) D of NHalo-RHBDL2 (RH2), EhROM1-HaloC (EhR1), NHalo-TvROM1 (TvR1), RHBDL3/Ventrhoid-HaloC (VRho), NHalo-iRhom2 (N-iR2), iRhom2-HaloC (iR2-C), and iRhom1-HaloC (iR1) in living HEK293T cells. Mean±s.d. of > 18,000 tracks (at 64Hz) from ≥5 cells is shown.

    Journal: Science (New York, N.Y.)

    Article Title: Rhomboid distorts lipids to break the viscosity-imposed speed limit of membrane diffusion

    doi: 10.1126/science.aao0076

    Figure Lengend Snippet: Experimental and evolutionary tuning of rhomboid diffusivity. ( A ) Tracks (colored by D ) of RHBDL2-HaloC molecule diffusion in HEK293T cell after being complexed with quantum dots. Mean±s.d. is shown. White scale bar indicates 5 μm. ( B ) Relative protease activity of RHBDL2-HaloC (extracellular tag) versus NHalo-RHBDL2 (cytoplasmic tag) on Ephrin B3 in HEK293T cells treated with extracellular quantum dots relative to buffer. ( C ) D of NHalo-RHBDL2 (RH2), EhROM1-HaloC (EhR1), NHalo-TvROM1 (TvR1), RHBDL3/Ventrhoid-HaloC (VRho), NHalo-iRhom2 (N-iR2), iRhom2-HaloC (iR2-C), and iRhom1-HaloC (iR1) in living HEK293T cells. Mean±s.d. of > 18,000 tracks (at 64Hz) from ≥5 cells is shown.

    Article Snippet: Human HEK293T cells (CRL-11268, purchased from American Type Culture Collection) were grown in DMEM (Life Technologies) supplemented with 2mM L-glutamine, 10mM Hepes pH 7, 10% fetal bovine serum (F4135, Sigma), and 10 μg/ml gentamicin at 37°C and humidified 5% CO2 .

    Techniques: Diffusion-based Assay, Activity Assay

    K101 acetylation promotes ECHS1 ubiquitination and degradation. a NAM treatment effects on endogenous ECHS1 levels in HEK293T cells were analyzed in the presence and absence of MG132. Mean values of quantitation with SD are reported. NS not significant; *** P

    Journal: Nature Communications

    Article Title: Enoyl-CoA hydratase-1 regulates mTOR signaling and apoptosis by sensing nutrients

    doi: 10.1038/s41467-017-00489-5

    Figure Lengend Snippet: K101 acetylation promotes ECHS1 ubiquitination and degradation. a NAM treatment effects on endogenous ECHS1 levels in HEK293T cells were analyzed in the presence and absence of MG132. Mean values of quantitation with SD are reported. NS not significant; *** P

    Article Snippet: Cell culture and treatments HEK293T cells (ATCC Number: CRL-11268), 786-O(ATCC Number: CRL-1932), ACHN renal cell cancer cells (ATCC Number: CRL-1611), SMMC7712 (CBP60210, Shanghai Cell Bank), Huh7 liver cancer cells (CBP60202, Shanghai Cell Bank), HCT 116 (ATCC Number: CCL-247), SW620 colon cancer cells (ATCC Number: CCL-227), LNCaP (ATCC Number: CRL-1740), PC3 prostate cancers cells(ATCC Number: CRL-1435), Chang cells (ATCC Number: CCL-13) were used in this study.

    Techniques: Quantitation Assay

    Nutrient induces FAs accumulation. a The conversion of 13 C-palmitate to 13 C-citrate was traced in HEK293T cells cultured in low and high glucose media, and in ECHS1 overexpressing HEK293T cells cultured in high glucose media. b Relative FAs levels in HEK293T cells, ECHS1 knocked down HEK293T cells and ECHS1 knocked down HEK293T cells overexpressing shRNA resistant ECHS1 (ECHS1-r) were detected. All levels were normalized to the total proteins. c The relative butyrate levels in Chang liver cells and ECHS1 knocked down Chang liver cells were determined ( n = 4). The butyrate levels in untreated Chang liver cells (25 μM) were set as 100%. d The relative FAs levels in HEK293T cells cultured in low glucose (−), high glucose (+), and high glucose with ECHS1 K101R overexpression were determined ( n = 3). e The FAs levels in the liver of wild type, Sirt3 −/− , and ECHS1 K/R overexpressing Sirt3 −/− 129/C57BL6 mice were assessed by oil red staining. Four mice of each group were analyzed. Relative FAs levels were determined by comparing intensity of signals, Sirt3 +/+ value was set as 100% arbitrarily ( right ). f The relative (to time 0) intracellular FAs levels in Chang cells that were maintained in high glucose (25 mM) and low glucose (1 mM) were detected ( n = 3) at time points as indicated. For all figures, mean values with SD are reported. NS not significant; * P

    Journal: Nature Communications

    Article Title: Enoyl-CoA hydratase-1 regulates mTOR signaling and apoptosis by sensing nutrients

    doi: 10.1038/s41467-017-00489-5

    Figure Lengend Snippet: Nutrient induces FAs accumulation. a The conversion of 13 C-palmitate to 13 C-citrate was traced in HEK293T cells cultured in low and high glucose media, and in ECHS1 overexpressing HEK293T cells cultured in high glucose media. b Relative FAs levels in HEK293T cells, ECHS1 knocked down HEK293T cells and ECHS1 knocked down HEK293T cells overexpressing shRNA resistant ECHS1 (ECHS1-r) were detected. All levels were normalized to the total proteins. c The relative butyrate levels in Chang liver cells and ECHS1 knocked down Chang liver cells were determined ( n = 4). The butyrate levels in untreated Chang liver cells (25 μM) were set as 100%. d The relative FAs levels in HEK293T cells cultured in low glucose (−), high glucose (+), and high glucose with ECHS1 K101R overexpression were determined ( n = 3). e The FAs levels in the liver of wild type, Sirt3 −/− , and ECHS1 K/R overexpressing Sirt3 −/− 129/C57BL6 mice were assessed by oil red staining. Four mice of each group were analyzed. Relative FAs levels were determined by comparing intensity of signals, Sirt3 +/+ value was set as 100% arbitrarily ( right ). f The relative (to time 0) intracellular FAs levels in Chang cells that were maintained in high glucose (25 mM) and low glucose (1 mM) were detected ( n = 3) at time points as indicated. For all figures, mean values with SD are reported. NS not significant; * P

    Article Snippet: Cell culture and treatments HEK293T cells (ATCC Number: CRL-11268), 786-O(ATCC Number: CRL-1932), ACHN renal cell cancer cells (ATCC Number: CRL-1611), SMMC7712 (CBP60210, Shanghai Cell Bank), Huh7 liver cancer cells (CBP60202, Shanghai Cell Bank), HCT 116 (ATCC Number: CCL-247), SW620 colon cancer cells (ATCC Number: CCL-227), LNCaP (ATCC Number: CRL-1740), PC3 prostate cancers cells(ATCC Number: CRL-1435), Chang cells (ATCC Number: CCL-13) were used in this study.

    Techniques: Cell Culture, shRNA, Over Expression, Mouse Assay, Staining

    K101 acetylation inhibits ECHS1 mitochondria translocation. a The K101 acetylation levels of ECHS1 in the cytosol, mitochondria membrane, and mitochondria matrix were detected after mouse primary hepatocytes were exposed to 25 mM glucose for 0, 2, 4, 6, and 8 h. b Flag-tagged ECHS1 was co-expressed with HA-tagged ubiquitin in HEK293T cells. ECHS1 ubiquitination levels in the cytosol, mitochondria membrane, and mitochondria matrix were detected under low and high glucose treatment for 4 h. GAPDH, COX IV and SOD2 were used as markers of cytosol, mitochondria membrane and mitochondria matrix, respectively. c ECHS1 levels in the cytosol, mitochondria membrane, and mitochondria matrix were analyzed ( n = 3) after cells were exposed to 25 mM glucose for 3, 6, 9, and 12 h. Representative western blots are presented. All levels were normalized to those of cells at time 0 and the average relative levels were quantified ( bottom ). d Schematic diagram of ECHS1 regulation by nutrients. Nutrient-induced acetylation inactivates ECHS1 and induces ubiquitination of ECHS1 that blocks ECHS1 mitochondrial translocation and promotes ECHS1 degradation. These effects synergistically inactivate ECHS1

    Journal: Nature Communications

    Article Title: Enoyl-CoA hydratase-1 regulates mTOR signaling and apoptosis by sensing nutrients

    doi: 10.1038/s41467-017-00489-5

    Figure Lengend Snippet: K101 acetylation inhibits ECHS1 mitochondria translocation. a The K101 acetylation levels of ECHS1 in the cytosol, mitochondria membrane, and mitochondria matrix were detected after mouse primary hepatocytes were exposed to 25 mM glucose for 0, 2, 4, 6, and 8 h. b Flag-tagged ECHS1 was co-expressed with HA-tagged ubiquitin in HEK293T cells. ECHS1 ubiquitination levels in the cytosol, mitochondria membrane, and mitochondria matrix were detected under low and high glucose treatment for 4 h. GAPDH, COX IV and SOD2 were used as markers of cytosol, mitochondria membrane and mitochondria matrix, respectively. c ECHS1 levels in the cytosol, mitochondria membrane, and mitochondria matrix were analyzed ( n = 3) after cells were exposed to 25 mM glucose for 3, 6, 9, and 12 h. Representative western blots are presented. All levels were normalized to those of cells at time 0 and the average relative levels were quantified ( bottom ). d Schematic diagram of ECHS1 regulation by nutrients. Nutrient-induced acetylation inactivates ECHS1 and induces ubiquitination of ECHS1 that blocks ECHS1 mitochondrial translocation and promotes ECHS1 degradation. These effects synergistically inactivate ECHS1

    Article Snippet: Cell culture and treatments HEK293T cells (ATCC Number: CRL-11268), 786-O(ATCC Number: CRL-1932), ACHN renal cell cancer cells (ATCC Number: CRL-1611), SMMC7712 (CBP60210, Shanghai Cell Bank), Huh7 liver cancer cells (CBP60202, Shanghai Cell Bank), HCT 116 (ATCC Number: CCL-247), SW620 colon cancer cells (ATCC Number: CCL-227), LNCaP (ATCC Number: CRL-1740), PC3 prostate cancers cells(ATCC Number: CRL-1435), Chang cells (ATCC Number: CCL-13) were used in this study.

    Techniques: Translocation Assay, Western Blot

    Nutrient activates mTOR signaling by accumulating BCAAs. a The conversion of Leucine-1,2- 13 C 2 to 13 C-labeled citrate was traced by targeted liquid-chromatography tandem mass spectrometry (LC–MS/MS) in HEK293T cells cultured in low (1 mM) and high (25 mM) glucose media, and in ECHS1 overexpressing HEK293T cells cultured in high glucose media. b The BCAA levels in HEK293T cells and ECHS1 knocked down HEK293T cells were determined ( n = 4). BCAA levels were normalized to those of HEK293T cells. c BCAA levels of HEK293T cells, NAM treated HEK293T cells, and NAM treated HEK293T cells with overexpression of ECHS1 K101R were determined by GC–MS ( n = 4). All levels were normalized to those of HEK293T cells. The expression of ECHS1 K/R was confirmed by western blot. d BCAAs levels in the liver of 129/C57BL6 mice, Sirt3 knockout 129/C57BL6 mice, and ECHS1 K101R overexpressing Sirt3 knockout 129/C57BL6 mice were determined ( n = 6). ECHS1 K/R expression was achieved by tail vein injection of the ECHS1 K/R plasmid, and the expression was confirmed by western blot. BCAA levels were normalized to those in the liver of isogenic control mice. e The levels of P-T389 on S6K were determined in HEK293T cells with and without ECHS1 knockdown. f The levels of P-T389 on S6K were determined in HEK293T cells with and without ECHS1 overexpression. g The levels of P-T389 on S6K were determined in the hepatocytes of Sirt3 KO and its isogenic control mice. h The effects of sh SIRT3 knockdown (for knockdown effects see Figure 4 ) on P-T389 of S6K were detected in HEK293T cells cultured with or without BCAA supplementation in the medium. i The cell size of Chang cells and ECHS1 or SIRT3 knocked down Chang cells were determined by measuring forward side scatter (FSC) in the presence or absence of rapamycin. j The percentage of Chang cells and ECHS1 or SIRT3 knocked down Chang cells in the G2/M phase was determined by fluorescence-activated cell sorter (FACS) in the presence or absence of rapamycin. For all figures, mean values with SD are reported. NS not significant; * P

    Journal: Nature Communications

    Article Title: Enoyl-CoA hydratase-1 regulates mTOR signaling and apoptosis by sensing nutrients

    doi: 10.1038/s41467-017-00489-5

    Figure Lengend Snippet: Nutrient activates mTOR signaling by accumulating BCAAs. a The conversion of Leucine-1,2- 13 C 2 to 13 C-labeled citrate was traced by targeted liquid-chromatography tandem mass spectrometry (LC–MS/MS) in HEK293T cells cultured in low (1 mM) and high (25 mM) glucose media, and in ECHS1 overexpressing HEK293T cells cultured in high glucose media. b The BCAA levels in HEK293T cells and ECHS1 knocked down HEK293T cells were determined ( n = 4). BCAA levels were normalized to those of HEK293T cells. c BCAA levels of HEK293T cells, NAM treated HEK293T cells, and NAM treated HEK293T cells with overexpression of ECHS1 K101R were determined by GC–MS ( n = 4). All levels were normalized to those of HEK293T cells. The expression of ECHS1 K/R was confirmed by western blot. d BCAAs levels in the liver of 129/C57BL6 mice, Sirt3 knockout 129/C57BL6 mice, and ECHS1 K101R overexpressing Sirt3 knockout 129/C57BL6 mice were determined ( n = 6). ECHS1 K/R expression was achieved by tail vein injection of the ECHS1 K/R plasmid, and the expression was confirmed by western blot. BCAA levels were normalized to those in the liver of isogenic control mice. e The levels of P-T389 on S6K were determined in HEK293T cells with and without ECHS1 knockdown. f The levels of P-T389 on S6K were determined in HEK293T cells with and without ECHS1 overexpression. g The levels of P-T389 on S6K were determined in the hepatocytes of Sirt3 KO and its isogenic control mice. h The effects of sh SIRT3 knockdown (for knockdown effects see Figure 4 ) on P-T389 of S6K were detected in HEK293T cells cultured with or without BCAA supplementation in the medium. i The cell size of Chang cells and ECHS1 or SIRT3 knocked down Chang cells were determined by measuring forward side scatter (FSC) in the presence or absence of rapamycin. j The percentage of Chang cells and ECHS1 or SIRT3 knocked down Chang cells in the G2/M phase was determined by fluorescence-activated cell sorter (FACS) in the presence or absence of rapamycin. For all figures, mean values with SD are reported. NS not significant; * P

    Article Snippet: Cell culture and treatments HEK293T cells (ATCC Number: CRL-11268), 786-O(ATCC Number: CRL-1932), ACHN renal cell cancer cells (ATCC Number: CRL-1611), SMMC7712 (CBP60210, Shanghai Cell Bank), Huh7 liver cancer cells (CBP60202, Shanghai Cell Bank), HCT 116 (ATCC Number: CCL-247), SW620 colon cancer cells (ATCC Number: CCL-227), LNCaP (ATCC Number: CRL-1740), PC3 prostate cancers cells(ATCC Number: CRL-1435), Chang cells (ATCC Number: CCL-13) were used in this study.

    Techniques: Labeling, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Cell Culture, Over Expression, Gas Chromatography-Mass Spectrometry, Expressing, Western Blot, Mouse Assay, Knock-Out, Injection, Plasmid Preparation, Fluorescence, FACS

    ECHS1 protein levels are decreased by nutrients. a Schematic diagram of catabolic pathways of FA ( black-colored ) and BCAA ( red-colored ) oxidation that involve ECHS1. Long chain FAs are oxidized to short-chained intermediates prior to being completely oxidized through ECHS1. BCAAs are oxidized through ECHS1. ACADL acyl-CoA dehydrogenase, long chain, ACADM medium-chain specific acyl-CoA dehydrogenase, mitochondrial, ACADS short-chain specific acyl-CoA dehydrogenase, ACSL acyl-CoA synthetase long-chain, BCAT branched-chain amino-acid transaminase, BCKDH branched-chain keto acid dehydrogenase, carnitine palmitoyltransferase, ECHS1 enoyl CoA hydratase, short chain 1, mitochondrial, EHHADH Enoyl-CoA, Hydratase/3-Hydroxyacyl CoA Dehydrogenase. b – d Endogenous ECHS1 levels were detected in HEK293T cells treated with different concentrations of glucose ( b ), FAs (linoleic acid + palmitic acid) ( c ), and amino acids (glutamate + aspartate) ( d ). Representative western blot results and quantitation (herein after) of triplicated western blot are shown. e HepG2 cells were exposed to indicated concentrations of glucose. Levels of endogenous β-oxidation enzymes were detected 4 h after glucose exposure. CPT1A Carnitine O-palmitoyltransferase 1, liver isoform, Carnitine O-palmitoyltransferase 2, mitochondrial, HADHA Trifunctional enzyme subunit alpha, mitochondrial, ACAA2 3-ketoacyl-CoA thiolase, mitochondrial. f Plasma ECHS1 levels of normal people ( blue , n = 14) and untreated patients with diabetes ( red , n = 12) were determined by western blots. Relative ECHS1 levels (normalized to the average ECHS1 level) were plotted against serum glucose levels. g Mitochondria of HEK293T cells cultured in DMEM base, DMEM base supplemented with glucose (25 mM), fatty acids (250 nM) and amino acids (8 mM), respectively, were isolated and the relative (to SOD2 and TOM40) mitochondrial ECHS1 levels were compared. For b – d , mean values of quantitation with SD are reported. * P

    Journal: Nature Communications

    Article Title: Enoyl-CoA hydratase-1 regulates mTOR signaling and apoptosis by sensing nutrients

    doi: 10.1038/s41467-017-00489-5

    Figure Lengend Snippet: ECHS1 protein levels are decreased by nutrients. a Schematic diagram of catabolic pathways of FA ( black-colored ) and BCAA ( red-colored ) oxidation that involve ECHS1. Long chain FAs are oxidized to short-chained intermediates prior to being completely oxidized through ECHS1. BCAAs are oxidized through ECHS1. ACADL acyl-CoA dehydrogenase, long chain, ACADM medium-chain specific acyl-CoA dehydrogenase, mitochondrial, ACADS short-chain specific acyl-CoA dehydrogenase, ACSL acyl-CoA synthetase long-chain, BCAT branched-chain amino-acid transaminase, BCKDH branched-chain keto acid dehydrogenase, carnitine palmitoyltransferase, ECHS1 enoyl CoA hydratase, short chain 1, mitochondrial, EHHADH Enoyl-CoA, Hydratase/3-Hydroxyacyl CoA Dehydrogenase. b – d Endogenous ECHS1 levels were detected in HEK293T cells treated with different concentrations of glucose ( b ), FAs (linoleic acid + palmitic acid) ( c ), and amino acids (glutamate + aspartate) ( d ). Representative western blot results and quantitation (herein after) of triplicated western blot are shown. e HepG2 cells were exposed to indicated concentrations of glucose. Levels of endogenous β-oxidation enzymes were detected 4 h after glucose exposure. CPT1A Carnitine O-palmitoyltransferase 1, liver isoform, Carnitine O-palmitoyltransferase 2, mitochondrial, HADHA Trifunctional enzyme subunit alpha, mitochondrial, ACAA2 3-ketoacyl-CoA thiolase, mitochondrial. f Plasma ECHS1 levels of normal people ( blue , n = 14) and untreated patients with diabetes ( red , n = 12) were determined by western blots. Relative ECHS1 levels (normalized to the average ECHS1 level) were plotted against serum glucose levels. g Mitochondria of HEK293T cells cultured in DMEM base, DMEM base supplemented with glucose (25 mM), fatty acids (250 nM) and amino acids (8 mM), respectively, were isolated and the relative (to SOD2 and TOM40) mitochondrial ECHS1 levels were compared. For b – d , mean values of quantitation with SD are reported. * P

    Article Snippet: Cell culture and treatments HEK293T cells (ATCC Number: CRL-11268), 786-O(ATCC Number: CRL-1932), ACHN renal cell cancer cells (ATCC Number: CRL-1611), SMMC7712 (CBP60210, Shanghai Cell Bank), Huh7 liver cancer cells (CBP60202, Shanghai Cell Bank), HCT 116 (ATCC Number: CCL-247), SW620 colon cancer cells (ATCC Number: CCL-227), LNCaP (ATCC Number: CRL-1740), PC3 prostate cancers cells(ATCC Number: CRL-1435), Chang cells (ATCC Number: CCL-13) were used in this study.

    Techniques: Western Blot, Quantitation Assay, Cell Culture, Isolation

    Nutrients promote ECHS1 K101 acetylation. a – c Western blot detection of acetylation levels of ectopically expressed ECHS1 in HEK293T cell cultured in the presence of excessive glucose ( a ), FAs ( b ), and amino acids ( c ) in the culture media. d Levels of endogenous ECHS1 in HEK293T and HepG2 cells were compared with and without the presence of deacetylases inhibitors NAM/TSA. e Effects of deacetylase inhibitors on the acetylation levels of affinity-purified endogenous ECHS1. f The MS spectrum that led to the identification of K101 acetylation containing tryptic peptide from endogenous ECHS1. g Western blot detection of proteins levels, AcK levels and AcK101 levels of endogenous ECHS1 in HEK293T cells that were cultured in DMEM base and DMEM base supplemented with glucose (25 mM), fatty acids (250 nM), and amino acids (8 mM). h Flag-tagged wildtype, K101R (K/R), and K101Q (K/Q) ECHS1 were overexpressed in HEK293T cells that were cultured in media with or without NAM/TSA. The relative acetylation levels of ECHS1 proteins purified from the above mentioned cells using Flag beads was determined. For b – e and h , mean values of quantitation with SD are reported. * P

    Journal: Nature Communications

    Article Title: Enoyl-CoA hydratase-1 regulates mTOR signaling and apoptosis by sensing nutrients

    doi: 10.1038/s41467-017-00489-5

    Figure Lengend Snippet: Nutrients promote ECHS1 K101 acetylation. a – c Western blot detection of acetylation levels of ectopically expressed ECHS1 in HEK293T cell cultured in the presence of excessive glucose ( a ), FAs ( b ), and amino acids ( c ) in the culture media. d Levels of endogenous ECHS1 in HEK293T and HepG2 cells were compared with and without the presence of deacetylases inhibitors NAM/TSA. e Effects of deacetylase inhibitors on the acetylation levels of affinity-purified endogenous ECHS1. f The MS spectrum that led to the identification of K101 acetylation containing tryptic peptide from endogenous ECHS1. g Western blot detection of proteins levels, AcK levels and AcK101 levels of endogenous ECHS1 in HEK293T cells that were cultured in DMEM base and DMEM base supplemented with glucose (25 mM), fatty acids (250 nM), and amino acids (8 mM). h Flag-tagged wildtype, K101R (K/R), and K101Q (K/Q) ECHS1 were overexpressed in HEK293T cells that were cultured in media with or without NAM/TSA. The relative acetylation levels of ECHS1 proteins purified from the above mentioned cells using Flag beads was determined. For b – e and h , mean values of quantitation with SD are reported. * P

    Article Snippet: Cell culture and treatments HEK293T cells (ATCC Number: CRL-11268), 786-O(ATCC Number: CRL-1932), ACHN renal cell cancer cells (ATCC Number: CRL-1611), SMMC7712 (CBP60210, Shanghai Cell Bank), Huh7 liver cancer cells (CBP60202, Shanghai Cell Bank), HCT 116 (ATCC Number: CCL-247), SW620 colon cancer cells (ATCC Number: CCL-227), LNCaP (ATCC Number: CRL-1740), PC3 prostate cancers cells(ATCC Number: CRL-1435), Chang cells (ATCC Number: CCL-13) were used in this study.

    Techniques: Western Blot, Cell Culture, Histone Deacetylase Assay, Affinity Purification, Mass Spectrometry, Purification, Quantitation Assay

    GCN5 acetylates and SIRT3 deacetylates ECHS1. a Myc-tagged GCN5 and Flag-tagged ECHS1 were co-expressed in HEK293T cells. The co-purified Myc-tagged GCN5 was detected in Flag beads purified proteins. b , c Western blot detection of endogenous ECHS1, and AcK and AcK101 levels of affinity-purified ECHS1 from HEK293T cells and GCN5 overexpressing HEK293T cells ( b ) and from HEK293T cells and GCN5 knockdown HEK293T cells ( c ). d Western blot detection of AcK and AcK101 levels of recombinant ECHS1 that before and after in vitro acetylation by GCN5. e The levels of endogenous ECHS1 were detected in HEK293T and GCN5 knockdown HEK293T cells that were cultured in the absence of glucose or presence of glucose. f The synthetic acetylated K101-containing ECHS1 peptide was deacetylated by recombinant SIRT3. The synthetic peptide (1047.5) and de-acetylated products (1005.4, red-circled ) were assayed by MS. g The levels of AcK and AcK101 of hyperacetylated ECHS1 (from NAM treated cells) were detected before and after in vitro treatment by SIRT3. h ECHS1 was co-expressed with SIRT3 or SIRT3 H248Y in HEK293T cells. The AcK101 levels of purified ECHS1 were determined. i The Ac101 levels of affinity-purified ECHS1 from liver of Sirt3 KO and control mice

    Journal: Nature Communications

    Article Title: Enoyl-CoA hydratase-1 regulates mTOR signaling and apoptosis by sensing nutrients

    doi: 10.1038/s41467-017-00489-5

    Figure Lengend Snippet: GCN5 acetylates and SIRT3 deacetylates ECHS1. a Myc-tagged GCN5 and Flag-tagged ECHS1 were co-expressed in HEK293T cells. The co-purified Myc-tagged GCN5 was detected in Flag beads purified proteins. b , c Western blot detection of endogenous ECHS1, and AcK and AcK101 levels of affinity-purified ECHS1 from HEK293T cells and GCN5 overexpressing HEK293T cells ( b ) and from HEK293T cells and GCN5 knockdown HEK293T cells ( c ). d Western blot detection of AcK and AcK101 levels of recombinant ECHS1 that before and after in vitro acetylation by GCN5. e The levels of endogenous ECHS1 were detected in HEK293T and GCN5 knockdown HEK293T cells that were cultured in the absence of glucose or presence of glucose. f The synthetic acetylated K101-containing ECHS1 peptide was deacetylated by recombinant SIRT3. The synthetic peptide (1047.5) and de-acetylated products (1005.4, red-circled ) were assayed by MS. g The levels of AcK and AcK101 of hyperacetylated ECHS1 (from NAM treated cells) were detected before and after in vitro treatment by SIRT3. h ECHS1 was co-expressed with SIRT3 or SIRT3 H248Y in HEK293T cells. The AcK101 levels of purified ECHS1 were determined. i The Ac101 levels of affinity-purified ECHS1 from liver of Sirt3 KO and control mice

    Article Snippet: Cell culture and treatments HEK293T cells (ATCC Number: CRL-11268), 786-O(ATCC Number: CRL-1932), ACHN renal cell cancer cells (ATCC Number: CRL-1611), SMMC7712 (CBP60210, Shanghai Cell Bank), Huh7 liver cancer cells (CBP60202, Shanghai Cell Bank), HCT 116 (ATCC Number: CCL-247), SW620 colon cancer cells (ATCC Number: CCL-227), LNCaP (ATCC Number: CRL-1740), PC3 prostate cancers cells(ATCC Number: CRL-1435), Chang cells (ATCC Number: CCL-13) were used in this study.

    Techniques: Purification, Western Blot, Affinity Purification, Recombinant, In Vitro, Cell Culture, Mass Spectrometry, Mouse Assay

    AMAV and TCRV GPs bind and use the TfR1 orthologs of their respective host species. (A) HEK293T cells were transfected with plasmids encoding human TfR1 (hTfR1), N. spinosus TfR1 (NsTfR1), or A. jamaicensis TfR1 (AjTfR1). Cells were incubated 48 hr later with an α-flag antibody or Ig-fusion proteins comprising the truncated GP1 subunits [32] of AMAV and TCRV (AMAV GP1Δ-Ig and TCRV GP1Δ-Ig, respectively). The association of these proteins with cells was measured by flow cytometry. The data shown are representative of two independent experiments, duplicated in each assay, with similar results. Mean fluorescence values for TfR1 ortholog expression were 435, 380, and 458 for human, Ns, and AjTfR1, respectively. Mean fluorescence values for AMAV GP1Δ-Ig binding to transfected cells were 1.5, 597, and 1.6, and those for TCRV GPΔ-Ig binding were 3.7, 425, and 553 for human, Ns, and AjTfR1, respectively. (B) HEK293T cells were transfected with plasmids encoding human, ZbTfR1, NsTfR1, AjTfR1 orthologs or vector alone. Cell surface expression of the TfR1 orthologs was determined as in Figure 3 . Aliquots of these cells were infected with AMAV, TCRV, or VSV pseudoviruses, and infection levels were assessed as in Figure 2 . (C) An experiment similar to the one performed in (B), except CHO cells were used for transfection and infected with MACV, JUNV, GTOV, or VSV pseudoviruses. Expression levels of the various TfR1 orthologs were normalized to that of human TfR1 (α-flag, left panels). Infection levels were normalized to that of mock-transfected cells.

    Journal: PLoS Pathogens

    Article Title: Host-Species Transferrin Receptor 1 Orthologs Are Cellular Receptors for Nonpathogenic New World Clade B Arenaviruses

    doi: 10.1371/journal.ppat.1000358

    Figure Lengend Snippet: AMAV and TCRV GPs bind and use the TfR1 orthologs of their respective host species. (A) HEK293T cells were transfected with plasmids encoding human TfR1 (hTfR1), N. spinosus TfR1 (NsTfR1), or A. jamaicensis TfR1 (AjTfR1). Cells were incubated 48 hr later with an α-flag antibody or Ig-fusion proteins comprising the truncated GP1 subunits [32] of AMAV and TCRV (AMAV GP1Δ-Ig and TCRV GP1Δ-Ig, respectively). The association of these proteins with cells was measured by flow cytometry. The data shown are representative of two independent experiments, duplicated in each assay, with similar results. Mean fluorescence values for TfR1 ortholog expression were 435, 380, and 458 for human, Ns, and AjTfR1, respectively. Mean fluorescence values for AMAV GP1Δ-Ig binding to transfected cells were 1.5, 597, and 1.6, and those for TCRV GPΔ-Ig binding were 3.7, 425, and 553 for human, Ns, and AjTfR1, respectively. (B) HEK293T cells were transfected with plasmids encoding human, ZbTfR1, NsTfR1, AjTfR1 orthologs or vector alone. Cell surface expression of the TfR1 orthologs was determined as in Figure 3 . Aliquots of these cells were infected with AMAV, TCRV, or VSV pseudoviruses, and infection levels were assessed as in Figure 2 . (C) An experiment similar to the one performed in (B), except CHO cells were used for transfection and infected with MACV, JUNV, GTOV, or VSV pseudoviruses. Expression levels of the various TfR1 orthologs were normalized to that of human TfR1 (α-flag, left panels). Infection levels were normalized to that of mock-transfected cells.

    Article Snippet: Cells and plasmids HEK293T cells (human embryonic kidney, ATCC, CRL-11268) were maintained in Dulbecco's modified Eagle's medium, and CHO cells (Chinese hamster ovary epithelial, ATCC, CCL-61) in F12 medium.

    Techniques: Transfection, Incubation, Flow Cytometry, Cytometry, Fluorescence, Expressing, Binding Assay, Plasmid Preparation, Infection

    AMAV and TCRV pseudovirus entry does not depend on human TfR1. HEK293T cells were infected with GTOV, AMAV, TCRV, or MACV pseudoviruses expressing GFP in the presence or absence of the indicated concentrations of an α-human TfR1 (BD Pharmingen) or a control (α-HLA) antibody (BD Pharmingen). Infection levels were assessed 48 hr later by measuring GFP expression by flow cytometry. Mean fluorescence values were normalized to that of cells infected in the absence of antibody. GFP mean fluorescence values for virus entry in the absence of antibody were 144.0, 89.7, 92.3, and 115.0 for GTOV, AMAV, TCRV, and MACV, respectively.

    Journal: PLoS Pathogens

    Article Title: Host-Species Transferrin Receptor 1 Orthologs Are Cellular Receptors for Nonpathogenic New World Clade B Arenaviruses

    doi: 10.1371/journal.ppat.1000358

    Figure Lengend Snippet: AMAV and TCRV pseudovirus entry does not depend on human TfR1. HEK293T cells were infected with GTOV, AMAV, TCRV, or MACV pseudoviruses expressing GFP in the presence or absence of the indicated concentrations of an α-human TfR1 (BD Pharmingen) or a control (α-HLA) antibody (BD Pharmingen). Infection levels were assessed 48 hr later by measuring GFP expression by flow cytometry. Mean fluorescence values were normalized to that of cells infected in the absence of antibody. GFP mean fluorescence values for virus entry in the absence of antibody were 144.0, 89.7, 92.3, and 115.0 for GTOV, AMAV, TCRV, and MACV, respectively.

    Article Snippet: Cells and plasmids HEK293T cells (human embryonic kidney, ATCC, CRL-11268) were maintained in Dulbecco's modified Eagle's medium, and CHO cells (Chinese hamster ovary epithelial, ATCC, CCL-61) in F12 medium.

    Techniques: Infection, Expressing, Flow Cytometry, Cytometry, Fluorescence

    AMAV and TCRV pseudoviruses can use animal orthologs of TfR1. CHO cells ((A), left panel) were transfected with vector alone (mock) or plasmids encoding human, mouse, rat, feline, canine, C. callosus (Cc), C. musculinus (Cm), and Z. brevicauda (Zb) TfR1 orthologs. HEK293T cells ((B), right panel) were transfected with the same plasmids with the exception of the one encoding canine TfR1. Cell surface expression was determined 48 hr later by flow cytometry using an antibody directed against a flag tag present at the C-terminus of each ortholog. In parallel, aliquots of these cells were infected with AMAV, TCRV, or LCMV pseudoviruses. Infection levels were assessed as in Figure 2 . Expression levels of various TfR1 were normalized to that of human TfR1 (α-flag, top panels). Infection levels were normalized to that of mock-transfected cells.

    Journal: PLoS Pathogens

    Article Title: Host-Species Transferrin Receptor 1 Orthologs Are Cellular Receptors for Nonpathogenic New World Clade B Arenaviruses

    doi: 10.1371/journal.ppat.1000358

    Figure Lengend Snippet: AMAV and TCRV pseudoviruses can use animal orthologs of TfR1. CHO cells ((A), left panel) were transfected with vector alone (mock) or plasmids encoding human, mouse, rat, feline, canine, C. callosus (Cc), C. musculinus (Cm), and Z. brevicauda (Zb) TfR1 orthologs. HEK293T cells ((B), right panel) were transfected with the same plasmids with the exception of the one encoding canine TfR1. Cell surface expression was determined 48 hr later by flow cytometry using an antibody directed against a flag tag present at the C-terminus of each ortholog. In parallel, aliquots of these cells were infected with AMAV, TCRV, or LCMV pseudoviruses. Infection levels were assessed as in Figure 2 . Expression levels of various TfR1 were normalized to that of human TfR1 (α-flag, top panels). Infection levels were normalized to that of mock-transfected cells.

    Article Snippet: Cells and plasmids HEK293T cells (human embryonic kidney, ATCC, CRL-11268) were maintained in Dulbecco's modified Eagle's medium, and CHO cells (Chinese hamster ovary epithelial, ATCC, CCL-61) in F12 medium.

    Techniques: Transfection, Plasmid Preparation, Expressing, Flow Cytometry, Cytometry, FLAG-tag, Infection

    Modest mutations convert human TfR1 into an efficient receptor for AMAV and TCRV. (A) The structure of the human TfR1 dimer is shown, oriented with the cell membrane at the bottom. The apical, protease-like, and helical domains are indicated in green, red, and yellow, respectively, on one monomer. The other monomer is shown in cyan. In the right panel, the TfR1 apical domain is enlarged; a loop comprising residues 202-212, implicated as a site of interaction with the GPs of NW clade B arenaviruses, is shown. The side chains of residues D204, K205, R208, V210, and Y211 are colored yellow. The image was rendered using PyMol [59] . (B) Sequence alignment of residues 195 through 216 of human TfR1 with analogous sequences of the TfR1 orthologs of Z. brevicauda (ZbTfR1), A. jamaicensis (AjTfR1), and N. spinosus (NsTfR1). Variants of human TfR1 containing sequence from Z. brevicauda (zh1 and zh2), A. jamaicensis (ah2 through ah5), and N. spinosus (nh2, nh4, nh5, nh7, and nh8) TfR1 were generated based on this sequence alignment. Zb, Aj, and NsTfR1 sequences are shown in green, blue, and yellow, respectively. The right panel summarizes the entry data. ND = not determined. (C–E) HEK293T cells were transfected with plasmids encoding human, Zb, Aj, or NsTfR1 along with the zh variants (C), the ah variants (D), or the nh variants (E). The expression level of each TfR1 variant was assessed as in Figure 3 . In parallel, cells were infected with AMAV, TCRV, or VSV pseudoviruses. The expression levels of the various TfR1 orthologs were normalized to that of human TfR1 (α-flag, left panels), and infection levels were normalized to that of mock-transfected cells.

    Journal: PLoS Pathogens

    Article Title: Host-Species Transferrin Receptor 1 Orthologs Are Cellular Receptors for Nonpathogenic New World Clade B Arenaviruses

    doi: 10.1371/journal.ppat.1000358

    Figure Lengend Snippet: Modest mutations convert human TfR1 into an efficient receptor for AMAV and TCRV. (A) The structure of the human TfR1 dimer is shown, oriented with the cell membrane at the bottom. The apical, protease-like, and helical domains are indicated in green, red, and yellow, respectively, on one monomer. The other monomer is shown in cyan. In the right panel, the TfR1 apical domain is enlarged; a loop comprising residues 202-212, implicated as a site of interaction with the GPs of NW clade B arenaviruses, is shown. The side chains of residues D204, K205, R208, V210, and Y211 are colored yellow. The image was rendered using PyMol [59] . (B) Sequence alignment of residues 195 through 216 of human TfR1 with analogous sequences of the TfR1 orthologs of Z. brevicauda (ZbTfR1), A. jamaicensis (AjTfR1), and N. spinosus (NsTfR1). Variants of human TfR1 containing sequence from Z. brevicauda (zh1 and zh2), A. jamaicensis (ah2 through ah5), and N. spinosus (nh2, nh4, nh5, nh7, and nh8) TfR1 were generated based on this sequence alignment. Zb, Aj, and NsTfR1 sequences are shown in green, blue, and yellow, respectively. The right panel summarizes the entry data. ND = not determined. (C–E) HEK293T cells were transfected with plasmids encoding human, Zb, Aj, or NsTfR1 along with the zh variants (C), the ah variants (D), or the nh variants (E). The expression level of each TfR1 variant was assessed as in Figure 3 . In parallel, cells were infected with AMAV, TCRV, or VSV pseudoviruses. The expression levels of the various TfR1 orthologs were normalized to that of human TfR1 (α-flag, left panels), and infection levels were normalized to that of mock-transfected cells.

    Article Snippet: Cells and plasmids HEK293T cells (human embryonic kidney, ATCC, CRL-11268) were maintained in Dulbecco's modified Eagle's medium, and CHO cells (Chinese hamster ovary epithelial, ATCC, CCL-61) in F12 medium.

    Techniques: Sequencing, Generated, Transfection, Expressing, Variant Assay, Infection

    Gβ selectivity of Nb5. a Multiple sequence alignment of key amino acid residues of Gβ that interact with Nb5. *The amino acid numbering of Gβ 5 has an off-set of −50. b Nb5-mediated Gβγ extraction from mouse brain. Solubilized and partially purified extracts from mouse brain (left) were subjected to either Nb5- (right, lanes 1 and 2) or Nb17- (right, lanes 3 and 4) mediated immobilized-metal affinity purification of Gβγ. c In-gel protein digestion of Gβ subtypes (band marked in b with asterisks) purified from mouse brain. Peptides were separated, analyzed and searched against a full mouse proteome to identify unique peptides from Gβ 1 , Gβ 2 , Gβ 3 , and Gβ 4 . d Schematic diagram of the effect of Gβγ-binding proteins on the BRET assay. Co-transfection of HEK293T/17 cells with Venus-Gβγ and masGRK3ct-Nluc-HA produced a high BRET signal through their direct interaction (left). Introduction of Gβγ-binding proteins (e.g., the Gα subunit) competed with masGRK3ct-Nluc-HA, lowering the BRET signal (right). e Effects of Nb5 on the interaction of Gβγ and the C-terminus of GRK3. The maximum BRET signal was determined by co-transfection of different Venus-Gβ subtypes + Gγ 2 pairs and masGRK3ct-Nluc-HA (grey). A minimum BRET signal also was determined after co-transfection of Venus-Gβ 1 γ 2 and masGRK3ct-Nluc-HA with an excess amount of Gα oA (pink). Effects of Nb5 and Nb17 were examined by co-transfection of Venus-Gβγ and masGRK3ct-Nluc-HA with either Nb5 (greencyan) or Nb17 (purple). Experiments were performed with Gβ subtypes 1–4 + Gγ 2 pairs. Each bar represents the mean of six replicates. Similar results were obtained in three independent experiments. Results are expressed as the mean ± SEM. One-way ANOVA with Tukey’s post hoc multiple comparison test relative to the Gβ 1 γ 2 /GRK3ct control, *** P ≤ 0.001, n = 6 replicates. f Western blot quantification of the expression levels of Gβ1–4, GRK3, Gα, Nb5, and Nb17 (Full blots are shown in Supplementary Figure 4a )

    Journal: Nature Communications

    Article Title: Targeting G protein-coupled receptor signaling at the G protein level with a selective nanobody inhibitor

    doi: 10.1038/s41467-018-04432-0

    Figure Lengend Snippet: Gβ selectivity of Nb5. a Multiple sequence alignment of key amino acid residues of Gβ that interact with Nb5. *The amino acid numbering of Gβ 5 has an off-set of −50. b Nb5-mediated Gβγ extraction from mouse brain. Solubilized and partially purified extracts from mouse brain (left) were subjected to either Nb5- (right, lanes 1 and 2) or Nb17- (right, lanes 3 and 4) mediated immobilized-metal affinity purification of Gβγ. c In-gel protein digestion of Gβ subtypes (band marked in b with asterisks) purified from mouse brain. Peptides were separated, analyzed and searched against a full mouse proteome to identify unique peptides from Gβ 1 , Gβ 2 , Gβ 3 , and Gβ 4 . d Schematic diagram of the effect of Gβγ-binding proteins on the BRET assay. Co-transfection of HEK293T/17 cells with Venus-Gβγ and masGRK3ct-Nluc-HA produced a high BRET signal through their direct interaction (left). Introduction of Gβγ-binding proteins (e.g., the Gα subunit) competed with masGRK3ct-Nluc-HA, lowering the BRET signal (right). e Effects of Nb5 on the interaction of Gβγ and the C-terminus of GRK3. The maximum BRET signal was determined by co-transfection of different Venus-Gβ subtypes + Gγ 2 pairs and masGRK3ct-Nluc-HA (grey). A minimum BRET signal also was determined after co-transfection of Venus-Gβ 1 γ 2 and masGRK3ct-Nluc-HA with an excess amount of Gα oA (pink). Effects of Nb5 and Nb17 were examined by co-transfection of Venus-Gβγ and masGRK3ct-Nluc-HA with either Nb5 (greencyan) or Nb17 (purple). Experiments were performed with Gβ subtypes 1–4 + Gγ 2 pairs. Each bar represents the mean of six replicates. Similar results were obtained in three independent experiments. Results are expressed as the mean ± SEM. One-way ANOVA with Tukey’s post hoc multiple comparison test relative to the Gβ 1 γ 2 /GRK3ct control, *** P ≤ 0.001, n = 6 replicates. f Western blot quantification of the expression levels of Gβ1–4, GRK3, Gα, Nb5, and Nb17 (Full blots are shown in Supplementary Figure 4a )

    Article Snippet: Gβ subunit selectivity of Nb5 HEK293T/17 cells (American Type Culture Collection, Manassas, VA) were chosen for this analysis because of their high transfectability .

    Techniques: Sequencing, Purification, Affinity Purification, Binding Assay, Bioluminescence Resonance Energy Transfer, Cotransfection, Produced, Western Blot, Expressing

    Levels of secreted IL-1β from reconstituted HEK293T cells Transfected components in each sample are indicated below the X-axis. 50 μl supernatants of each HEK293T cell culture sample were used for the IL-1β ELISA. Representative data was graphed using GraphPad Prism 6 software.

    Journal: Bio-protocol

    Article Title: Reconstruction of the Mouse Inflammasome System in HEK293T Cells

    doi: 10.21769/BioProtoc.1986

    Figure Lengend Snippet: Levels of secreted IL-1β from reconstituted HEK293T cells Transfected components in each sample are indicated below the X-axis. 50 μl supernatants of each HEK293T cell culture sample were used for the IL-1β ELISA. Representative data was graphed using GraphPad Prism 6 software.

    Article Snippet: Costar 24 well clear TC-treated multiple well plate (Corning, Costar® , catalog number: 3527) 1.5 ml Eppendorf tubes HEK293T cells (ATCC, catalog number: CRL-11268) Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, Gibco™ , catalog number: 10569-044) Fetal bovine serum (FBS) (Gemini Bio Products, catalog number: 100-106) Penicillin-streptomycin (10,000 U/ml; 10,000 μg/ml) (Thermo Fisher Scientific, Gibco™ , catalog number: 15140-163) Pro-IL-1β-Flag, NLRP3-Flag, ASC1-Flag, pro-caspase-1-Flag and NEK7-HA plasmids Note: Pro-IL-1β, NLRP3, ASC1, pro-caspase-1, and NEK7 were amplified by standard PCR techniques and were subsequently inserted into mammalian expression vectors using the InFusion ® HD cloning kit per manufacturer’s instructions (click here for a detailed protocol and ).

    Techniques: Transfection, Cell Culture, Enzyme-linked Immunosorbent Assay, Software

    Optimization of linear-DNA based transfection. (A) Optimization of the amount of linear-DNA based transfection. 0.05 , 0.1, 0.2 linear and 0.2 μg of plasmid DNA encoding AtCRY2 were transfected with the optimized Lipofectamine 2000, respectively. After 24-h incubation, total protein of each sample was probed by the antibody to AtCRY2, stripped and re-probed by the antibody to Actin. (B) Efficiency testing of different transfection reagents. 0.2 μl linear DNA encoding AtCRY2 were transfected with Calcium Phosphate, Lipofectamine ® 2000 or Lipofectamine ® 3000, the optimized protocols were applied for those transfection reagents, respectively. After transfection and incubation, the immunoblot was performed as described in (A) . (C) Efficiency testing of co-transfection with different transfection reagents. 0.3 μg linear DNA encoding AtCRY2 and Myc-AtCIB1 were co-transfected with different transfection reagent. The expression level of AtCRY2 and Myc-AtCIB1, transfected with indicated transfection reagent, was shown. The immunoblot was performed as described in (A) . (D) The microscopy of HEK293T cells transfected with AtCRY2–GFP. Cells were transfected and then incubated with indicated time before microscopy. The GFP and BF (Bright field) channels were merged with Zeiss Zen2 software. (E) The efficiency testing of different post-transfection incubation time. After transfection and incubation for indicated time, cells were detached by Recombinant trypsin and re-suspended with PBS (pH 7.2). The efficiency of AtCRY2–GFP was calculated with the following equation: [cell with GFP signal/total counted cell] × 100%.

    Journal: Frontiers in Plant Science

    Article Title: Using HEK293T Expression System to Study Photoactive Plant Cryptochromes

    doi: 10.3389/fpls.2016.00940

    Figure Lengend Snippet: Optimization of linear-DNA based transfection. (A) Optimization of the amount of linear-DNA based transfection. 0.05 , 0.1, 0.2 linear and 0.2 μg of plasmid DNA encoding AtCRY2 were transfected with the optimized Lipofectamine 2000, respectively. After 24-h incubation, total protein of each sample was probed by the antibody to AtCRY2, stripped and re-probed by the antibody to Actin. (B) Efficiency testing of different transfection reagents. 0.2 μl linear DNA encoding AtCRY2 were transfected with Calcium Phosphate, Lipofectamine ® 2000 or Lipofectamine ® 3000, the optimized protocols were applied for those transfection reagents, respectively. After transfection and incubation, the immunoblot was performed as described in (A) . (C) Efficiency testing of co-transfection with different transfection reagents. 0.3 μg linear DNA encoding AtCRY2 and Myc-AtCIB1 were co-transfected with different transfection reagent. The expression level of AtCRY2 and Myc-AtCIB1, transfected with indicated transfection reagent, was shown. The immunoblot was performed as described in (A) . (D) The microscopy of HEK293T cells transfected with AtCRY2–GFP. Cells were transfected and then incubated with indicated time before microscopy. The GFP and BF (Bright field) channels were merged with Zeiss Zen2 software. (E) The efficiency testing of different post-transfection incubation time. After transfection and incubation for indicated time, cells were detached by Recombinant trypsin and re-suspended with PBS (pH 7.2). The efficiency of AtCRY2–GFP was calculated with the following equation: [cell with GFP signal/total counted cell] × 100%.

    Article Snippet: HEK293T Cell Culture HEK293T (ATCC® CRL-11268TM ) cells were maintained in DMEM (Thermo, 10569-044) supplemented with 10% (v/v) FBS (Thermo, 10100147), 100 U/ml penicillin, and 100 mg/ml streptomycin (Hyclone, SV30010) in Cell Culture Flask, T75 (Eppendorf, 0030711.122).

    Techniques: Transfection, Plasmid Preparation, Incubation, Cotransfection, Expressing, Microscopy, Software, Recombinant

    Workflow of PCR product based HEK293 cell transfection (96-well-plate format with Lipofectamine 2000). (1) HEK293T cells were cultured in DMEM medium supplemented with 10% FBS and 100 U/ml penicillin, and 100 mg/ml streptomycin under the condition of 37°C, 5% CO 2 . Cells were seeded into 96-well-plate for ∼24 h before transfection. At the same day, SV40 promoter and plant ORF were amplified, respectively, with primers that include 15 bp of overlap sequence. The linear DNA including SV40 promoter and plant ORF were then generated with overlap PCR, purified and quantified for transfection. (2) HEK293T cells were cultured until 80–90% confluence is reached. Added 10 μl Opti-MEM into a sterile tube. (3) Added 0.2 μg linear DNA and vortex. (4) Added 0.6 μl Lipofectamine 2000 and vortex. (5) Incubated at room temperature for 5 min. (6) Added the mixture to 96-well-plate, 10 μl per well, and shook the plate gently. (7) After incubation for 16–32 h, cells were then collected and lysed for Biochemistry assay.

    Journal: Frontiers in Plant Science

    Article Title: Using HEK293T Expression System to Study Photoactive Plant Cryptochromes

    doi: 10.3389/fpls.2016.00940

    Figure Lengend Snippet: Workflow of PCR product based HEK293 cell transfection (96-well-plate format with Lipofectamine 2000). (1) HEK293T cells were cultured in DMEM medium supplemented with 10% FBS and 100 U/ml penicillin, and 100 mg/ml streptomycin under the condition of 37°C, 5% CO 2 . Cells were seeded into 96-well-plate for ∼24 h before transfection. At the same day, SV40 promoter and plant ORF were amplified, respectively, with primers that include 15 bp of overlap sequence. The linear DNA including SV40 promoter and plant ORF were then generated with overlap PCR, purified and quantified for transfection. (2) HEK293T cells were cultured until 80–90% confluence is reached. Added 10 μl Opti-MEM into a sterile tube. (3) Added 0.2 μg linear DNA and vortex. (4) Added 0.6 μl Lipofectamine 2000 and vortex. (5) Incubated at room temperature for 5 min. (6) Added the mixture to 96-well-plate, 10 μl per well, and shook the plate gently. (7) After incubation for 16–32 h, cells were then collected and lysed for Biochemistry assay.

    Article Snippet: HEK293T Cell Culture HEK293T (ATCC® CRL-11268TM ) cells were maintained in DMEM (Thermo, 10569-044) supplemented with 10% (v/v) FBS (Thermo, 10100147), 100 U/ml penicillin, and 100 mg/ml streptomycin (Hyclone, SV30010) in Cell Culture Flask, T75 (Eppendorf, 0030711.122).

    Techniques: Polymerase Chain Reaction, Transfection, Cell Culture, Amplification, Sequencing, Generated, Purification, Incubation

    Biochemical analysis of Arabidopsis CRY2 expressed in HEK293T cell. (A) For expression and purification of the recombinant protein, 6xHis-AtCRY2 and 6xHis-AtCRY2 D387A were transfected with Calcium Phosphate, respectively. After 24 hours of incubation, cells were collected and lysed. Recombinant AtCRY2 and AtCRY2 D387A were purified with Ni-NTA agarose beads. The AtCRY2 exhibited a faint yellow color after enrichment. (B) The absorption spectrum of recombinant AtCRY2 and AtCRY2 D387A were detected by a full-length spectrophotometer, and the related absorption curve was calculated by the following equation: [absorption of CRY2 - absorption of CRY2 D387A ]. (C,D) Co-IP analysis showing blue light dependent interaction of AtCRY2–AtCIB1 (C) and AtCRY2–AtSPA1 (D) in mammalian cells. The HEK293T cells co-transfected with AtCRY2–AtCIB1 or AtCRY2–AtSPA1 were incubated in dark condition. Cells were irradiated with blue light (50 μmol m -2 s -1 ) for indicated time before harvest. Total protein extraction and immunoprecipitation (IP) product prepared by AtCRY2 antibody were first probed with AtCRY2 antibody, stripped, and re-probed with Myc antibody.

    Journal: Frontiers in Plant Science

    Article Title: Using HEK293T Expression System to Study Photoactive Plant Cryptochromes

    doi: 10.3389/fpls.2016.00940

    Figure Lengend Snippet: Biochemical analysis of Arabidopsis CRY2 expressed in HEK293T cell. (A) For expression and purification of the recombinant protein, 6xHis-AtCRY2 and 6xHis-AtCRY2 D387A were transfected with Calcium Phosphate, respectively. After 24 hours of incubation, cells were collected and lysed. Recombinant AtCRY2 and AtCRY2 D387A were purified with Ni-NTA agarose beads. The AtCRY2 exhibited a faint yellow color after enrichment. (B) The absorption spectrum of recombinant AtCRY2 and AtCRY2 D387A were detected by a full-length spectrophotometer, and the related absorption curve was calculated by the following equation: [absorption of CRY2 - absorption of CRY2 D387A ]. (C,D) Co-IP analysis showing blue light dependent interaction of AtCRY2–AtCIB1 (C) and AtCRY2–AtSPA1 (D) in mammalian cells. The HEK293T cells co-transfected with AtCRY2–AtCIB1 or AtCRY2–AtSPA1 were incubated in dark condition. Cells were irradiated with blue light (50 μmol m -2 s -1 ) for indicated time before harvest. Total protein extraction and immunoprecipitation (IP) product prepared by AtCRY2 antibody were first probed with AtCRY2 antibody, stripped, and re-probed with Myc antibody.

    Article Snippet: HEK293T Cell Culture HEK293T (ATCC® CRL-11268TM ) cells were maintained in DMEM (Thermo, 10569-044) supplemented with 10% (v/v) FBS (Thermo, 10100147), 100 U/ml penicillin, and 100 mg/ml streptomycin (Hyclone, SV30010) in Cell Culture Flask, T75 (Eppendorf, 0030711.122).

    Techniques: Expressing, Purification, Recombinant, Transfection, Incubation, Spectrophotometry, Co-Immunoprecipitation Assay, Irradiation, Protein Extraction, Immunoprecipitation

    Tandem mass-spectrometry identification of −GG signature peptides from ubiquitinated TCR α on Lys-118, Lys-144, and Lys-178. HEK293T cells were transiently transfected with WT-TCR α or vector alone, incubated with MG132, and then lysed. Lysates were subjected to immunoprecipitation with an anti-HA antibody and examined by SDS-PAGE followed by Coomassie staining. Four regions of the gel were excised and trypsinized as shown in (a). Samples were analyzed by tandem mass-spectrometry and the results are summarized in (b). Peptide spectral matches to −GG signature peptides (ggSP) at Lys-118, Lys-144, or Lys-178 in each region of the gel are indicated by red shading. Representative MS/MS spectra for each of the ggSP are shown: (c) Lys-118, (d) Lys-144, and (e) Lys-178. The peaks are labeled with the fragment ion and its charge state. For example, y14-1 is the singly charged version of y14. b- and y-fragment ions are denoted by blue and red, respectively. Green squares denote precursor ions, multiply charged fragment ions, or predictable neutral loss ions accounted for during manual inspection of MS/MS data.

    Journal: International Journal of Proteomics

    Article Title: A Novel Peptide-Based SILAC Method to Identify the Posttranslational Modifications Provides Evidence for Unconventional Ubiquitination in the ER-Associated Degradation Pathway

    doi: 10.1155/2013/857918

    Figure Lengend Snippet: Tandem mass-spectrometry identification of −GG signature peptides from ubiquitinated TCR α on Lys-118, Lys-144, and Lys-178. HEK293T cells were transiently transfected with WT-TCR α or vector alone, incubated with MG132, and then lysed. Lysates were subjected to immunoprecipitation with an anti-HA antibody and examined by SDS-PAGE followed by Coomassie staining. Four regions of the gel were excised and trypsinized as shown in (a). Samples were analyzed by tandem mass-spectrometry and the results are summarized in (b). Peptide spectral matches to −GG signature peptides (ggSP) at Lys-118, Lys-144, or Lys-178 in each region of the gel are indicated by red shading. Representative MS/MS spectra for each of the ggSP are shown: (c) Lys-118, (d) Lys-144, and (e) Lys-178. The peaks are labeled with the fragment ion and its charge state. For example, y14-1 is the singly charged version of y14. b- and y-fragment ions are denoted by blue and red, respectively. Green squares denote precursor ions, multiply charged fragment ions, or predictable neutral loss ions accounted for during manual inspection of MS/MS data.

    Article Snippet: Cell Culture and Reagents HEK293T cells (number CRL-11268; American Tissue Culture Collection) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 μ g/mL penicillin/streptomycin and transfected using Fugene HD (Roche) according to the manufacturer's instructions.

    Techniques: Mass Spectrometry, Transfection, Plasmid Preparation, Incubation, Immunoprecipitation, SDS Page, Staining, Labeling

    KR-TCR α peptide VAGFNLLMTLR (aa 254–264) is modified. HEK293T cells were incubated in either heavy or light SILAC media for five days and then transiently transfected with KR-TCR α or vector alone, incubated with MG132 and then lysed. Lysates were subjected to immunoprecipitation with an anti-HA antibody and examined by SDS-PAGE followed by Coomassie staining. (a) Gel pieces containing heavy, unmodified TCR α were combined with gel pieces containing light SILAC labeled, ubiquitin-modified KR-TCR α . Heavy and light proteins were digested with trypsin and analyzed by LC-MS/MS. (b) Seven narrow range full-MS spectra for representative KR-TCR α peptides are shown. Data were collected in high-resolution on an LTQ-Orbitrap, with light ions shown in red and heavy ions in blue. (c) HEK293T cells were incubated in either heavy or light SILAC media for five days, transfected with KR-TCR α or KR-TCR α -T262A, and the same experimental procedure was followed as in Table 1 . The relative abundance of six peptides are quantified and represented in the bar graph. Numbers represent the area under the curve for the peptide in the high molecular weight region (corresponding to modified TCR α ) divided by the peak height of the same peptide in the low molecular weight region (corresponding to unmodified TCR α ).

    Journal: International Journal of Proteomics

    Article Title: A Novel Peptide-Based SILAC Method to Identify the Posttranslational Modifications Provides Evidence for Unconventional Ubiquitination in the ER-Associated Degradation Pathway

    doi: 10.1155/2013/857918

    Figure Lengend Snippet: KR-TCR α peptide VAGFNLLMTLR (aa 254–264) is modified. HEK293T cells were incubated in either heavy or light SILAC media for five days and then transiently transfected with KR-TCR α or vector alone, incubated with MG132 and then lysed. Lysates were subjected to immunoprecipitation with an anti-HA antibody and examined by SDS-PAGE followed by Coomassie staining. (a) Gel pieces containing heavy, unmodified TCR α were combined with gel pieces containing light SILAC labeled, ubiquitin-modified KR-TCR α . Heavy and light proteins were digested with trypsin and analyzed by LC-MS/MS. (b) Seven narrow range full-MS spectra for representative KR-TCR α peptides are shown. Data were collected in high-resolution on an LTQ-Orbitrap, with light ions shown in red and heavy ions in blue. (c) HEK293T cells were incubated in either heavy or light SILAC media for five days, transfected with KR-TCR α or KR-TCR α -T262A, and the same experimental procedure was followed as in Table 1 . The relative abundance of six peptides are quantified and represented in the bar graph. Numbers represent the area under the curve for the peptide in the high molecular weight region (corresponding to modified TCR α ) divided by the peak height of the same peptide in the low molecular weight region (corresponding to unmodified TCR α ).

    Article Snippet: Cell Culture and Reagents HEK293T cells (number CRL-11268; American Tissue Culture Collection) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 μ g/mL penicillin/streptomycin and transfected using Fugene HD (Roche) according to the manufacturer's instructions.

    Techniques: Modification, Incubation, Transfection, Plasmid Preparation, Immunoprecipitation, SDS Page, Staining, Labeling, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Molecular Weight

    KR-TCR α is ubiquitinated and degraded by the proteasome. (a) HEK293T cells were transfected with a vector expressing WT-TCR α , or a vector expressing KR-TCR α . Cells were either untreated or incubated with MG132 before lysis. Cellular lysates were immunoprecipitated using an anti-HA antibody and the ubiquitination status of TCR α was analyzed by Western blot analysis using an anti-ubiquitin antibody (top panel). Membranes were stripped and reanalyzed using an anti-HA antibody (bottom panel). (b) HEK293T cells were cotransfected with 6XHis-Ub and either a vector expressing WT-TCR α or KR-TCR α . Cells were either untreated or incubated with MG132 before lysis. Cellular lysates were immunoprecipitated using an anti-HA antibody and the ubiquitination status of TCR α was analyzed by Western blot analysis using an anti-His antibody (top panel). Membranes were stripped and reanalyzed using an anti-HA antibody (bottom panel). Molecular weight markers (kD) of marker proteins are indicated.

    Journal: International Journal of Proteomics

    Article Title: A Novel Peptide-Based SILAC Method to Identify the Posttranslational Modifications Provides Evidence for Unconventional Ubiquitination in the ER-Associated Degradation Pathway

    doi: 10.1155/2013/857918

    Figure Lengend Snippet: KR-TCR α is ubiquitinated and degraded by the proteasome. (a) HEK293T cells were transfected with a vector expressing WT-TCR α , or a vector expressing KR-TCR α . Cells were either untreated or incubated with MG132 before lysis. Cellular lysates were immunoprecipitated using an anti-HA antibody and the ubiquitination status of TCR α was analyzed by Western blot analysis using an anti-ubiquitin antibody (top panel). Membranes were stripped and reanalyzed using an anti-HA antibody (bottom panel). (b) HEK293T cells were cotransfected with 6XHis-Ub and either a vector expressing WT-TCR α or KR-TCR α . Cells were either untreated or incubated with MG132 before lysis. Cellular lysates were immunoprecipitated using an anti-HA antibody and the ubiquitination status of TCR α was analyzed by Western blot analysis using an anti-His antibody (top panel). Membranes were stripped and reanalyzed using an anti-HA antibody (bottom panel). Molecular weight markers (kD) of marker proteins are indicated.

    Article Snippet: Cell Culture and Reagents HEK293T cells (number CRL-11268; American Tissue Culture Collection) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 μ g/mL penicillin/streptomycin and transfected using Fugene HD (Roche) according to the manufacturer's instructions.

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

    Site-specific incorporation of 3 into proteins at diverse codons and specific proteome labelling using SORT-M in human cells. (a) Western blot analysis demonstrates the efficient amino acid dependant expression of an mCherry-EGFP fusion protein separated by an amber stop codon bearing a C-terminal HA-tag (mCh-TAG-EGFP-HA) in HEK293T cells. Anti-FLAG detected tagged PylRS (b) Specific labelling of mCh-TAG-EGFP-HA (immunoprecipitated from 10 6 cells) with 4a (20μM in 50μL PBS, 1h, RT) confirms the incorporation of 3 into protein in HEK293 cells. (c) SORT-M labelling of 3 that is statistically incorporated into newly synthesised proteins across the whole proteome of mammalian cells directed by six different PylRS/PyltRNA XXX mutants using 0.5 mM 3 . Labeling with 4g (20μM in PBS, 1h, RT, as above). The amino acids in parentheses are the natural amino acids encoded by the endogenous tRNA bearing the corresponding anti-codon.

    Journal: Nature biotechnology

    Article Title: Proteome labelling and protein identification in specific tissues and at specific developmental stages in an animal

    doi: 10.1038/nbt.2860

    Figure Lengend Snippet: Site-specific incorporation of 3 into proteins at diverse codons and specific proteome labelling using SORT-M in human cells. (a) Western blot analysis demonstrates the efficient amino acid dependant expression of an mCherry-EGFP fusion protein separated by an amber stop codon bearing a C-terminal HA-tag (mCh-TAG-EGFP-HA) in HEK293T cells. Anti-FLAG detected tagged PylRS (b) Specific labelling of mCh-TAG-EGFP-HA (immunoprecipitated from 10 6 cells) with 4a (20μM in 50μL PBS, 1h, RT) confirms the incorporation of 3 into protein in HEK293 cells. (c) SORT-M labelling of 3 that is statistically incorporated into newly synthesised proteins across the whole proteome of mammalian cells directed by six different PylRS/PyltRNA XXX mutants using 0.5 mM 3 . Labeling with 4g (20μM in PBS, 1h, RT, as above). The amino acids in parentheses are the natural amino acids encoded by the endogenous tRNA bearing the corresponding anti-codon.

    Article Snippet: Adherent HEK293T cells (ATCC CRL-11268; 4×106 per immunoprecipitation) were transfected with 7.5 μg p4CMVE-U6-PylT and 7.5ug pPylRS-mCherry-TAG-EFGP-HA using TransIT-293 transfection reagent according to the manufacturer’s protocol and cultured for 48 hours in DMEM/10%FBS, supplemented with 0.5 mM 1 or 2 mM 3 where indicated.

    Techniques: Western Blot, Expressing, Immunoprecipitation, Labeling

    Photophysical properties, kinetics and electrical properties of photoactivated QuasAr3 (paQuasAr3). (a) Photoactivation by blue light. Red lines: fluorescence of HEK cells expressing paQuasAr3 during voltage steps under constant red illumination (10 W/mm 2 ) and variable blue illumination (average of n = 8 cells). Grey lines: same experiment in HEK cells expressing QuasAr3 (average of n = 7 cells). (b) PaQuasAr3 fluorescence vs. blue light intensity at two membrane voltages ( n = 8 cells, mean ± s.e.m.). Photoactivation showed saturation behavior, with 50% maximum enhancement at I 488 = 27 mW/mm 2 . (c) Voltage-dependent near infrared fluorescence of paQuasAr3 and QuasAr3 with and without blue light (150 mW/mm 2 ). All fluorescence values are normalized to fluorescence with red only illumination at V m = −75 mV. (d) Same data as (c) but each fluorescence trace was normalized to its value F 0 at V m = −75 mV. Blue illumination enhanced the absolute fluorescence and the absolute voltage sensitivity, but the fractional voltage sensitivity (ΔF/F 0 ) was the same between QuasAr3 and paQuasAr3 and was not affected by blue illumination ( n = 7 cells in each condition, p = 0.91, one-way ANOVA). (e) Kinetics of QuasAr3 and paQuasAr3 measured in HEK293T cells. Cells were subjected to a square wave from −60 mV to +40 mV at 5 Hz (see panel a). Response transients were fit to a double exponential. QuasAr3, n = 5 cells, paQuasAr3, n = 9 cells. All values are mean ± s.e.m. Red intensity, 10 W/mm 2 , blue intensity, 150 mW/mm 2 . (f) Response of paQuasAr3 and QuasAr3 to steps of blue illumination. The blue light enhancement arose with a 50 ± 14 ms time-constant and subsided with a 167 ± 26 ms time-constant (mean ± s.d.). Blue light activated paQuasAr3 was ~2-fold brighter than QuasAr3 (mean of n = 10 cells). (g) Action spectrum for photosensitization, measured in E. coli expressing paQuasAr3. Fluorescence was excited at λ exc = 640 nm and emission was collected from λ em = 660 nm – 740 nm. The activation wavelength was scanned from λ act = 450 – 650 nm. Peak activation was at λ act = 470 nm. (h) Fluorescence excitation spectra ± blue sensitization (40 mW/mm 2 ). The sensitized state of paQuasAr3 had a fluorescence excitation spectrum similar to QuasAr3, with peak excitation at λ exc = 580 nm. (i) SNR of single spikes in acute slices for QuasAr3 ( n = 10 cells) and paQuasAr3 (n=10 cells) with either red only or red and blue illumination (mean ± s.d. paQuasAr3 ± blue, paired t -test, paQuasAr3 red and blue vs. QuasAr3 red only, t -test). (j) Voltage clamp recordings in CA1 pyramidal cell expressing paQuasAr3-s showed no photocurrents in response to illumination with red light (640 nm, 12 W/mm 2 ), blue light (488 nm, 90 mW/mm 2 ) or combination of the two, both when the cell was held at −70 mV and at 0 mV (repeated in n = 2 cells). (k) Electrical properties measured by patch clamp in acute slices. QuasAr3 ( n = 11 cells, 6 slices, 2 mice) and paQuasAr3 ( n = 9 cells, 6 slices, 3 mice) were expressed in the visual cortex and compared with non-expressing cortical cells ( n = 9 cells, 7 slices, 7 mice). paQuasAr3-s ( n = 7 cells, 7 slices, 5 mice) was expressed in CA1 pyramidal cell layer and compared with non-expressing cells in that layer ( n = 8 cells, 6 slices, 5 mice). Error bars are mean ± s.d.

    Journal: Nature

    Article Title: Voltage imaging and optogenetics reveal behavior dependent changes in hippocampal dynamics

    doi: 10.1038/s41586-019-1166-7

    Figure Lengend Snippet: Photophysical properties, kinetics and electrical properties of photoactivated QuasAr3 (paQuasAr3). (a) Photoactivation by blue light. Red lines: fluorescence of HEK cells expressing paQuasAr3 during voltage steps under constant red illumination (10 W/mm 2 ) and variable blue illumination (average of n = 8 cells). Grey lines: same experiment in HEK cells expressing QuasAr3 (average of n = 7 cells). (b) PaQuasAr3 fluorescence vs. blue light intensity at two membrane voltages ( n = 8 cells, mean ± s.e.m.). Photoactivation showed saturation behavior, with 50% maximum enhancement at I 488 = 27 mW/mm 2 . (c) Voltage-dependent near infrared fluorescence of paQuasAr3 and QuasAr3 with and without blue light (150 mW/mm 2 ). All fluorescence values are normalized to fluorescence with red only illumination at V m = −75 mV. (d) Same data as (c) but each fluorescence trace was normalized to its value F 0 at V m = −75 mV. Blue illumination enhanced the absolute fluorescence and the absolute voltage sensitivity, but the fractional voltage sensitivity (ΔF/F 0 ) was the same between QuasAr3 and paQuasAr3 and was not affected by blue illumination ( n = 7 cells in each condition, p = 0.91, one-way ANOVA). (e) Kinetics of QuasAr3 and paQuasAr3 measured in HEK293T cells. Cells were subjected to a square wave from −60 mV to +40 mV at 5 Hz (see panel a). Response transients were fit to a double exponential. QuasAr3, n = 5 cells, paQuasAr3, n = 9 cells. All values are mean ± s.e.m. Red intensity, 10 W/mm 2 , blue intensity, 150 mW/mm 2 . (f) Response of paQuasAr3 and QuasAr3 to steps of blue illumination. The blue light enhancement arose with a 50 ± 14 ms time-constant and subsided with a 167 ± 26 ms time-constant (mean ± s.d.). Blue light activated paQuasAr3 was ~2-fold brighter than QuasAr3 (mean of n = 10 cells). (g) Action spectrum for photosensitization, measured in E. coli expressing paQuasAr3. Fluorescence was excited at λ exc = 640 nm and emission was collected from λ em = 660 nm – 740 nm. The activation wavelength was scanned from λ act = 450 – 650 nm. Peak activation was at λ act = 470 nm. (h) Fluorescence excitation spectra ± blue sensitization (40 mW/mm 2 ). The sensitized state of paQuasAr3 had a fluorescence excitation spectrum similar to QuasAr3, with peak excitation at λ exc = 580 nm. (i) SNR of single spikes in acute slices for QuasAr3 ( n = 10 cells) and paQuasAr3 (n=10 cells) with either red only or red and blue illumination (mean ± s.d. paQuasAr3 ± blue, paired t -test, paQuasAr3 red and blue vs. QuasAr3 red only, t -test). (j) Voltage clamp recordings in CA1 pyramidal cell expressing paQuasAr3-s showed no photocurrents in response to illumination with red light (640 nm, 12 W/mm 2 ), blue light (488 nm, 90 mW/mm 2 ) or combination of the two, both when the cell was held at −70 mV and at 0 mV (repeated in n = 2 cells). (k) Electrical properties measured by patch clamp in acute slices. QuasAr3 ( n = 11 cells, 6 slices, 2 mice) and paQuasAr3 ( n = 9 cells, 6 slices, 3 mice) were expressed in the visual cortex and compared with non-expressing cortical cells ( n = 9 cells, 7 slices, 7 mice). paQuasAr3-s ( n = 7 cells, 7 slices, 5 mice) was expressed in CA1 pyramidal cell layer and compared with non-expressing cells in that layer ( n = 8 cells, 6 slices, 5 mice). Error bars are mean ± s.d.

    Article Snippet: Imaging and electrophysiology in HEK cells and primary neurons HEK293T cells (ATCC; CRL-11268) were cultured and transfected as described before .

    Techniques: Fluorescence, Expressing, Mass Spectrometry, Activation Assay, Activated Clotting Time Assay, Patch Clamp, Mouse Assay

    Shrew-1 protein isoforms 2 and 3 are targeted to the secretory pathway. (A) Constructs encoding iso1-shrew-1-myc and iso2-shrew-1-myc were expressed in HEK293T cells and detected with an anti-shrew-1 antibody (Nanotools, green). Nuclei stained with DAPI (blue). Both shrew-1 protein isoforms predominantly localized at the plasma membrane. Scale bar: 10 μm. (B) HEK293T cells expressing iso1-shrew-1-myc or iso2-shrew-1-myc were probed with biotin, pulled down with NeutrAvidin beads and analyzed by SDS-PAGE and immunoblot (beads) alongside 13% each of the pull-down input (input) and supernatant (sup) and visualized with an anti-shrew-1 antibody (Nanotools). Comparable pull-down efficiencies and purity of the cell surface samples was confirmed with Pan-cadherin and GAPDH detection. Both shrew-1 protein isoforms are O-glycosylated (arrowhead; Fig. S4 and main text), but also exhibit a fraction of premature protein (bracket). (C) Co-transfection of MCF-7 cells with iso3-shrew-1-myc (Genovac F, red) and Golgi-GFP showed a co-localization of iso3-shrew-1-myc with the trans cisternae of the Golgi apparatus (yellow). The nuclei were stained with DAPI (blue). Scale bar: 10 μm. (B) Ectopic Shrew-1 isoform 3 expression in HEK293T cells revealed the presence of double bands at the estimated size of 13.4 kDa (asterisk) and at about 17 kDa (arrowhead) compared to the empty vector control (bracket). Shrew-1 isoform 3 fused with a myc-tag (1.2 kDa) exhibited for both bands a delayed migration. (D) Cytosol-microsomal fractionation of transfected HEK293T cells was performed followed by immunoblot analysis of both fractions (cytosol, microsomes) as well as whole cell lysate samples (total). As markers for the distinct fractions GAPDH (cytosol) and Grp94 (microsomes) were visualized. Shrew-1 isoform 3 was detected in the microsomal fraction as a double band. (E) The isolated microsomal membranes of iso3-shrew-1 or empty vector transfected HEK293T cells were subjected to a proteinase K-protection assay. Whereas Grp94, the ER-lumenal protein, was protected by microsomal membranes from the proteolytic digestion, iso3-shrew-1 and β-catenin, associated with the membrane at the cytosolic side, were not. (F) By addition of Triton X-100 the microsomal membranes were disrupted, resulting in the degradation of Grp94. These results suggest that shrew-1 isoform 3 is associated with the membrane at the cytosolic side.

    Journal: Biology Open

    Article Title: Alternative exon usage creates novel transcript variants of tumor suppressor SHREW-1 gene with differential tissue expression profile

    doi: 10.1242/bio.019463

    Figure Lengend Snippet: Shrew-1 protein isoforms 2 and 3 are targeted to the secretory pathway. (A) Constructs encoding iso1-shrew-1-myc and iso2-shrew-1-myc were expressed in HEK293T cells and detected with an anti-shrew-1 antibody (Nanotools, green). Nuclei stained with DAPI (blue). Both shrew-1 protein isoforms predominantly localized at the plasma membrane. Scale bar: 10 μm. (B) HEK293T cells expressing iso1-shrew-1-myc or iso2-shrew-1-myc were probed with biotin, pulled down with NeutrAvidin beads and analyzed by SDS-PAGE and immunoblot (beads) alongside 13% each of the pull-down input (input) and supernatant (sup) and visualized with an anti-shrew-1 antibody (Nanotools). Comparable pull-down efficiencies and purity of the cell surface samples was confirmed with Pan-cadherin and GAPDH detection. Both shrew-1 protein isoforms are O-glycosylated (arrowhead; Fig. S4 and main text), but also exhibit a fraction of premature protein (bracket). (C) Co-transfection of MCF-7 cells with iso3-shrew-1-myc (Genovac F, red) and Golgi-GFP showed a co-localization of iso3-shrew-1-myc with the trans cisternae of the Golgi apparatus (yellow). The nuclei were stained with DAPI (blue). Scale bar: 10 μm. (B) Ectopic Shrew-1 isoform 3 expression in HEK293T cells revealed the presence of double bands at the estimated size of 13.4 kDa (asterisk) and at about 17 kDa (arrowhead) compared to the empty vector control (bracket). Shrew-1 isoform 3 fused with a myc-tag (1.2 kDa) exhibited for both bands a delayed migration. (D) Cytosol-microsomal fractionation of transfected HEK293T cells was performed followed by immunoblot analysis of both fractions (cytosol, microsomes) as well as whole cell lysate samples (total). As markers for the distinct fractions GAPDH (cytosol) and Grp94 (microsomes) were visualized. Shrew-1 isoform 3 was detected in the microsomal fraction as a double band. (E) The isolated microsomal membranes of iso3-shrew-1 or empty vector transfected HEK293T cells were subjected to a proteinase K-protection assay. Whereas Grp94, the ER-lumenal protein, was protected by microsomal membranes from the proteolytic digestion, iso3-shrew-1 and β-catenin, associated with the membrane at the cytosolic side, were not. (F) By addition of Triton X-100 the microsomal membranes were disrupted, resulting in the degradation of Grp94. These results suggest that shrew-1 isoform 3 is associated with the membrane at the cytosolic side.

    Article Snippet: Mice, cell lines, culture and lactogenic differentiation HEK293T cells (CRL-11268; ATCC, Manassas, VA, USA) and MCF-7 cells (European Collection of Cell Cultures, Salisbury, UK) were cultured in DMEM high glucose supplemented with 10% fetal calf serum (FCS), 100 U ml−1 penicillin and 100 µg ml−1 streptomycin (Sigma-Aldrich, Munich, Germany) at 37°C with 5% CO2 .

    Techniques: Construct, Staining, Expressing, SDS Page, Cotransfection, Plasmid Preparation, Migration, Fractionation, Transfection, Isolation

    Detection of shrew-1 isoforms in mouse organs. (A) Protein lysates of several murine organs were subjected to SDS-PAGE and analyzed by immunoblot using antibodies recognizing the CD of shrew-1, Sigma and Genovac F, respectively. Shrew-1 isoform 3 exogenously expressed in HEK293T was used as positive control and GAPDH as loading control. Both the Genovac F antibody as well as the Sigma antibody recognized the double bands of iso3-shrew-1 (13.4 kDa, asterisks; 17 kDa, arrowhead). Interestingly, also in the organs (spleen, kidney, embryo head, and to a lesser amount in the liver and the brain) protein species at about 17 kDa (arrowhead) were detected by both antibodies, suggesting the existence of a shrew-1 protein with the size of isoform 3 in vivo . (B) Schematic overview of mammary gland differentiation cycle and intracellular localization of shrew-1 isoforms by histological analysis. (C) Whole fat pad lysates of murine mammary gland at different developmental stages were subjected to SDS-PAGE and analyzed by immunoblot using antibodies recognizing the CD of shrew-1 to visualize all known shrew-1 isoforms (abcam and Genovac F). The antibody against β-casein was used as differentiation marker and probing against GAPDH as loading control. A double protein band at approx.13 kDa representing isoform 3 could be detected with the shrew-1 abcam antibody (long exposure) and additional bands ranging from 34 to 72 kDa possibly representing isoform 1 expression. The Genovac F antibody recognized a double band at approx. 26 kDa. d, days; V, virgin; Inv, involution.

    Journal: Biology Open

    Article Title: Alternative exon usage creates novel transcript variants of tumor suppressor SHREW-1 gene with differential tissue expression profile

    doi: 10.1242/bio.019463

    Figure Lengend Snippet: Detection of shrew-1 isoforms in mouse organs. (A) Protein lysates of several murine organs were subjected to SDS-PAGE and analyzed by immunoblot using antibodies recognizing the CD of shrew-1, Sigma and Genovac F, respectively. Shrew-1 isoform 3 exogenously expressed in HEK293T was used as positive control and GAPDH as loading control. Both the Genovac F antibody as well as the Sigma antibody recognized the double bands of iso3-shrew-1 (13.4 kDa, asterisks; 17 kDa, arrowhead). Interestingly, also in the organs (spleen, kidney, embryo head, and to a lesser amount in the liver and the brain) protein species at about 17 kDa (arrowhead) were detected by both antibodies, suggesting the existence of a shrew-1 protein with the size of isoform 3 in vivo . (B) Schematic overview of mammary gland differentiation cycle and intracellular localization of shrew-1 isoforms by histological analysis. (C) Whole fat pad lysates of murine mammary gland at different developmental stages were subjected to SDS-PAGE and analyzed by immunoblot using antibodies recognizing the CD of shrew-1 to visualize all known shrew-1 isoforms (abcam and Genovac F). The antibody against β-casein was used as differentiation marker and probing against GAPDH as loading control. A double protein band at approx.13 kDa representing isoform 3 could be detected with the shrew-1 abcam antibody (long exposure) and additional bands ranging from 34 to 72 kDa possibly representing isoform 1 expression. The Genovac F antibody recognized a double band at approx. 26 kDa. d, days; V, virgin; Inv, involution.

    Article Snippet: Mice, cell lines, culture and lactogenic differentiation HEK293T cells (CRL-11268; ATCC, Manassas, VA, USA) and MCF-7 cells (European Collection of Cell Cultures, Salisbury, UK) were cultured in DMEM high glucose supplemented with 10% fetal calf serum (FCS), 100 U ml−1 penicillin and 100 µg ml−1 streptomycin (Sigma-Aldrich, Munich, Germany) at 37°C with 5% CO2 .

    Techniques: SDS Page, Positive Control, In Vivo, Marker, Expressing

    USP15 interacts with BARD1 BRCT domain through its C-terminal region. a Reciprocal endogenous immunoprecipitation (IP) between USP15 and BARD1 were performed in HEK293T cells. (Left panel) IP with anti-BARD1 antibody and blot with anti-BARD1 or USP15 antibody, respectively. (Right panel) IP with anti-USP15 antibody and blot with anti-USP15 or BARD1 antibody, respectively. b Co-immunoprecipitation (co-IP) assays were performed to check the interaction between USP15 and BARD1 upon DNA damage. HEK293T cells treated as indicated were lysed and immunoprecipitated with anti-BARD1 antibody, and Western blot was performed with indicated antibodies. c Schematic representation of USP15-truncated mutants used in this study (upper panel). Plasmids encoding HA-tagged full-length or deletion mutants of USP15 were co-transfected with plasmids encoding FLAG-tagged full-length BARD1 into 293T cells. Immunoprecipitation and immunoblotting were performed 48 h post transfection as indicated. d Schematic representation of BARD1-truncated mutants used in this study (upper panel). Plasmids encoding FLAG-tagged full-length or deletion mutants of BARD1 were co-transfected with plasmids encoding HA-tagged full-length USP15 into 293T cells. Immunoprecipitation and immunoblotting were performed 48 h post transfection as indicated. e USP15-knockout U2OS direct repeat green fluorescent protein (DR-GFP) cells were reconstituted with HA-USP15 wild-type (WT) or D3 mutant and homologous recombination (HR) efficiency were determined. Data are presented as mean±SD of three independent experiments. Two-tailed Student's t test, * P

    Journal: Nature Communications

    Article Title: The deubiquitylating enzyme USP15 regulates homologous recombination repair and cancer cell response to PARP inhibitors

    doi: 10.1038/s41467-019-09232-8

    Figure Lengend Snippet: USP15 interacts with BARD1 BRCT domain through its C-terminal region. a Reciprocal endogenous immunoprecipitation (IP) between USP15 and BARD1 were performed in HEK293T cells. (Left panel) IP with anti-BARD1 antibody and blot with anti-BARD1 or USP15 antibody, respectively. (Right panel) IP with anti-USP15 antibody and blot with anti-USP15 or BARD1 antibody, respectively. b Co-immunoprecipitation (co-IP) assays were performed to check the interaction between USP15 and BARD1 upon DNA damage. HEK293T cells treated as indicated were lysed and immunoprecipitated with anti-BARD1 antibody, and Western blot was performed with indicated antibodies. c Schematic representation of USP15-truncated mutants used in this study (upper panel). Plasmids encoding HA-tagged full-length or deletion mutants of USP15 were co-transfected with plasmids encoding FLAG-tagged full-length BARD1 into 293T cells. Immunoprecipitation and immunoblotting were performed 48 h post transfection as indicated. d Schematic representation of BARD1-truncated mutants used in this study (upper panel). Plasmids encoding FLAG-tagged full-length or deletion mutants of BARD1 were co-transfected with plasmids encoding HA-tagged full-length USP15 into 293T cells. Immunoprecipitation and immunoblotting were performed 48 h post transfection as indicated. e USP15-knockout U2OS direct repeat green fluorescent protein (DR-GFP) cells were reconstituted with HA-USP15 wild-type (WT) or D3 mutant and homologous recombination (HR) efficiency were determined. Data are presented as mean±SD of three independent experiments. Two-tailed Student's t test, * P

    Article Snippet: Cell culture HEK293T (ATCC, CRL-11268), U2OS (ATCC, HTB-96), and MCF7 (ATCC, HTB-22) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplied with 10% fetal bovine serum (FBS) and penicillin/streptomycin (P/S) at 37 °C with 5% CO2 .

    Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Transfection, Knock-Out, Mutagenesis, Homologous Recombination, Two Tailed Test

    Identification of novel MATR3 interactors. ( A ) FLAG-MATR3 was expressed in HEK293T cells and immunoprecipitated using anti-FLAG conjugated beads. Immune complexes were separated by SDS-PAGE and visualized with silver staining. Cells with empty vector allowed discrimination between specific and non-specific immunoprecipitation. Bands that appeared specific were identified using mass spectrometry. ( B ) FLAG-MATR3 was expressed in HEK293T cells and immunoprecipitated using FLAG-conjugated beads. The immune complexes were blotted with the indicated antibodies. ( C ) Endogenous MATR3 was immunoprecipitated from HEK293T cells, and the immune complexes were treated with RNase A and blotted with antibodies against the indicated proteins.

    Journal: PLoS ONE

    Article Title: Matrin 3 Binds and Stabilizes mRNA

    doi: 10.1371/journal.pone.0023882

    Figure Lengend Snippet: Identification of novel MATR3 interactors. ( A ) FLAG-MATR3 was expressed in HEK293T cells and immunoprecipitated using anti-FLAG conjugated beads. Immune complexes were separated by SDS-PAGE and visualized with silver staining. Cells with empty vector allowed discrimination between specific and non-specific immunoprecipitation. Bands that appeared specific were identified using mass spectrometry. ( B ) FLAG-MATR3 was expressed in HEK293T cells and immunoprecipitated using FLAG-conjugated beads. The immune complexes were blotted with the indicated antibodies. ( C ) Endogenous MATR3 was immunoprecipitated from HEK293T cells, and the immune complexes were treated with RNase A and blotted with antibodies against the indicated proteins.

    Article Snippet: Cell lines HEK293T (ATCC Number: CRL-11268) and U2OS (ATCC Number: HTB-96) cells were grown in DMEM supplemented with 10% fetal bovine serum, at 37°C and 5% CO2 atmosphere.

    Techniques: Immunoprecipitation, SDS Page, Silver Staining, Plasmid Preparation, Mass Spectrometry

    Validation of RIP-seq results. ( A ) HEK293T cells were transfected with the indicated MATR3 constructs and the ectopic MATR3 proteins were immunoprecipitated using FLAG-conjugated beads and blotted with the indicated antibodies. ( B ) RNA was extracted from the immune complexes, yeast Phe-tRNA was added, qPCR was carried out on reverse transcription products and the Relative Quantification (RQ) is shown as fold-change of signal compared to ΔRRM2 minus the background of the empty vector. The plot represents the mean of three independent experiments and error bars represent SD (*P≤0.05, **P≤0.01, t test).

    Journal: PLoS ONE

    Article Title: Matrin 3 Binds and Stabilizes mRNA

    doi: 10.1371/journal.pone.0023882

    Figure Lengend Snippet: Validation of RIP-seq results. ( A ) HEK293T cells were transfected with the indicated MATR3 constructs and the ectopic MATR3 proteins were immunoprecipitated using FLAG-conjugated beads and blotted with the indicated antibodies. ( B ) RNA was extracted from the immune complexes, yeast Phe-tRNA was added, qPCR was carried out on reverse transcription products and the Relative Quantification (RQ) is shown as fold-change of signal compared to ΔRRM2 minus the background of the empty vector. The plot represents the mean of three independent experiments and error bars represent SD (*P≤0.05, **P≤0.01, t test).

    Article Snippet: Cell lines HEK293T (ATCC Number: CRL-11268) and U2OS (ATCC Number: HTB-96) cells were grown in DMEM supplemented with 10% fetal bovine serum, at 37°C and 5% CO2 atmosphere.

    Techniques: Transfection, Construct, Immunoprecipitation, Real-time Polymerase Chain Reaction, Plasmid Preparation

    MATR3 binds RNA via its RRM2 domain. ( A ) Schematic presentation of MATR3 domains and deletion of specific domains in different constructs used in this study. ( B ) HEK293T cells were transfected with the indicated MATR3 expression constructs. FLAG-conjugated beads were used for immunoprecipitation and the immune complexes were blotted with the indicated antibodies.

    Journal: PLoS ONE

    Article Title: Matrin 3 Binds and Stabilizes mRNA

    doi: 10.1371/journal.pone.0023882

    Figure Lengend Snippet: MATR3 binds RNA via its RRM2 domain. ( A ) Schematic presentation of MATR3 domains and deletion of specific domains in different constructs used in this study. ( B ) HEK293T cells were transfected with the indicated MATR3 expression constructs. FLAG-conjugated beads were used for immunoprecipitation and the immune complexes were blotted with the indicated antibodies.

    Article Snippet: Cell lines HEK293T (ATCC Number: CRL-11268) and U2OS (ATCC Number: HTB-96) cells were grown in DMEM supplemented with 10% fetal bovine serum, at 37°C and 5% CO2 atmosphere.

    Techniques: Construct, Transfection, Expressing, Immunoprecipitation

    NMI stimulates macrophages through the TLR4 pathway. a Western blot analysis of mNMI in the BMDM cell lysate after 1 h incubation with recombinant mNMI. The BMDM cells were isolated from Nmi −/− mice and pretreated with macrophage colony-stimulating factor (MCSF). b , c TNF and IL-6 released by BMDMs from WT (C57BL/6) mice, pretreated with bafilomycin A1 (10 nM), TAK-242 (100 nM) or dimethyl sulphoxide (DMSO) for 2 h and stimulated with mNMI (5 μg ml −1 ) or LPS (100 ng ml −1 ) for 8 h. d – g TNF and IL-6 levels in the supernatants of BMDMs from WT, Tlr4 −/− and Tlr2 −/− mice were analyzed using ELISA 4 h post activation by different stimulus. h After incubation with mNMI-GFP for 1 h, the percentage of GFP labeled CD11b + F4/80 + cells was determined by Flow cytometric analysis. The cells were isolated from spleen in WT or Tlr4 −/− mice. i NMI in the human THP1 cell (ATCC TIB-202™) lysates interacts with hTLR4. Ni-NTA beads coupled with 2 μg His-hTLR4 and/or His-hMD2 fusion proteins were used as bait. j The luciferase activity of HEK293T cells (ATCC CRL-11268™) are shown after stimulated with 5 μg ml −1 mNMI for 4 h (in the presence or absence of 25 μg ml −1 polymyxin B (PMB)). The cells were pre-transfected with mTLR4-MD2-CD14 and NF-κB promoter with luciferase activity. 100 ng ml −1 LPS was administrated as positive control. In b – e and g , error bars indicate ± s.e.m. from 3 biological replicates. Significance was tested by one-way ANOVA followed by Student–Newman–Keuls test. ** P

    Journal: Nature Communications

    Article Title: NMI and IFP35 serve as proinflammatory DAMPs during cellular infection and injury

    doi: 10.1038/s41467-017-00930-9

    Figure Lengend Snippet: NMI stimulates macrophages through the TLR4 pathway. a Western blot analysis of mNMI in the BMDM cell lysate after 1 h incubation with recombinant mNMI. The BMDM cells were isolated from Nmi −/− mice and pretreated with macrophage colony-stimulating factor (MCSF). b , c TNF and IL-6 released by BMDMs from WT (C57BL/6) mice, pretreated with bafilomycin A1 (10 nM), TAK-242 (100 nM) or dimethyl sulphoxide (DMSO) for 2 h and stimulated with mNMI (5 μg ml −1 ) or LPS (100 ng ml −1 ) for 8 h. d – g TNF and IL-6 levels in the supernatants of BMDMs from WT, Tlr4 −/− and Tlr2 −/− mice were analyzed using ELISA 4 h post activation by different stimulus. h After incubation with mNMI-GFP for 1 h, the percentage of GFP labeled CD11b + F4/80 + cells was determined by Flow cytometric analysis. The cells were isolated from spleen in WT or Tlr4 −/− mice. i NMI in the human THP1 cell (ATCC TIB-202™) lysates interacts with hTLR4. Ni-NTA beads coupled with 2 μg His-hTLR4 and/or His-hMD2 fusion proteins were used as bait. j The luciferase activity of HEK293T cells (ATCC CRL-11268™) are shown after stimulated with 5 μg ml −1 mNMI for 4 h (in the presence or absence of 25 μg ml −1 polymyxin B (PMB)). The cells were pre-transfected with mTLR4-MD2-CD14 and NF-κB promoter with luciferase activity. 100 ng ml −1 LPS was administrated as positive control. In b – e and g , error bars indicate ± s.e.m. from 3 biological replicates. Significance was tested by one-way ANOVA followed by Student–Newman–Keuls test. ** P

    Article Snippet: Luciferase assay HEK293T cells (ATCC CRL -11268™) were seeded in 6-well plates at a density of 2 × 106 cells per well and grew overnight.

    Techniques: Western Blot, Incubation, Recombinant, Isolation, Mouse Assay, Enzyme-linked Immunosorbent Assay, Activation Assay, Labeling, Flow Cytometry, Luciferase, Activity Assay, Transfection, Positive Control

    Assessment of mitochondrial protein levels, respiration and MICOS/MIB complex integrity in ArmC1 knockout cells. (A) Mitochondria were isolated from wild type and ArmC1 knockout HeLa cells and 25 and 50 μg of mitochondrial protein was analyzed by SDS-PAGE and western blot with designated antibodies. Asterisk indicates a non-specific band. (B) Oxygen consumption rate (OCR) of wildtype and ArmC1 knockout cells was analyzed by Seahorse Flux Analyzer. Basal respiration, the ATP production, the maximal respiration and the non-mitochondrial respiration were determined by sequentially injecting 2 μM oligomycin, 1 μM FCCP, and 0.5 μM rotenone and antimycin A. (C) Mitochondria from wildtype HeLa cells, two different knockdown cell lines of ArmC1 and HEK293T cells transfected with FLAG-ArmC1 pCDNA3 construct were isolated and analyzed by BN-PAGE and western blot, using antibodies against Mi60/Mitofilin, ArmC1 and Sam50. Sam50, sorting and assembly machinery 50, Tom, translocase of the outer mitochondrial membrane, NDUFS1, NADH dehydrogenase [ubiquinone] iron-sulfur protein 1, SDHA, Succinate dehydrogenase complex subunit A, UQCRFS1, Ubiquinol-cytochrome c reductase iron-sulfur subunit 1, Cox, cytochrome oxidase, F1β, ATP synthase subunit beta, Hsp60, heat shock protein 60.

    Journal: PLoS ONE

    Article Title: Armadillo repeat-containing protein 1 is a dual localization protein associated with mitochondrial intermembrane space bridging complex

    doi: 10.1371/journal.pone.0218303

    Figure Lengend Snippet: Assessment of mitochondrial protein levels, respiration and MICOS/MIB complex integrity in ArmC1 knockout cells. (A) Mitochondria were isolated from wild type and ArmC1 knockout HeLa cells and 25 and 50 μg of mitochondrial protein was analyzed by SDS-PAGE and western blot with designated antibodies. Asterisk indicates a non-specific band. (B) Oxygen consumption rate (OCR) of wildtype and ArmC1 knockout cells was analyzed by Seahorse Flux Analyzer. Basal respiration, the ATP production, the maximal respiration and the non-mitochondrial respiration were determined by sequentially injecting 2 μM oligomycin, 1 μM FCCP, and 0.5 μM rotenone and antimycin A. (C) Mitochondria from wildtype HeLa cells, two different knockdown cell lines of ArmC1 and HEK293T cells transfected with FLAG-ArmC1 pCDNA3 construct were isolated and analyzed by BN-PAGE and western blot, using antibodies against Mi60/Mitofilin, ArmC1 and Sam50. Sam50, sorting and assembly machinery 50, Tom, translocase of the outer mitochondrial membrane, NDUFS1, NADH dehydrogenase [ubiquinone] iron-sulfur protein 1, SDHA, Succinate dehydrogenase complex subunit A, UQCRFS1, Ubiquinol-cytochrome c reductase iron-sulfur subunit 1, Cox, cytochrome oxidase, F1β, ATP synthase subunit beta, Hsp60, heat shock protein 60.

    Article Snippet: Cell lines and cell culture Human embryonic kidney epithelial (HEK293T) cells (ATCC CRL-11268) and human cervical carcinoma (HeLa) cells (ATCC CCL-227) were obtained from the American Type Culture Collection. sam50kd-2 , mflkd-2 , chchd3kd-2 , chchd6kd-3 and apookd-4 [ ], as well as the cell line stably expressing mitochondria-targeted GFP (CoxVa presequence fused to the GFP), were generated from HeLa cells using lentiviral-based shRNA expression system [ ].

    Techniques: Knock-Out, Isolation, SDS Page, Western Blot, Transfection, Construct, Polyacrylamide Gel Electrophoresis

    Analysis of mitochondrial association of endogenous ArmC1. (A) HEK293T cells were transfected with pCDNA3 construct encoding for N-terminally FLAG-tagged ArmC1 (FLAG-ArmC1). Transfected and non-transfected HEK293T cells were fractionated into mitochondrial and cytosolic fraction and analyzed by SDS-PAGE and western blot, using antibodies against Tom20, ArmC1, FLAG, tubulin and actin. (B) Mitochondria were isolated from HEK293T and HeLa cells and subjected to swelling in hypotonic buffer or lysis with 1% Triton X-100, followed by the treatment with 50 μg/ml protease K. Samples were analyzed by SDS-PAGE and western blot, using antibodies against Tom20, Mic60/Mitofilin, Hsp60 and ArmC1. (C) Mitochondria as in B were subjected to carbonate extraction in 100 mM Na 2 CO 3 , pH 10.8. Total mitochondria, membrane pellet and cytosolic fraction were analyzed by SDS-PAGE and western blot using antibodies against Hsp60, Tom20 and ArmC1. (D) Amino acid sequence of ArmC1 with predicted armadillo repeat and the conserved C-terminal domain in gray boxes. Tom20 –translocase of the outer membrane 20, Hsp60 –heat shock protein 60.

    Journal: PLoS ONE

    Article Title: Armadillo repeat-containing protein 1 is a dual localization protein associated with mitochondrial intermembrane space bridging complex

    doi: 10.1371/journal.pone.0218303

    Figure Lengend Snippet: Analysis of mitochondrial association of endogenous ArmC1. (A) HEK293T cells were transfected with pCDNA3 construct encoding for N-terminally FLAG-tagged ArmC1 (FLAG-ArmC1). Transfected and non-transfected HEK293T cells were fractionated into mitochondrial and cytosolic fraction and analyzed by SDS-PAGE and western blot, using antibodies against Tom20, ArmC1, FLAG, tubulin and actin. (B) Mitochondria were isolated from HEK293T and HeLa cells and subjected to swelling in hypotonic buffer or lysis with 1% Triton X-100, followed by the treatment with 50 μg/ml protease K. Samples were analyzed by SDS-PAGE and western blot, using antibodies against Tom20, Mic60/Mitofilin, Hsp60 and ArmC1. (C) Mitochondria as in B were subjected to carbonate extraction in 100 mM Na 2 CO 3 , pH 10.8. Total mitochondria, membrane pellet and cytosolic fraction were analyzed by SDS-PAGE and western blot using antibodies against Hsp60, Tom20 and ArmC1. (D) Amino acid sequence of ArmC1 with predicted armadillo repeat and the conserved C-terminal domain in gray boxes. Tom20 –translocase of the outer membrane 20, Hsp60 –heat shock protein 60.

    Article Snippet: Cell lines and cell culture Human embryonic kidney epithelial (HEK293T) cells (ATCC CRL-11268) and human cervical carcinoma (HeLa) cells (ATCC CCL-227) were obtained from the American Type Culture Collection. sam50kd-2 , mflkd-2 , chchd3kd-2 , chchd6kd-3 and apookd-4 [ ], as well as the cell line stably expressing mitochondria-targeted GFP (CoxVa presequence fused to the GFP), were generated from HeLa cells using lentiviral-based shRNA expression system [ ].

    Techniques: Transfection, Construct, SDS Page, Western Blot, Isolation, Lysis, Sequencing

    Immunoprecipitation and BN-PAGE analysis of ArmC1. (A) Mitochondria were isolated from HEK293T cells transfected with an empty pCDNA3 plasmid or with the FLAG-ArmC1 pCDNA3 construct, lysed in the buffer containing 0.5% digitonin and 1 mM PMSF, and incubated with Anti-FLAG M2 affinity gel. Eluted proteins were separated using SDS-PAGE and after colloidal coomassie G-250 staining specific bands were excised from the FLAG-ArmC1 sample and analyzed by mass spectrometry in parallel with the respective regions of the gel from the control empty vector sample. Proteins detected in the FLAG-ArmC1 but not in the control sample are listed in the adjacent table. (B) Immunoprecipitation was performed as in A and samples were analyzed by SDS-PAGE and western blot, using antibodies against Mic60/Mitofilin, Mic10/MINOS1, Sam50, Metaxin 1, Actin, FLAG and Hsp60. (C) Mitochondria were isolated from non-transfected HEK293T cells and cells where FLAG-ArmC1 has been expressed with the help of transient transfection of the FLAG-ArmC1 pCDNA3 plasmid. After solubilization with 1% digitonin buffer, samples were analyzed by BN-PAGE and western blot, using antibodies against Mic60/Mitofilin, ArmC1 and Sam50. MINOS1—mitochondrial inner membrane organizing system 1, Sam50—sorting and assembly machinery 50, Hsp60 –heat shock protein 60.

    Journal: PLoS ONE

    Article Title: Armadillo repeat-containing protein 1 is a dual localization protein associated with mitochondrial intermembrane space bridging complex

    doi: 10.1371/journal.pone.0218303

    Figure Lengend Snippet: Immunoprecipitation and BN-PAGE analysis of ArmC1. (A) Mitochondria were isolated from HEK293T cells transfected with an empty pCDNA3 plasmid or with the FLAG-ArmC1 pCDNA3 construct, lysed in the buffer containing 0.5% digitonin and 1 mM PMSF, and incubated with Anti-FLAG M2 affinity gel. Eluted proteins were separated using SDS-PAGE and after colloidal coomassie G-250 staining specific bands were excised from the FLAG-ArmC1 sample and analyzed by mass spectrometry in parallel with the respective regions of the gel from the control empty vector sample. Proteins detected in the FLAG-ArmC1 but not in the control sample are listed in the adjacent table. (B) Immunoprecipitation was performed as in A and samples were analyzed by SDS-PAGE and western blot, using antibodies against Mic60/Mitofilin, Mic10/MINOS1, Sam50, Metaxin 1, Actin, FLAG and Hsp60. (C) Mitochondria were isolated from non-transfected HEK293T cells and cells where FLAG-ArmC1 has been expressed with the help of transient transfection of the FLAG-ArmC1 pCDNA3 plasmid. After solubilization with 1% digitonin buffer, samples were analyzed by BN-PAGE and western blot, using antibodies against Mic60/Mitofilin, ArmC1 and Sam50. MINOS1—mitochondrial inner membrane organizing system 1, Sam50—sorting and assembly machinery 50, Hsp60 –heat shock protein 60.

    Article Snippet: Cell lines and cell culture Human embryonic kidney epithelial (HEK293T) cells (ATCC CRL-11268) and human cervical carcinoma (HeLa) cells (ATCC CCL-227) were obtained from the American Type Culture Collection. sam50kd-2 , mflkd-2 , chchd3kd-2 , chchd6kd-3 and apookd-4 [ ], as well as the cell line stably expressing mitochondria-targeted GFP (CoxVa presequence fused to the GFP), were generated from HeLa cells using lentiviral-based shRNA expression system [ ].

    Techniques: Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Isolation, Transfection, Plasmid Preparation, Construct, Incubation, SDS Page, Staining, Mass Spectrometry, Western Blot

    Images of fluorescent microscopic samples through unfixed human colon tissue. Fluorescent beads of 400 nm in diameter and fixed HEK293T/17-GFP cells were imaged through 250 and 200 μm of human colon tissue, respectively. ( A and C ) Images taken from the reference imaging system under uniform TF illumination across the FOV. Camera binning in (A) was set to 4 × 4, and exposure time was 5 s. No camera binning was used in (C), and exposure time was 15 s. ( B and D ) Images obtained with TRAFIX using a Hadamard basis containing 1024 and 4096 illumination patterns, respectively. All patterns were used for image reconstruction (CR = 1). Camera binning for each Hadamard pattern was set to 64 × 64, and exposure time values were (B) 1 s and (D) 0.75 s. The spacing between beads and the diameter of cells were measured to assess image quality (tables S3 and S2, respectively). The SBR is shown for all reconstructed images. Scale bars, 10 μm.

    Journal: Science Advances

    Article Title: Wide-field multiphoton imaging through scattering media without correction

    doi: 10.1126/sciadv.aau1338

    Figure Lengend Snippet: Images of fluorescent microscopic samples through unfixed human colon tissue. Fluorescent beads of 400 nm in diameter and fixed HEK293T/17-GFP cells were imaged through 250 and 200 μm of human colon tissue, respectively. ( A and C ) Images taken from the reference imaging system under uniform TF illumination across the FOV. Camera binning in (A) was set to 4 × 4, and exposure time was 5 s. No camera binning was used in (C), and exposure time was 15 s. ( B and D ) Images obtained with TRAFIX using a Hadamard basis containing 1024 and 4096 illumination patterns, respectively. All patterns were used for image reconstruction (CR = 1). Camera binning for each Hadamard pattern was set to 64 × 64, and exposure time values were (B) 1 s and (D) 0.75 s. The spacing between beads and the diameter of cells were measured to assess image quality (tables S3 and S2, respectively). The SBR is shown for all reconstructed images. Scale bars, 10 μm.

    Article Snippet: HEK293T/17 cell line obtained from American Type Culture Collection was cultured in Dulbecco’s modified Eagle’s medium GlutaMAX-I supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin and was transfected using TransIT-LT1 transfection reagent with the Vesicular Stomatitis Virus glycoprotein (VSV-G) pseudotyped lentivirus vector and the packaging plasmid psPAX2 to deliver the plasmid pLenti-GFP-Puro.

    Techniques: Imaging

    Images of fluorescent microscopic samples through scattering phantoms. Fluorescent beads of 400 nm in diameter and fixed HEK293T/17-GFP cells were imaged through 500- and 540-μm of scattering phantoms, respectively. ( A and F ) Images taken from the reference imaging system under uniform TF illumination across the FOV. Exposure time was set to 20 s, and camera binning was 4 × 4 (beads) and 2 × 2 (cells). ( B to E , G , and H ) Images obtained in epifluorescence configuration with TRAFIX using a Hadamard basis containing 4096 illumination patterns. They were reconstructed with different CRs corresponding to 100% (CR = 1), 50% (CR = 2), 25% (CR = 4), or 12.5% (CR = 8) of the total patterns. Each measurement under individual illumination patterns was taken with a binning of 64 × 64 and an exposure time of 0.5 s. The spacing between beads was measured in all five images obtaining deviations smaller than 3% from the reference image (table S3). The diameters of the cells in (F) were measured to be 20.7 and 14.3 μm, respectively, and their values in (G) and (H) differ less than 4 and 12% from the reference value (table S2). The SBR is shown for all reconstructed images. Scale bars, 10 μm.

    Journal: Science Advances

    Article Title: Wide-field multiphoton imaging through scattering media without correction

    doi: 10.1126/sciadv.aau1338

    Figure Lengend Snippet: Images of fluorescent microscopic samples through scattering phantoms. Fluorescent beads of 400 nm in diameter and fixed HEK293T/17-GFP cells were imaged through 500- and 540-μm of scattering phantoms, respectively. ( A and F ) Images taken from the reference imaging system under uniform TF illumination across the FOV. Exposure time was set to 20 s, and camera binning was 4 × 4 (beads) and 2 × 2 (cells). ( B to E , G , and H ) Images obtained in epifluorescence configuration with TRAFIX using a Hadamard basis containing 4096 illumination patterns. They were reconstructed with different CRs corresponding to 100% (CR = 1), 50% (CR = 2), 25% (CR = 4), or 12.5% (CR = 8) of the total patterns. Each measurement under individual illumination patterns was taken with a binning of 64 × 64 and an exposure time of 0.5 s. The spacing between beads was measured in all five images obtaining deviations smaller than 3% from the reference image (table S3). The diameters of the cells in (F) were measured to be 20.7 and 14.3 μm, respectively, and their values in (G) and (H) differ less than 4 and 12% from the reference value (table S2). The SBR is shown for all reconstructed images. Scale bars, 10 μm.

    Article Snippet: HEK293T/17 cell line obtained from American Type Culture Collection was cultured in Dulbecco’s modified Eagle’s medium GlutaMAX-I supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin and was transfected using TransIT-LT1 transfection reagent with the Vesicular Stomatitis Virus glycoprotein (VSV-G) pseudotyped lentivirus vector and the packaging plasmid psPAX2 to deliver the plasmid pLenti-GFP-Puro.

    Techniques: Imaging

    Physiological role and subcellular localization of DTWD1 and DTWD2. a Growth curves of WT HEK293T (open square), DTWD1 KO#1 (open circle), DTWD1 KO#2 (closed circle) and DTWD2 KO (open rhombus) strains (left panel), and WT HEK293T (open square), DTWD1 / DTWD2 double-KO#1 (open circle) and DTWD1 / DTWD2 double-KO#2 (closed circle) strains (right panel), cultured in DMEM medium. Fluorescence was calculated as the average values of biological replicates ( n = 6), with s.d. ** P

    Journal: Nature Communications

    Article Title: Biogenesis and functions of aminocarboxypropyluridine in tRNA

    doi: 10.1038/s41467-019-13525-3

    Figure Lengend Snippet: Physiological role and subcellular localization of DTWD1 and DTWD2. a Growth curves of WT HEK293T (open square), DTWD1 KO#1 (open circle), DTWD1 KO#2 (closed circle) and DTWD2 KO (open rhombus) strains (left panel), and WT HEK293T (open square), DTWD1 / DTWD2 double-KO#1 (open circle) and DTWD1 / DTWD2 double-KO#2 (closed circle) strains (right panel), cultured in DMEM medium. Fluorescence was calculated as the average values of biological replicates ( n = 6), with s.d. ** P

    Article Snippet: Human cell culture and measurement of cell proliferation HEK293T (CRL-11268, ATCC) and Hela (CCL-2, ATCC) cells were cultured in high-glucose DMEM (D5796; Sigma) supplemented with 5% FBS (Gibco) and 1% penicillin–streptomycin (Fujifilm Wako Pure Chemical Corporation), at 37 °C in a humidified atmosphere containing 5% CO2 .

    Techniques: Cell Culture, Fluorescence

    Expression of full-length antibodies from recombinant AAV vectors. Levels of expressed IgG or immunoadhesin were analyzed by Western Blot after transfection of HEK293T cells with equal amounts of plasmid DNA (0.5 μg + 0.5 μg or 1 μg). Comparison of secreted ( A ) 4L6 IgGs or ( B ) 5L7 IgGs from co-transfection of heavy and light chain vectors (two vector approach) vs . transfection of bicistronic vectors (one vector approach). The two vector approach yielded slightly higher levels of secreted antibodies than the one vector approach. IgG1 versions of the 4L6 and 5L7 full-length antibodies expressed better than IgG2 versions.

    Journal: PLoS ONE

    Article Title: Recombinant AAV Vectors for Enhanced Expression of Authentic IgG

    doi: 10.1371/journal.pone.0158009

    Figure Lengend Snippet: Expression of full-length antibodies from recombinant AAV vectors. Levels of expressed IgG or immunoadhesin were analyzed by Western Blot after transfection of HEK293T cells with equal amounts of plasmid DNA (0.5 μg + 0.5 μg or 1 μg). Comparison of secreted ( A ) 4L6 IgGs or ( B ) 5L7 IgGs from co-transfection of heavy and light chain vectors (two vector approach) vs . transfection of bicistronic vectors (one vector approach). The two vector approach yielded slightly higher levels of secreted antibodies than the one vector approach. IgG1 versions of the 4L6 and 5L7 full-length antibodies expressed better than IgG2 versions.

    Article Snippet: Cell culture and DNA transfection HEK293T/17 cells (ATCC) were maintained in complete D10 growth medium: DMEM supplemented with 10% ultra-low IgG FBS, 25 mM HEPES, 2 mM L-glutamine (all Gibco, Thermo Fisher) and 100 μg/ml Primocin (InvivoGen).

    Techniques: Expressing, Recombinant, Western Blot, Transfection, Plasmid Preparation, Cotransfection

    Metabolic labeling of LRRK2 depends on an intact ROC domain. A–B. Influence of guanine nucleotides on phosphorylation of LRRK2 in cellular lysates. HEK293T cells expressing LRRK2 were lysed and incubated with ATP- 32 P for 30 minutes at 30°C without additions (control) or in the presence of 10 µM GDP, GTP or the non hydrolyzable GTP analogues GTPγS and GMPPCP. LRRK2 was subsequently IP purified and submitted to SDS-PAGE and blotting to a PVDF membrane A. Shown here are the representative blot autoradiograms, immunoblot detection and ponceau staining of the phospholabeled samples. B. Quantification of A. C–D. Metabolic labeling of LRRK2 wt, GTP binding deficient LRRK2 mutants (K1347A, T1348N), and LRRK2 C-terminal (972–2527, encompassing ROC domain and lacking most cellular phosphorylation sites) and N-terminal (1–1245, encompassing cellular phosphorylation sites and lacking ROC domain) fragments. C. Representative blot autoradiograms and blot immunodetection of the metabolically labeled samples. D. Quantification of C. Data are representative of 4 experiments. Statistical differences of results in panels B and D were tested by one-way ANOVA as described in the materials and methods section. *** P

    Journal: PLoS ONE

    Article Title: LRRK2 Kinase Activity Is Dependent on LRRK2 GTP Binding Capacity but Independent of LRRK2 GTP Binding

    doi: 10.1371/journal.pone.0023207

    Figure Lengend Snippet: Metabolic labeling of LRRK2 depends on an intact ROC domain. A–B. Influence of guanine nucleotides on phosphorylation of LRRK2 in cellular lysates. HEK293T cells expressing LRRK2 were lysed and incubated with ATP- 32 P for 30 minutes at 30°C without additions (control) or in the presence of 10 µM GDP, GTP or the non hydrolyzable GTP analogues GTPγS and GMPPCP. LRRK2 was subsequently IP purified and submitted to SDS-PAGE and blotting to a PVDF membrane A. Shown here are the representative blot autoradiograms, immunoblot detection and ponceau staining of the phospholabeled samples. B. Quantification of A. C–D. Metabolic labeling of LRRK2 wt, GTP binding deficient LRRK2 mutants (K1347A, T1348N), and LRRK2 C-terminal (972–2527, encompassing ROC domain and lacking most cellular phosphorylation sites) and N-terminal (1–1245, encompassing cellular phosphorylation sites and lacking ROC domain) fragments. C. Representative blot autoradiograms and blot immunodetection of the metabolically labeled samples. D. Quantification of C. Data are representative of 4 experiments. Statistical differences of results in panels B and D were tested by one-way ANOVA as described in the materials and methods section. *** P

    Article Snippet: Expression and purification of recombinant LRRK2 protein HEK293T cells (ATCC CRL-11268) were transfected with pCHMWS-3xflag-LRRK2 plasmid using polyethyleneimine and lysed after 48–72 hours in lysis buffer (Tris 20 mM pH 7.5, NaCl 150 mM, EDTA 1 mM, Triton 1%, Glycerol 10%, protease inhibitor cocktail (Roche, Vilvoorde, Belgium)).

    Techniques: Labeling, Expressing, Incubation, Purification, SDS Page, Staining, Binding Assay, Immunodetection, Metabolic Labelling

    Influence of guanine nucleotides on macromolecular properties of LRRK2. A–D. Size exclusion chromatography elution profiles of LRRK2 in cell lysates or purified LRRK2 in the presence of GDP or GTPγS. 3xflag LRRK2 was expressed by transient transfection in HEK293T cells. Cleared cell lysates (A C) or purified protein (B D) were prepared as described in Materials and Methods and submitted to SEC in the presence of 10 µM of either GDP (A B) or GTPγS (C D). LRRK2 elution profiles were obtained by measuring LRRK2 levels in each elution fraction via immuno dot blot and are displayed as relative signal intensity in function of the elution volume, as described in Materials and Methods . The elution peaks of the protein standards are indicated above the dot blot of panel A. E–F. Native PAGE of LRRK2 purified from lysates loaded with different concentrations of nucleotides. 3xflag tagged LRRK2 was expressed in HEK293T cells via transient transfection. Cells were lysed at 48 h post-transfection and incubated with varying concentrations of GDP, GTPγS or GMPPCP (10, 100 and 500 µM) for 30 minutes at 30°C. Treated lysates were purified as described in materials and methods and separated via native PAGE. Gels were silver stained (top panel) or blotted onto PVDF membranes to detect flag immunoreactivity E. LRRK2 protein amounts visualized via silver staining on SDS-PAGE are shown under the native PAGE images. F. Signal intensity plotted against migration distance for each lane. The arrow marks the peak corresponding to the band which migrates at the predicted size of a LRRK2 dimer. Data are representative of 3 experiments.

    Journal: PLoS ONE

    Article Title: LRRK2 Kinase Activity Is Dependent on LRRK2 GTP Binding Capacity but Independent of LRRK2 GTP Binding

    doi: 10.1371/journal.pone.0023207

    Figure Lengend Snippet: Influence of guanine nucleotides on macromolecular properties of LRRK2. A–D. Size exclusion chromatography elution profiles of LRRK2 in cell lysates or purified LRRK2 in the presence of GDP or GTPγS. 3xflag LRRK2 was expressed by transient transfection in HEK293T cells. Cleared cell lysates (A C) or purified protein (B D) were prepared as described in Materials and Methods and submitted to SEC in the presence of 10 µM of either GDP (A B) or GTPγS (C D). LRRK2 elution profiles were obtained by measuring LRRK2 levels in each elution fraction via immuno dot blot and are displayed as relative signal intensity in function of the elution volume, as described in Materials and Methods . The elution peaks of the protein standards are indicated above the dot blot of panel A. E–F. Native PAGE of LRRK2 purified from lysates loaded with different concentrations of nucleotides. 3xflag tagged LRRK2 was expressed in HEK293T cells via transient transfection. Cells were lysed at 48 h post-transfection and incubated with varying concentrations of GDP, GTPγS or GMPPCP (10, 100 and 500 µM) for 30 minutes at 30°C. Treated lysates were purified as described in materials and methods and separated via native PAGE. Gels were silver stained (top panel) or blotted onto PVDF membranes to detect flag immunoreactivity E. LRRK2 protein amounts visualized via silver staining on SDS-PAGE are shown under the native PAGE images. F. Signal intensity plotted against migration distance for each lane. The arrow marks the peak corresponding to the band which migrates at the predicted size of a LRRK2 dimer. Data are representative of 3 experiments.

    Article Snippet: Expression and purification of recombinant LRRK2 protein HEK293T cells (ATCC CRL-11268) were transfected with pCHMWS-3xflag-LRRK2 plasmid using polyethyleneimine and lysed after 48–72 hours in lysis buffer (Tris 20 mM pH 7.5, NaCl 150 mM, EDTA 1 mM, Triton 1%, Glycerol 10%, protease inhibitor cocktail (Roche, Vilvoorde, Belgium)).

    Techniques: Size-exclusion Chromatography, Purification, Transfection, Dot Blot, Clear Native PAGE, Incubation, Staining, Silver Staining, SDS Page, Migration

    Concentration response curves of BPA on (A) ERα-bla and (B) AR-bla assays. GeneBLAzer β-lactamase HEK293 cell lines that co-express Gal4-reporter system with Gal4DBD-linked ERα-LBD or AR-LBD were tested in 12-24 concentrations

    Journal: Chemico-biological interactions

    Article Title: Bisphenol A affects androgen receptor function via multiple mechanisms

    doi: 10.1016/j.cbi.2013.03.013

    Figure Lengend Snippet: Concentration response curves of BPA on (A) ERα-bla and (B) AR-bla assays. GeneBLAzer β-lactamase HEK293 cell lines that co-express Gal4-reporter system with Gal4DBD-linked ERα-LBD or AR-LBD were tested in 12-24 concentrations

    Article Snippet: GeneBLAzer β-lactamase (bla) HEK293T cell lines that co-express the Gal4-reporter system with Gal4DBD-linked LBD of related human NRs and Gal4 response element-bla reporter were obtained from Invitrogen (Carlsbad, CA).

    Techniques: Concentration Assay

    Kv2.1 P404W increases Cav1.2 single channel activity. ( A, B ) Maximum z -projections of TIRF images of Cav1.2-mediated Ca 2+ sparklets in a representative HEK293T cell transfected with Cav1.2 and auxiliary subunits and loaded with Fluo-5F via the patch pipette, before ( A ) and after ( B ) treatment with 500 nM Bay K8644 (scale bar: 5 µm). ( C ) Maximum z -projection of TIRF images of DsRed-Kv2.1 in a representative HEK293T cell cotransfected with Cav1.2 and auxiliary subunits (scale bar: 5 µm). ( D, F ) Maximum z -projections of TIRF images of sparklets in a representative HEK293T cell transfected with DsRed-Kv2.1, Cav1.2, and auxiliary subunits and loaded with Fluo-5F via the patch pipette, before ( D ) and after ( F ) treatment with 500 nM Bay K8644. ( E, G ) Merged images of panels C and D ( E ), or panels C and F ( G ). ( H ) Fluorescence intensity profiles of representative sparklets recorded in 20 mM external Ca 2+ in a control cell (upper panel, ROI depicted in A) or in a cell additionally expressing Kv2.1 (lower panel, ROI depicted in D). ( I ) Fluorescence intensity profiles of representative sparklets recorded in 20 mM external Ca 2+ and treated with Bay K8644 in a control cell (upper panel, ROI depicted in B) or in a cell additionally expressing Kv2.1 (lower panel, ROI depicted in F). ( J ) Summary data of sparklet site n P s measured from n = 6 cells expressing Cav1.2 alone and n = 7 cells coexpressing Cav1.2 and Kv2.1. Each point represents a single sparklet site (vehicle: *p=0.0367; Bay K: p=0.9224; two-tailed Mann-Whitney test). ( K ) Summary data of sparklet site nearest neighbor distance (NND) measured from n = 6 cells expressing Cav1.2 alone and n = 7 cells coexpressing Cav1.2 and Kv2.1. Each point represents a single sparklet site (vehicle: *p=0.0214; Bay K: p

    Journal: eLife

    Article Title: Kv2.1 mediates spatial and functional coupling of L-type calcium channels and ryanodine receptors in mammalian neurons

    doi: 10.7554/eLife.49953

    Figure Lengend Snippet: Kv2.1 P404W increases Cav1.2 single channel activity. ( A, B ) Maximum z -projections of TIRF images of Cav1.2-mediated Ca 2+ sparklets in a representative HEK293T cell transfected with Cav1.2 and auxiliary subunits and loaded with Fluo-5F via the patch pipette, before ( A ) and after ( B ) treatment with 500 nM Bay K8644 (scale bar: 5 µm). ( C ) Maximum z -projection of TIRF images of DsRed-Kv2.1 in a representative HEK293T cell cotransfected with Cav1.2 and auxiliary subunits (scale bar: 5 µm). ( D, F ) Maximum z -projections of TIRF images of sparklets in a representative HEK293T cell transfected with DsRed-Kv2.1, Cav1.2, and auxiliary subunits and loaded with Fluo-5F via the patch pipette, before ( D ) and after ( F ) treatment with 500 nM Bay K8644. ( E, G ) Merged images of panels C and D ( E ), or panels C and F ( G ). ( H ) Fluorescence intensity profiles of representative sparklets recorded in 20 mM external Ca 2+ in a control cell (upper panel, ROI depicted in A) or in a cell additionally expressing Kv2.1 (lower panel, ROI depicted in D). ( I ) Fluorescence intensity profiles of representative sparklets recorded in 20 mM external Ca 2+ and treated with Bay K8644 in a control cell (upper panel, ROI depicted in B) or in a cell additionally expressing Kv2.1 (lower panel, ROI depicted in F). ( J ) Summary data of sparklet site n P s measured from n = 6 cells expressing Cav1.2 alone and n = 7 cells coexpressing Cav1.2 and Kv2.1. Each point represents a single sparklet site (vehicle: *p=0.0367; Bay K: p=0.9224; two-tailed Mann-Whitney test). ( K ) Summary data of sparklet site nearest neighbor distance (NND) measured from n = 6 cells expressing Cav1.2 alone and n = 7 cells coexpressing Cav1.2 and Kv2.1. Each point represents a single sparklet site (vehicle: *p=0.0214; Bay K: p

    Article Snippet: HEK293T cell culture HEK293T cells were obtained from ATCC (Cat# CRL-3216).

    Techniques: Activity Assay, Transfection, Transferring, Fluorescence, Expressing, Two Tailed Test, MANN-WHITNEY

    Kv2.1 increases the frequency of Cav1.3s and RyR-mediated sparks reconstituted in HEK293T cells. ( A ) TIRF image of HEK293T cell expressing the short isoform of Cav1.3 (Cav1.3s), RyR2, STAC1, and the LTCC auxiliary subunits β3 and α2δ1, and loaded with Cal-590 AM (scale bar: 10 µm and holds for panels A-C). ( B ) TIRF images of HEK293T cells additionally coexpressing STAC1. ( C ) TIRF images of HEK293T cells additionally coexpressing STAC1 and Kv2.1. ( D–F ) Kymographs showing the localized Ca 2+ release events detected in the ROI on the cell in panels A-C, respectively. ( G–H ) Data from cells expressing Cav1.3, RyR2 and auxiliary subunits without (white bars) or with coexpression of STAC1 (blue bars) or STAC1 + Kv2.1 (red bars). ( G ) Expression of STAC1 reduces the duration of Cav1.3s- and RyR2-mediated CICR events reconstituted in HEK293T cells. (*p=0.0339; **p=0.0026; ANOVA followed by Dunnett’s test). ( H ) Summary data of the amplitude, frequency, and spatial spread (width) of all sparks recorded. Each point corresponds to a single cell (**p=0.0081; ***p=0.0001; one-way ANOVA followed by Dunnett’s post-hoc test vs. control).

    Journal: eLife

    Article Title: Kv2.1 mediates spatial and functional coupling of L-type calcium channels and ryanodine receptors in mammalian neurons

    doi: 10.7554/eLife.49953

    Figure Lengend Snippet: Kv2.1 increases the frequency of Cav1.3s and RyR-mediated sparks reconstituted in HEK293T cells. ( A ) TIRF image of HEK293T cell expressing the short isoform of Cav1.3 (Cav1.3s), RyR2, STAC1, and the LTCC auxiliary subunits β3 and α2δ1, and loaded with Cal-590 AM (scale bar: 10 µm and holds for panels A-C). ( B ) TIRF images of HEK293T cells additionally coexpressing STAC1. ( C ) TIRF images of HEK293T cells additionally coexpressing STAC1 and Kv2.1. ( D–F ) Kymographs showing the localized Ca 2+ release events detected in the ROI on the cell in panels A-C, respectively. ( G–H ) Data from cells expressing Cav1.3, RyR2 and auxiliary subunits without (white bars) or with coexpression of STAC1 (blue bars) or STAC1 + Kv2.1 (red bars). ( G ) Expression of STAC1 reduces the duration of Cav1.3s- and RyR2-mediated CICR events reconstituted in HEK293T cells. (*p=0.0339; **p=0.0026; ANOVA followed by Dunnett’s test). ( H ) Summary data of the amplitude, frequency, and spatial spread (width) of all sparks recorded. Each point corresponds to a single cell (**p=0.0081; ***p=0.0001; one-way ANOVA followed by Dunnett’s post-hoc test vs. control).

    Article Snippet: HEK293T cell culture HEK293T cells were obtained from ATCC (Cat# CRL-3216).

    Techniques: Expressing

    Cav1.2 channel activity is increased in cells coexpressing STAC1 upon coexpression with Kv2.1 P404W . ( A-D ) Data recorded from HEK293T cells transfected with Cav1.2-GFP and auxiliary subunits Cavβ3, Cavα 2 δ 1 , and STAC1, without (+pCDNA3, in black) or with Kv2.1 P404W (in red). ( A ) Representative Ca 2+ current traces at +10 mV. ( B ) Normalized I-V relationship of whole-cell I Ca recorded from n = 8 (Cav1.2 + pcDNA3) and n = 9 (Cav1.2 + Kv2.1 P404W ) cells. ( C ) Voltage-dependence of whole-cell Cav1.2 conductance G / G max . For the conductance-voltage relationships, the half-maximal activation voltage V 1/2 =1.6±2.0 [pcDNA3] vs. −9.5 ± 2.9 [+Kv2.1 P404W ] mV, p=0.0166; slope factor k = 8.8 ± 1.2 [pcDNA3] vs. 6.1 ± 0.6 [+Kv2.1 P404W ], p=0.0490; Student’s t -test). ( D ) Comparison of r 250 values (fraction of peak current remaining after 250 ms of depolarization) at the indicated potentials.

    Journal: eLife

    Article Title: Kv2.1 mediates spatial and functional coupling of L-type calcium channels and ryanodine receptors in mammalian neurons

    doi: 10.7554/eLife.49953

    Figure Lengend Snippet: Cav1.2 channel activity is increased in cells coexpressing STAC1 upon coexpression with Kv2.1 P404W . ( A-D ) Data recorded from HEK293T cells transfected with Cav1.2-GFP and auxiliary subunits Cavβ3, Cavα 2 δ 1 , and STAC1, without (+pCDNA3, in black) or with Kv2.1 P404W (in red). ( A ) Representative Ca 2+ current traces at +10 mV. ( B ) Normalized I-V relationship of whole-cell I Ca recorded from n = 8 (Cav1.2 + pcDNA3) and n = 9 (Cav1.2 + Kv2.1 P404W ) cells. ( C ) Voltage-dependence of whole-cell Cav1.2 conductance G / G max . For the conductance-voltage relationships, the half-maximal activation voltage V 1/2 =1.6±2.0 [pcDNA3] vs. −9.5 ± 2.9 [+Kv2.1 P404W ] mV, p=0.0166; slope factor k = 8.8 ± 1.2 [pcDNA3] vs. 6.1 ± 0.6 [+Kv2.1 P404W ], p=0.0490; Student’s t -test). ( D ) Comparison of r 250 values (fraction of peak current remaining after 250 ms of depolarization) at the indicated potentials.

    Article Snippet: HEK293T cell culture HEK293T cells were obtained from ATCC (Cat# CRL-3216).

    Techniques: Activity Assay, Transfection, Activation Assay, Mass Spectrometry

    Kv2.1 increases clustering of surface Cav1.2 channels. ( A ) Optical sections of HEK293T cells transfected with and immunolabeled for cell surface Cav1.2-HA and Kv1.5 (upper panels) or Kv2.1 (lower panels) (scale bar: 10 μm and holds for all panels). ( B ) Line scan of pixel intensities from the ROI depicted in the merged image of the upper images in panel A. ( C ) Line scan of pixel intensities from the ROI depicted in the merged image of the lower images in panel A. ( D ) Summary graph of CV values of Cav1.2-HA fluorescent signal intensity measured from HEK293T cells cotransfected with Kv1.5 or Kv2.1. Each point corresponds to a single cell (*p=0.0348 versus Kv1.5, two-tailed t -test). ( E ) Pearson’s correlation coefficient (PCC) values of cell surface Cav1.2 and Kv1.5 or Kv2.1 immunolabeling (each point represents a single cell; ***p=0.0002; Student’s t -test).

    Journal: eLife

    Article Title: Kv2.1 mediates spatial and functional coupling of L-type calcium channels and ryanodine receptors in mammalian neurons

    doi: 10.7554/eLife.49953

    Figure Lengend Snippet: Kv2.1 increases clustering of surface Cav1.2 channels. ( A ) Optical sections of HEK293T cells transfected with and immunolabeled for cell surface Cav1.2-HA and Kv1.5 (upper panels) or Kv2.1 (lower panels) (scale bar: 10 μm and holds for all panels). ( B ) Line scan of pixel intensities from the ROI depicted in the merged image of the upper images in panel A. ( C ) Line scan of pixel intensities from the ROI depicted in the merged image of the lower images in panel A. ( D ) Summary graph of CV values of Cav1.2-HA fluorescent signal intensity measured from HEK293T cells cotransfected with Kv1.5 or Kv2.1. Each point corresponds to a single cell (*p=0.0348 versus Kv1.5, two-tailed t -test). ( E ) Pearson’s correlation coefficient (PCC) values of cell surface Cav1.2 and Kv1.5 or Kv2.1 immunolabeling (each point represents a single cell; ***p=0.0002; Student’s t -test).

    Article Snippet: HEK293T cell culture HEK293T cells were obtained from ATCC (Cat# CRL-3216).

    Techniques: Transfection, Immunolabeling, Two Tailed Test, Periodic Counter-current Chromatography

    GCaMP3-Kv2.1 WT and GCaMP3-Kv2.1 P404W show comparable PM expression in HEK293T cells. ( A ) TIRF images of a HEK293T cell transfected with GCaMP3-Kv2.1 WT . GxTX-633 labeling is shown in red and GCaMP3 fluorescence is shown in green in the merge panel (scale bar: 10 µm). ( B ) As in A but obtained from cells expressing GCaMP3-Kv2.1 P404W . ( C ) Line scan of pixel intensities from the ROI depicted in the merged image in panel A. ( D ) Line scan of pixel intensities from the ROI depicted in the merged image in panel B. ( E ) Mean fluorescence intensity of GxTX-633 surface labeling obtained from cells expressing GCaMP3-Kv2.1 WT or -Kv2.1 P404W . Each point represents a single cell (AU: arbitrary units; p=0.2353, Mann-Whitney test). ( F ) Mean fluorescence intensity of GCaMP3 obtained from cells expressing GCaMP3-Kv2.1 WT or -Kv2.1 P404W . Each point represents a single cell (p=0.0575, Mann-Whitney test).

    Journal: eLife

    Article Title: Kv2.1 mediates spatial and functional coupling of L-type calcium channels and ryanodine receptors in mammalian neurons

    doi: 10.7554/eLife.49953

    Figure Lengend Snippet: GCaMP3-Kv2.1 WT and GCaMP3-Kv2.1 P404W show comparable PM expression in HEK293T cells. ( A ) TIRF images of a HEK293T cell transfected with GCaMP3-Kv2.1 WT . GxTX-633 labeling is shown in red and GCaMP3 fluorescence is shown in green in the merge panel (scale bar: 10 µm). ( B ) As in A but obtained from cells expressing GCaMP3-Kv2.1 P404W . ( C ) Line scan of pixel intensities from the ROI depicted in the merged image in panel A. ( D ) Line scan of pixel intensities from the ROI depicted in the merged image in panel B. ( E ) Mean fluorescence intensity of GxTX-633 surface labeling obtained from cells expressing GCaMP3-Kv2.1 WT or -Kv2.1 P404W . Each point represents a single cell (AU: arbitrary units; p=0.2353, Mann-Whitney test). ( F ) Mean fluorescence intensity of GCaMP3 obtained from cells expressing GCaMP3-Kv2.1 WT or -Kv2.1 P404W . Each point represents a single cell (p=0.0575, Mann-Whitney test).

    Article Snippet: HEK293T cell culture HEK293T cells were obtained from ATCC (Cat# CRL-3216).

    Techniques: Expressing, Transfection, Labeling, Fluorescence, MANN-WHITNEY

    Cav1.3s is recruited to Kv2-induced EPJs. ( A ) TIRF images of a HEK293T cell cotransfected with the short isoform of Cav1.3 (GFP-Cav1.3 (green), BFP-SEC61β (blue) and auxiliary subunits Cavβ3 and Cavα2δ (not shown). Scalebar is 10 μm and holds for all large panels in figure. Pseudocolored intensity profiles of GFP-Cav1.3 and BFP-SEC61β, from the boxed area in the merged image, are shown to the right of merged image. (scale bar: 2.5 μm and holds for all pseudocolored intensity profiles in figure). ( B ) TIRF images of HEK293T cells cotransfected with DsRed-Kv2.1 (red), GFP-Cav1.3 (green), BFP-SEC61β (blue) and auxiliary subunits Cavβ3 and Cavα2δ (not shown). Pseudocolored intensity profiles of DsRed-Kv2.1, GFP-Cav1.3 and BFP-SEC61β, from the boxed area in the merged image, are shown to the right of merged image. ( C ) TIRF images of a HEK293T cell cotransfected with DsRed-Kv2.2 (red), GFP-Cav1.3 (green), BFP-SEC61β (blue) and auxiliary subunits Cavβ3 and Cavα2δ (not shown). Pseudocolored intensity profiles of DsRed-Kv2.2, GFP-Cav1.3 and BFP-SEC61β from the boxed area in the merged image, are shown to the right of merged image. ( D ) Optical sections of HEK293T cells transfected with and immunolabeled for Cav3.1 alone (upper panel) or with Kv2.1 (lower panels) (scale bar: 10 μm and holds for all panels). ( E ) Pearson’s correlation coefficient (PCC) values of Cav3.1 and Kv2.1 immunolabeling or Cav1.3-GFP and DsRed-Kv2.1 fluorescence (each point represents a single cell; ****p

    Journal: eLife

    Article Title: Kv2.1 mediates spatial and functional coupling of L-type calcium channels and ryanodine receptors in mammalian neurons

    doi: 10.7554/eLife.49953

    Figure Lengend Snippet: Cav1.3s is recruited to Kv2-induced EPJs. ( A ) TIRF images of a HEK293T cell cotransfected with the short isoform of Cav1.3 (GFP-Cav1.3 (green), BFP-SEC61β (blue) and auxiliary subunits Cavβ3 and Cavα2δ (not shown). Scalebar is 10 μm and holds for all large panels in figure. Pseudocolored intensity profiles of GFP-Cav1.3 and BFP-SEC61β, from the boxed area in the merged image, are shown to the right of merged image. (scale bar: 2.5 μm and holds for all pseudocolored intensity profiles in figure). ( B ) TIRF images of HEK293T cells cotransfected with DsRed-Kv2.1 (red), GFP-Cav1.3 (green), BFP-SEC61β (blue) and auxiliary subunits Cavβ3 and Cavα2δ (not shown). Pseudocolored intensity profiles of DsRed-Kv2.1, GFP-Cav1.3 and BFP-SEC61β, from the boxed area in the merged image, are shown to the right of merged image. ( C ) TIRF images of a HEK293T cell cotransfected with DsRed-Kv2.2 (red), GFP-Cav1.3 (green), BFP-SEC61β (blue) and auxiliary subunits Cavβ3 and Cavα2δ (not shown). Pseudocolored intensity profiles of DsRed-Kv2.2, GFP-Cav1.3 and BFP-SEC61β from the boxed area in the merged image, are shown to the right of merged image. ( D ) Optical sections of HEK293T cells transfected with and immunolabeled for Cav3.1 alone (upper panel) or with Kv2.1 (lower panels) (scale bar: 10 μm and holds for all panels). ( E ) Pearson’s correlation coefficient (PCC) values of Cav3.1 and Kv2.1 immunolabeling or Cav1.3-GFP and DsRed-Kv2.1 fluorescence (each point represents a single cell; ****p

    Article Snippet: HEK293T cell culture HEK293T cells were obtained from ATCC (Cat# CRL-3216).

    Techniques: Transfection, Immunolabeling, Periodic Counter-current Chromatography, Fluorescence

    Ubiquitination of Stat3 at K180 by Hectd3 is essential for RORγt + IL-17A hi Th17 cell generation. a HEK293T cells were co-transfected with HA-Ub, Flag-Stat3, and Xpress-Hectd3. Extracts were immunoprecipitated with anti-Flag antibodies, followed by trypsin digestion and tandem mass spectrometry, as described in Material and methods. A fragmentation spectrum of ubiquitinated TLkSQGDMQDLNGNNQSVTR peptide (ubiquitinated K180 residue) of Stat3. Parent ion corresponding to TLkSQGDMQDLNGNNQSVTR peptide mass (774.0342, z = +3, retention time t = 38.1356 min) has been subjected to higher-energy collisional dissociation in mass spectrometer. The detected b- and y-fragment ion series have been annotated and mass difference corresponding to GG tag (114.04293 Da) has been assigned to a K3 residue as indicated (K180-GG) by the difference between the y17 and y18 fragment ion masses. b Representative immunoblot of HA, Flag, Xpress, and GAPDH following Flag immunoprecipitation of protein extracts from HEK293T cells co-transfected as indicated with HA-Ub, Flag-Stat3, Flag-Stat3 K180R, and Xpress-Hectd3; data are representative of three independent experiments. c Flow cytometry analysis of intracellular IL-17A, and intranuclear RORγt and Stat3 in GFP + Stat3 −/− CD4 + T cells transduced with MSCV-Stat3 or MSCV-Stat3 K180R retroviruses and in vitro polarized under Th17 conditions. Representative of three independent experiments. Gating strategy was first on GFP + T cells. d MFI of pStat3 Y705 and Stat3 in GFP + Stat3 −/− CD4 + T cells transduced with indicated retroviruses normalized to those transduced with MSCV empty vector and in vitro polarized under Th17 conditions. Data ( n = 6) are mean of three (pStat3 Y705) and two (Stat3) independent experiments and are presented as mean ± SEM; p value was obtained from Student’s t test. Source data are provided as a Source Data file

    Journal: Nature Communications

    Article Title: Hectd3 promotes pathogenic Th17 lineage through Stat3 activation and Malt1 signaling in neuroinflammation

    doi: 10.1038/s41467-019-08605-3

    Figure Lengend Snippet: Ubiquitination of Stat3 at K180 by Hectd3 is essential for RORγt + IL-17A hi Th17 cell generation. a HEK293T cells were co-transfected with HA-Ub, Flag-Stat3, and Xpress-Hectd3. Extracts were immunoprecipitated with anti-Flag antibodies, followed by trypsin digestion and tandem mass spectrometry, as described in Material and methods. A fragmentation spectrum of ubiquitinated TLkSQGDMQDLNGNNQSVTR peptide (ubiquitinated K180 residue) of Stat3. Parent ion corresponding to TLkSQGDMQDLNGNNQSVTR peptide mass (774.0342, z = +3, retention time t = 38.1356 min) has been subjected to higher-energy collisional dissociation in mass spectrometer. The detected b- and y-fragment ion series have been annotated and mass difference corresponding to GG tag (114.04293 Da) has been assigned to a K3 residue as indicated (K180-GG) by the difference between the y17 and y18 fragment ion masses. b Representative immunoblot of HA, Flag, Xpress, and GAPDH following Flag immunoprecipitation of protein extracts from HEK293T cells co-transfected as indicated with HA-Ub, Flag-Stat3, Flag-Stat3 K180R, and Xpress-Hectd3; data are representative of three independent experiments. c Flow cytometry analysis of intracellular IL-17A, and intranuclear RORγt and Stat3 in GFP + Stat3 −/− CD4 + T cells transduced with MSCV-Stat3 or MSCV-Stat3 K180R retroviruses and in vitro polarized under Th17 conditions. Representative of three independent experiments. Gating strategy was first on GFP + T cells. d MFI of pStat3 Y705 and Stat3 in GFP + Stat3 −/− CD4 + T cells transduced with indicated retroviruses normalized to those transduced with MSCV empty vector and in vitro polarized under Th17 conditions. Data ( n = 6) are mean of three (pStat3 Y705) and two (Stat3) independent experiments and are presented as mean ± SEM; p value was obtained from Student’s t test. Source data are provided as a Source Data file

    Article Snippet: Cells HEK293T cells (Cat#CRL-3216, ATCC, VA, USA), were grown in DMEM supplemented with 1% l -glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 1% penicillin/streptomycin, 0.01 M HEPES, 55 μM 2-mercaptoethanol, and 10% FBS (10% DMEM).

    Techniques: Transfection, Immunoprecipitation, Mass Spectrometry, Flow Cytometry, Cytometry, Transduction, In Vitro, Plasmid Preparation

    Stat3 linker region and Hectd3 DOC domain mediate the interaction between Stat3 and Hectd3. a Immunoblot of Xpress and Flag following Flag or Xpress immunoprecipitation of protein extracts from HEK293T cells co-transfected as indicated with HA-Ub, Flag-Stat3, and Xpress-Hectd3; data are representative of three independent experiments. b Stat3 and Hectd3 domains representation. c Immunoblot of Xpress, Flag, and GAPDH in the Flag-immunoprecipitate or input of protein extracts from HEK293T cells co-transfected as indicated with Flag-Stat3, Flag-Stat3 ΔND, Flag-Stat3 ΔCCD, Flag-Stat3 ΔDBD, Flag-Stat3 ΔLD, Flag-Stat3 ΔSH2D, or Flag-Stat3 ΔTAD, and Xpress-Hectd3; data are representative of two independent experiments. * denotes non-specific bands. d Immunoblot of Flag, Xpress, and GAPDH in the Xpress-immunoprecipitate or input of protein extracts from HEK293T cells co-transfected as indicated, with Xpress-Hectd3, Xpress-Hectd3 ΔND, Xpress-Hectd3 ΔDOC, Xpress-Hectd3 ΔLD, or Xpress-Hectd3 ΔHECT, and Flag-Stat3; data are representative of two independent experiments. Source data are provided as a Source Data file

    Journal: Nature Communications

    Article Title: Hectd3 promotes pathogenic Th17 lineage through Stat3 activation and Malt1 signaling in neuroinflammation

    doi: 10.1038/s41467-019-08605-3

    Figure Lengend Snippet: Stat3 linker region and Hectd3 DOC domain mediate the interaction between Stat3 and Hectd3. a Immunoblot of Xpress and Flag following Flag or Xpress immunoprecipitation of protein extracts from HEK293T cells co-transfected as indicated with HA-Ub, Flag-Stat3, and Xpress-Hectd3; data are representative of three independent experiments. b Stat3 and Hectd3 domains representation. c Immunoblot of Xpress, Flag, and GAPDH in the Flag-immunoprecipitate or input of protein extracts from HEK293T cells co-transfected as indicated with Flag-Stat3, Flag-Stat3 ΔND, Flag-Stat3 ΔCCD, Flag-Stat3 ΔDBD, Flag-Stat3 ΔLD, Flag-Stat3 ΔSH2D, or Flag-Stat3 ΔTAD, and Xpress-Hectd3; data are representative of two independent experiments. * denotes non-specific bands. d Immunoblot of Flag, Xpress, and GAPDH in the Xpress-immunoprecipitate or input of protein extracts from HEK293T cells co-transfected as indicated, with Xpress-Hectd3, Xpress-Hectd3 ΔND, Xpress-Hectd3 ΔDOC, Xpress-Hectd3 ΔLD, or Xpress-Hectd3 ΔHECT, and Flag-Stat3; data are representative of two independent experiments. Source data are provided as a Source Data file

    Article Snippet: Cells HEK293T cells (Cat#CRL-3216, ATCC, VA, USA), were grown in DMEM supplemented with 1% l -glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 1% penicillin/streptomycin, 0.01 M HEPES, 55 μM 2-mercaptoethanol, and 10% FBS (10% DMEM).

    Techniques: Immunoprecipitation, Transfection

    Hectd3 interacts and promotes non-degradative K27- and K29-linked polyubiquitination on Malt1 and NF-κB activation in CD4 + T cells. a Immunoblot of Hectd3, Malt1, and GAPDH following Malt1 or IgG immunoprecipitation of protein extracts from draining lymph node CD4 + T cells of WT mice 13 days following EAE induction. b Immunoblot of Malt1 following polyubiquitinated protein enrichment of extract from draining lymph node CD4 + T cells of wild-type Hectd3 −/− or (WT) mice 13 days following EAE induction. About 1.5× the amount of total protein from Hectd3 −/− CD4 + T cells, compared to WT CD4 + T cells, was used to normalize for the reduction in Malt1 protein level in Hectd3 −/− CD4 + T cells. At least 700 μg of total protein from mouse primary CD4 + T cells were enriched for ubiquitinated protein with Ubiquitinated Protein Enrichment Kit or Anti-Ub TUBE2, Agarose following manufacturer’s protocol. c , d Percentage of p65 or RelB nuclear translocation using p65/DAPI similarity analysis from ImageStream, in CD4 + T cells isolated from draining lymph nodes of Hectd3 −/− and WT mice, 13 days following EAE induction. e , f ImageStream fluorescence imaging of PMA/ionomycin-induced p65 nuclear translocation ( e ) or RelB ( f ) in CD4 + T cells isolated from draining lymph nodes of Hectd3 −/− or WT mice, 13 days following EAE induction. g Immunoblot of HA, Flag, and Xpress following two-step Flag immunoprecipitation of protein extracts from HEK293T cells co-transfected with the indicated HA-Ub K only mutants, Flag-Malt1A, and Xpress-Hectd3. HA-Ub K only mutant denote that the only lysine in the HA-tagged ubiquitin is at the indicated residue, and all other lysine residues are mutated to arginine. HA-Ub K0 mutant indicate that all seven lysine residues are mutated to arginine. a – g Immunoblots, ImageStream similarity analyses, and ImageStream fluorescence imaging are representative of at least three independent experiments. Source data are provided as a Source Data file

    Journal: Nature Communications

    Article Title: Hectd3 promotes pathogenic Th17 lineage through Stat3 activation and Malt1 signaling in neuroinflammation

    doi: 10.1038/s41467-019-08605-3

    Figure Lengend Snippet: Hectd3 interacts and promotes non-degradative K27- and K29-linked polyubiquitination on Malt1 and NF-κB activation in CD4 + T cells. a Immunoblot of Hectd3, Malt1, and GAPDH following Malt1 or IgG immunoprecipitation of protein extracts from draining lymph node CD4 + T cells of WT mice 13 days following EAE induction. b Immunoblot of Malt1 following polyubiquitinated protein enrichment of extract from draining lymph node CD4 + T cells of wild-type Hectd3 −/− or (WT) mice 13 days following EAE induction. About 1.5× the amount of total protein from Hectd3 −/− CD4 + T cells, compared to WT CD4 + T cells, was used to normalize for the reduction in Malt1 protein level in Hectd3 −/− CD4 + T cells. At least 700 μg of total protein from mouse primary CD4 + T cells were enriched for ubiquitinated protein with Ubiquitinated Protein Enrichment Kit or Anti-Ub TUBE2, Agarose following manufacturer’s protocol. c , d Percentage of p65 or RelB nuclear translocation using p65/DAPI similarity analysis from ImageStream, in CD4 + T cells isolated from draining lymph nodes of Hectd3 −/− and WT mice, 13 days following EAE induction. e , f ImageStream fluorescence imaging of PMA/ionomycin-induced p65 nuclear translocation ( e ) or RelB ( f ) in CD4 + T cells isolated from draining lymph nodes of Hectd3 −/− or WT mice, 13 days following EAE induction. g Immunoblot of HA, Flag, and Xpress following two-step Flag immunoprecipitation of protein extracts from HEK293T cells co-transfected with the indicated HA-Ub K only mutants, Flag-Malt1A, and Xpress-Hectd3. HA-Ub K only mutant denote that the only lysine in the HA-tagged ubiquitin is at the indicated residue, and all other lysine residues are mutated to arginine. HA-Ub K0 mutant indicate that all seven lysine residues are mutated to arginine. a – g Immunoblots, ImageStream similarity analyses, and ImageStream fluorescence imaging are representative of at least three independent experiments. Source data are provided as a Source Data file

    Article Snippet: Cells HEK293T cells (Cat#CRL-3216, ATCC, VA, USA), were grown in DMEM supplemented with 1% l -glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 1% penicillin/streptomycin, 0.01 M HEPES, 55 μM 2-mercaptoethanol, and 10% FBS (10% DMEM).

    Techniques: Activation Assay, Immunoprecipitation, Mouse Assay, Protein Enrichment, Translocation Assay, Isolation, Fluorescence, Imaging, Transfection, Mutagenesis, Western Blot

    Hectd3 promotes non-degradative K27-linked polyubiquitin chains on Stat3. a Immunoblot of HA, Flag, Xpress, and GAPDH following Flag immunoprecipitation of protein extracts from HEK293T cells co-transfected as indicated with HA-Ub, Flag-Stat3, and Xpress-Hectd3. b Immunoblot of HA and Flag following immunoprecipitation of protein extracts from HEK293T cells co-transfected as indicated with HA-Ub, Flag-Stat3, and Xpress-Hectd3 vectors and treated for 4 h with 20 μM MG132 prior to protein extraction. c Immunoblot of HA and Flag following two-step Flag immunoprecipitation of protein extracts from HEK293T cells co-transfected with the indicated HA-Ub K only mutants, Flag-Stat3, and Xpress-Hectd3. HA-Ub K only mutants denote that the only lysine in the HA-tagged ubiquitin is at the indicated residue, and all other lysine residues are mutated to arginine. HA-Ub K0 mutant has all seven lysine residues mutated to arginine. a – c Immunoblots are representative of three independent experiments. Source data are provided as a Source Data file

    Journal: Nature Communications

    Article Title: Hectd3 promotes pathogenic Th17 lineage through Stat3 activation and Malt1 signaling in neuroinflammation

    doi: 10.1038/s41467-019-08605-3

    Figure Lengend Snippet: Hectd3 promotes non-degradative K27-linked polyubiquitin chains on Stat3. a Immunoblot of HA, Flag, Xpress, and GAPDH following Flag immunoprecipitation of protein extracts from HEK293T cells co-transfected as indicated with HA-Ub, Flag-Stat3, and Xpress-Hectd3. b Immunoblot of HA and Flag following immunoprecipitation of protein extracts from HEK293T cells co-transfected as indicated with HA-Ub, Flag-Stat3, and Xpress-Hectd3 vectors and treated for 4 h with 20 μM MG132 prior to protein extraction. c Immunoblot of HA and Flag following two-step Flag immunoprecipitation of protein extracts from HEK293T cells co-transfected with the indicated HA-Ub K only mutants, Flag-Stat3, and Xpress-Hectd3. HA-Ub K only mutants denote that the only lysine in the HA-tagged ubiquitin is at the indicated residue, and all other lysine residues are mutated to arginine. HA-Ub K0 mutant has all seven lysine residues mutated to arginine. a – c Immunoblots are representative of three independent experiments. Source data are provided as a Source Data file

    Article Snippet: Cells HEK293T cells (Cat#CRL-3216, ATCC, VA, USA), were grown in DMEM supplemented with 1% l -glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 1% penicillin/streptomycin, 0.01 M HEPES, 55 μM 2-mercaptoethanol, and 10% FBS (10% DMEM).

    Techniques: Immunoprecipitation, Transfection, Protein Extraction, Mutagenesis, Western Blot

    Ubiquitination of Malt1A K648 by Hectd3 is essential for RORγt + IL-17A hi Th17 cell generation. a HEK293T cells were co-transfected with HA-Ub, Flag-Malt1A, and Xpress-Hectd3. Extracts were immunoprecipitated with anti-Flag antibodies, followed by trypsin digestion and tandem mass spectrometry, as described in Material and methods. A fragmentation spectrum of ubiquitinated DANKGTPEETGSYLVSK peptide (ubiquitinated K648 residue) of Malt1A. Parent ion corresponding to DANkGTPEETGSYLVSK peptide mass has been subjected to higher-energy collisional dissociation in mass spectrometer. The detected b- and y-fragment ion series have been annotated. b Representative immunoblot of Flag and ubiquitin following polyubiquitinated protein enrichment of extracts using TUBE2 from CD90.1+ sorted EL4 cells transduced with MSCV-CD90.1-Flag-Malt1A or MSCV-CD90.1-Flag-Malt1A K648R retrovirus. c Flow cytometry analysis of intracellular IL-17A and intranuclear RORγt in CD90.1 + Malt1 −/− CD4 + T cells transduced with indicated retrovirus and in vitro polarized under Th17 conditions. Representative of three independent experiments. Gating strategy was first on CD90.1 + T cells. d MFI of Malt1A in CD90.1 + Malt1 −/− CD4 + T cells transduced with indicated retroviruses and in vitro polarized under Th17 condition. Data ( n = 6) are mean of three independent experiments and are presented as mean ± SEM; p value was obtained from Student’s t test. Source data are provided as a Source Data file

    Journal: Nature Communications

    Article Title: Hectd3 promotes pathogenic Th17 lineage through Stat3 activation and Malt1 signaling in neuroinflammation

    doi: 10.1038/s41467-019-08605-3

    Figure Lengend Snippet: Ubiquitination of Malt1A K648 by Hectd3 is essential for RORγt + IL-17A hi Th17 cell generation. a HEK293T cells were co-transfected with HA-Ub, Flag-Malt1A, and Xpress-Hectd3. Extracts were immunoprecipitated with anti-Flag antibodies, followed by trypsin digestion and tandem mass spectrometry, as described in Material and methods. A fragmentation spectrum of ubiquitinated DANKGTPEETGSYLVSK peptide (ubiquitinated K648 residue) of Malt1A. Parent ion corresponding to DANkGTPEETGSYLVSK peptide mass has been subjected to higher-energy collisional dissociation in mass spectrometer. The detected b- and y-fragment ion series have been annotated. b Representative immunoblot of Flag and ubiquitin following polyubiquitinated protein enrichment of extracts using TUBE2 from CD90.1+ sorted EL4 cells transduced with MSCV-CD90.1-Flag-Malt1A or MSCV-CD90.1-Flag-Malt1A K648R retrovirus. c Flow cytometry analysis of intracellular IL-17A and intranuclear RORγt in CD90.1 + Malt1 −/− CD4 + T cells transduced with indicated retrovirus and in vitro polarized under Th17 conditions. Representative of three independent experiments. Gating strategy was first on CD90.1 + T cells. d MFI of Malt1A in CD90.1 + Malt1 −/− CD4 + T cells transduced with indicated retroviruses and in vitro polarized under Th17 condition. Data ( n = 6) are mean of three independent experiments and are presented as mean ± SEM; p value was obtained from Student’s t test. Source data are provided as a Source Data file

    Article Snippet: Cells HEK293T cells (Cat#CRL-3216, ATCC, VA, USA), were grown in DMEM supplemented with 1% l -glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 1% penicillin/streptomycin, 0.01 M HEPES, 55 μM 2-mercaptoethanol, and 10% FBS (10% DMEM).

    Techniques: Transfection, Immunoprecipitation, Mass Spectrometry, Protein Enrichment, Transduction, Flow Cytometry, Cytometry, In Vitro

    IL-1β induces the expression of miR-31, which affects E-selectin abundance a. The monolayer of HUVEC endothelial cells was treated with IL-1β (20ng/ml) or TNFα (10ng/ml) for indicated hours. Western blotting monitored the expression of E-selectin and Actin used as loading control. b. The miR-31 level was measured by quantitative reverse transcription-PCR and the snRNA U6 was used as the normalization control. c. Endothelial cells were transfected with 30nM of either anti-miR-31 (+) or corresponding inhibitor negative control (−) before the addition of IL-1β. The Western blots shown are representative of three independent experiments. The endogenous GAPDH was used as loading control. d. Top: The representation of miR-31 base-pairing with either E-selectin wild-type (wt) or mutated (mut) 3′ UTR sequence. The nucleotides in the gray box represent the seed region of miR-31, region important for target interaction. Bottom: E-selectin 3′UTR mediated reporter assays. Vector expressing luciferase reporter under the regulation of either wild-type (black bars) or mutated (white bars) E-selectin 3′UTR were transfected into HEK293T cells. 48 hours after transfection, relative luciferase activities were measured. Another set of reporters transfected cells were also transfected with 30nM of either anti-miR-31 or corresponding inhibitor negative control (ctl) to further test the contribution of miR-31 in E-selectin regulation. The error bars shown in panel b and d represent standard errors of three and six independent experiments, respectively. The significance was analyzed using a Student's t -test (* p

    Journal: Oncotarget

    Article Title: p38 and JNK pathways control E-selectin-dependent extravasation of colon cancer cells by modulating miR-31 transcription

    doi: 10.18632/oncotarget.13779

    Figure Lengend Snippet: IL-1β induces the expression of miR-31, which affects E-selectin abundance a. The monolayer of HUVEC endothelial cells was treated with IL-1β (20ng/ml) or TNFα (10ng/ml) for indicated hours. Western blotting monitored the expression of E-selectin and Actin used as loading control. b. The miR-31 level was measured by quantitative reverse transcription-PCR and the snRNA U6 was used as the normalization control. c. Endothelial cells were transfected with 30nM of either anti-miR-31 (+) or corresponding inhibitor negative control (−) before the addition of IL-1β. The Western blots shown are representative of three independent experiments. The endogenous GAPDH was used as loading control. d. Top: The representation of miR-31 base-pairing with either E-selectin wild-type (wt) or mutated (mut) 3′ UTR sequence. The nucleotides in the gray box represent the seed region of miR-31, region important for target interaction. Bottom: E-selectin 3′UTR mediated reporter assays. Vector expressing luciferase reporter under the regulation of either wild-type (black bars) or mutated (white bars) E-selectin 3′UTR were transfected into HEK293T cells. 48 hours after transfection, relative luciferase activities were measured. Another set of reporters transfected cells were also transfected with 30nM of either anti-miR-31 or corresponding inhibitor negative control (ctl) to further test the contribution of miR-31 in E-selectin regulation. The error bars shown in panel b and d represent standard errors of three and six independent experiments, respectively. The significance was analyzed using a Student's t -test (* p

    Article Snippet: HEK293T human embryonic kidney cells (ATCC) were cultivated in DMEM (Lonza, Allendale, NJ) supplemented with 10% FBS.

    Techniques: Expressing, Western Blot, Polymerase Chain Reaction, Transfection, Negative Control, Sequencing, Plasmid Preparation, Luciferase, CTL Assay

    CYP26A1 gene expression is sensitive to RA treatment in HNF4α-containing HepG2 cells. HepG2 or HEK293T cells were grown in 6-well plates and treated with either ethanol as the vehicle control or RA in triplicate for 4 h and then collected for

    Journal: Journal of cellular biochemistry

    Article Title: Hepatocyte Nuclear Factor 4α (HNF4α) in Coordination With Retinoic Acid Receptors Increases all-trans-Retinoic Acid-Dependent CYP26A1 Gene Expression in HepG2 Human Hepatocytes

    doi: 10.1002/jcb.24839

    Figure Lengend Snippet: CYP26A1 gene expression is sensitive to RA treatment in HNF4α-containing HepG2 cells. HepG2 or HEK293T cells were grown in 6-well plates and treated with either ethanol as the vehicle control or RA in triplicate for 4 h and then collected for

    Article Snippet: HepG2 cells, and human embryonic kidney HEK293T cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) and 0.5% penicillin-streptomycin at 37 °C in a 5% CO2 -air incubator.

    Techniques: Expressing

    HNF4α enhances CYP26A1 promoter activity in HepG2 cells but not in HEK293T cells treated with RA. Cells grown in 24-well plates were cotransfected in triplicate with p GL3-Basic- luc -hCYP26A1 promoter containing full-length human CYP26A1 promoter

    Journal: Journal of cellular biochemistry

    Article Title: Hepatocyte Nuclear Factor 4α (HNF4α) in Coordination With Retinoic Acid Receptors Increases all-trans-Retinoic Acid-Dependent CYP26A1 Gene Expression in HepG2 Human Hepatocytes

    doi: 10.1002/jcb.24839

    Figure Lengend Snippet: HNF4α enhances CYP26A1 promoter activity in HepG2 cells but not in HEK293T cells treated with RA. Cells grown in 24-well plates were cotransfected in triplicate with p GL3-Basic- luc -hCYP26A1 promoter containing full-length human CYP26A1 promoter

    Article Snippet: HepG2 cells, and human embryonic kidney HEK293T cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) and 0.5% penicillin-streptomycin at 37 °C in a 5% CO2 -air incubator.

    Techniques: Activity Assay

    LRRC25 Inhibits NF-κB Activation and Impairs the Inflammatory Response. ( A , B , D ) HEK293T WT or 293T LRRC25 KO cells were transfected with a NF-κB-luc reporter plasmid, a TLR4 plasmid (for LPS treatment), and an empty vector or LRRC25 construct and analyzed for NF-κB luciferase activity after treatment with 10 μg/ml LPS for 12 h ( A , D ) and 20 ng/ml TNF-α for 6 h ( B ). ( C ) Protein extracts of HEK293T WT and 293T LRRC25 KO cells, THP-1 WT or THP-1 LRRC25 KO cells were subjected to immunoblot with anti-LRRC25 antibody, with the sequence alignment of 293T LRRC25 KO with 293T WT , and THP-1 LRRC25 KO cells with WT. Unprocessed original scans of blots are shown in Supplementary Fig. S3 . ( E ) THP-1 WT or THP-1 LRRC25 KO cells were treated with LPS (200 ng/ml) for 0, 2, 4, 6 h. Total RNAs from the treated cells were harvested at the indicated time points and mRNA level of TNF- α, IL-1 β, and IL-6 were determined by real-time PCR analysis. ( F ) THP-1 WT or THP-1 LRRC25 KO cells were treated with or without TNF-α (20 ng/ml) for the indicated time. The mRNA levels of TNF- α, IL-1 β and IL-6 were detected by real-time PCR analysis. ( G ) THP-1 WT or THP-1 LRRC25 KO cells were treated with LPS or Pam3csk4 for 24 h. Cells supernatant were then collected to measure the IL-6 and TNF-α production by ELISA. Data in figure ( A – G ) are means ± SEM (n = 3) of three independent experiments (*p

    Journal: Scientific Reports

    Article Title: LRRC25 Functions as an Inhibitor of NF-κB Signaling Pathway by Promoting p65/RelA for Autophagic Degradation

    doi: 10.1038/s41598-017-12573-3

    Figure Lengend Snippet: LRRC25 Inhibits NF-κB Activation and Impairs the Inflammatory Response. ( A , B , D ) HEK293T WT or 293T LRRC25 KO cells were transfected with a NF-κB-luc reporter plasmid, a TLR4 plasmid (for LPS treatment), and an empty vector or LRRC25 construct and analyzed for NF-κB luciferase activity after treatment with 10 μg/ml LPS for 12 h ( A , D ) and 20 ng/ml TNF-α for 6 h ( B ). ( C ) Protein extracts of HEK293T WT and 293T LRRC25 KO cells, THP-1 WT or THP-1 LRRC25 KO cells were subjected to immunoblot with anti-LRRC25 antibody, with the sequence alignment of 293T LRRC25 KO with 293T WT , and THP-1 LRRC25 KO cells with WT. Unprocessed original scans of blots are shown in Supplementary Fig. S3 . ( E ) THP-1 WT or THP-1 LRRC25 KO cells were treated with LPS (200 ng/ml) for 0, 2, 4, 6 h. Total RNAs from the treated cells were harvested at the indicated time points and mRNA level of TNF- α, IL-1 β, and IL-6 were determined by real-time PCR analysis. ( F ) THP-1 WT or THP-1 LRRC25 KO cells were treated with or without TNF-α (20 ng/ml) for the indicated time. The mRNA levels of TNF- α, IL-1 β and IL-6 were detected by real-time PCR analysis. ( G ) THP-1 WT or THP-1 LRRC25 KO cells were treated with LPS or Pam3csk4 for 24 h. Cells supernatant were then collected to measure the IL-6 and TNF-α production by ELISA. Data in figure ( A – G ) are means ± SEM (n = 3) of three independent experiments (*p

    Article Snippet: Cell culture and transfection THP-1 cells, HeLa cells, and Human embryonic kidney 293T (HEK293T) cells were purchased from American Type Culture Collection.

    Techniques: Activation Assay, Transfection, Plasmid Preparation, Construct, Luciferase, Activity Assay, Sequencing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    LRRC25 Inhibits NF-κB Activation at p65/RelA Level. ( A ) HEK293T cells were transfected with a NF-κB-luc reporter and plasmids encoding MyD88, IRAK1, TRAF2, TRAF6, TAK1 + TAB1, IKKα, IKKβ or p65/RelA, along with empty vector (no wedge) and increasing amount (wedge) of expression vector for LRRC25 (0, 50, 100 ng) for 24 h and analyzed for NF-κB luciferase activity. ( B ) HEK293T WT and 293T LRRC25 KO cells were transfected with a NF-κB-luc reporter, plus plasmids of MyD88, IRAK1, TRAF2, TRAF6, TAK1 + TAB1, IKKα, IKKβ or p65/RelA for 24 h and analyzed for NF-κB luciferase activity. Data in (A-B) are means ± SEM of three independent experiments (*p

    Journal: Scientific Reports

    Article Title: LRRC25 Functions as an Inhibitor of NF-κB Signaling Pathway by Promoting p65/RelA for Autophagic Degradation

    doi: 10.1038/s41598-017-12573-3

    Figure Lengend Snippet: LRRC25 Inhibits NF-κB Activation at p65/RelA Level. ( A ) HEK293T cells were transfected with a NF-κB-luc reporter and plasmids encoding MyD88, IRAK1, TRAF2, TRAF6, TAK1 + TAB1, IKKα, IKKβ or p65/RelA, along with empty vector (no wedge) and increasing amount (wedge) of expression vector for LRRC25 (0, 50, 100 ng) for 24 h and analyzed for NF-κB luciferase activity. ( B ) HEK293T WT and 293T LRRC25 KO cells were transfected with a NF-κB-luc reporter, plus plasmids of MyD88, IRAK1, TRAF2, TRAF6, TAK1 + TAB1, IKKα, IKKβ or p65/RelA for 24 h and analyzed for NF-κB luciferase activity. Data in (A-B) are means ± SEM of three independent experiments (*p

    Article Snippet: Cell culture and transfection THP-1 cells, HeLa cells, and Human embryonic kidney 293T (HEK293T) cells were purchased from American Type Culture Collection.

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

    LRRC25 Interacts with p65/RelA. ( A ) Flag-p65/RelA and Myc-LRRC25 expression plasmids were transfected into HEK293T cells. Cells extracts were harvested 24 h after transfection and subjected to co-immunoprecipitation (Co-IP) and immunoblot (IB) analysis. ( B ) PBMCs were treated with 200 ng/ml LPS for 0, 60, and 120 min, and collected at the indicated time points. Cell extracts were harvested for Co-IP with anti-p65/RelA, followed by IB analysis with anti-LRRC25 antibody. Cell extracts without treatment were used as IgG control (Isotype ctrl). ( C ) Domain structures of p65/RelA and domain deletions. RHD, Rel homology domain; TAD, transactivation domain. ( D ) Flag-p65/RelA full length (FL) and deletion mutants were co-transfected with Myc-LRRC25 into HEK293T cells. Cell extracts were immunoprecipitation with anti-Flag and immunoblotted for Myc-LRRC25. ( E ) The structure of LRRC25 and its truncation mutants. ( F ) HEK293T cells were transfected with HA-p65/RelA and Flag-LRRC25 or its truncation mutants. Cells extracts were immunoprecipitation with anti-Flag and immunoblotted for HA-p65/RelA. Repeated experiment was shown in Supplementary Fig. S4 . ( G ) Immunoblot analysis of protein extracts of 293T cells transfected with empty vector or vector for Flag-LRRC25 or truncation mutants, along with expression plasmids of HA-p65/RelA. ( H ) HEK293T cells were transfected with a NF-κB-luc reporter plasmid, together with Flag-p65/RelA, an empty vector or LRRC25 (FL) or its truncation mutants. Cells were harvested 24 h after transfection and were subjected to luciferase activity analysis. Data are means ± SEM of three independent experiments (*p

    Journal: Scientific Reports

    Article Title: LRRC25 Functions as an Inhibitor of NF-κB Signaling Pathway by Promoting p65/RelA for Autophagic Degradation

    doi: 10.1038/s41598-017-12573-3

    Figure Lengend Snippet: LRRC25 Interacts with p65/RelA. ( A ) Flag-p65/RelA and Myc-LRRC25 expression plasmids were transfected into HEK293T cells. Cells extracts were harvested 24 h after transfection and subjected to co-immunoprecipitation (Co-IP) and immunoblot (IB) analysis. ( B ) PBMCs were treated with 200 ng/ml LPS for 0, 60, and 120 min, and collected at the indicated time points. Cell extracts were harvested for Co-IP with anti-p65/RelA, followed by IB analysis with anti-LRRC25 antibody. Cell extracts without treatment were used as IgG control (Isotype ctrl). ( C ) Domain structures of p65/RelA and domain deletions. RHD, Rel homology domain; TAD, transactivation domain. ( D ) Flag-p65/RelA full length (FL) and deletion mutants were co-transfected with Myc-LRRC25 into HEK293T cells. Cell extracts were immunoprecipitation with anti-Flag and immunoblotted for Myc-LRRC25. ( E ) The structure of LRRC25 and its truncation mutants. ( F ) HEK293T cells were transfected with HA-p65/RelA and Flag-LRRC25 or its truncation mutants. Cells extracts were immunoprecipitation with anti-Flag and immunoblotted for HA-p65/RelA. Repeated experiment was shown in Supplementary Fig. S4 . ( G ) Immunoblot analysis of protein extracts of 293T cells transfected with empty vector or vector for Flag-LRRC25 or truncation mutants, along with expression plasmids of HA-p65/RelA. ( H ) HEK293T cells were transfected with a NF-κB-luc reporter plasmid, together with Flag-p65/RelA, an empty vector or LRRC25 (FL) or its truncation mutants. Cells were harvested 24 h after transfection and were subjected to luciferase activity analysis. Data are means ± SEM of three independent experiments (*p

    Article Snippet: Cell culture and transfection THP-1 cells, HeLa cells, and Human embryonic kidney 293T (HEK293T) cells were purchased from American Type Culture Collection.

    Techniques: Expressing, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Plasmid Preparation, Luciferase, Activity Assay

    LRRC25 Promotes the Degradation of p65/RelA through Autophagy. ( A ) The expression plasmids of Flag-p65/RelA and HA-LRRC25 were transfected into HEK293T for 24 h. Cells were either untreated or pretreated for 6 h with the proteasome inhibitor MG132 (5 μM), and then cells lysates were subjected to immunoblot analysis. ( B ) HEK293T cells were transfected with the expression plasmids of Flag-p65/RelA and Myc-LRRC25 and either untreated or treated for 6 hrs with Bafilomycin A1 (Baf A1) (100 μM). Cells were harvested 24 h after transfection and analyzed by immunoblot. ( C ) 293T WT cells or ATG5 KO 293T cells were transfected with Myc-LRRC25, along with TNF-α treatment. Cells were harvested 24 h after transfection and analyzed by immunoblot with the indicated antibodies. (D) THP-1 cells were treated with or without LPS and cells extracts were harvested for Co-IP with anti-p65/RelA, followed by IB analysis with anti-LRRC25 or anti-p62 antibodies. Cell extracts without treatment were used as IgG control. ( E ) Flag-p65/RelA, HA-p62 and Myc-LRRC25 expression plasmids were transfected into HEK293T cells. Cells extracts were harvested 24 h after transfection and subjected to immunoprecipitation (IP) and immunoblot (IB) analysis. ( F ) 293T cells were transfected with HA-p62 or GFP-LRRC25 and RFP-p65 for 24 h, and then stained with anti-HA-tag DyLight 650 antibody. DAPI (blue) was used for nuclear staining, Scale bar: 10 μm. ( G ) WT and SQSTM1 KO cells were transfected with a NF-κB-luc reporter plasmid, together with Flag-p65/RelA, an empty vector or HA-LRRC25 and analyzed for NF-κB luciferase activity. Values are means ± SEM of three independent experiments (*p

    Journal: Scientific Reports

    Article Title: LRRC25 Functions as an Inhibitor of NF-κB Signaling Pathway by Promoting p65/RelA for Autophagic Degradation

    doi: 10.1038/s41598-017-12573-3

    Figure Lengend Snippet: LRRC25 Promotes the Degradation of p65/RelA through Autophagy. ( A ) The expression plasmids of Flag-p65/RelA and HA-LRRC25 were transfected into HEK293T for 24 h. Cells were either untreated or pretreated for 6 h with the proteasome inhibitor MG132 (5 μM), and then cells lysates were subjected to immunoblot analysis. ( B ) HEK293T cells were transfected with the expression plasmids of Flag-p65/RelA and Myc-LRRC25 and either untreated or treated for 6 hrs with Bafilomycin A1 (Baf A1) (100 μM). Cells were harvested 24 h after transfection and analyzed by immunoblot. ( C ) 293T WT cells or ATG5 KO 293T cells were transfected with Myc-LRRC25, along with TNF-α treatment. Cells were harvested 24 h after transfection and analyzed by immunoblot with the indicated antibodies. (D) THP-1 cells were treated with or without LPS and cells extracts were harvested for Co-IP with anti-p65/RelA, followed by IB analysis with anti-LRRC25 or anti-p62 antibodies. Cell extracts without treatment were used as IgG control. ( E ) Flag-p65/RelA, HA-p62 and Myc-LRRC25 expression plasmids were transfected into HEK293T cells. Cells extracts were harvested 24 h after transfection and subjected to immunoprecipitation (IP) and immunoblot (IB) analysis. ( F ) 293T cells were transfected with HA-p62 or GFP-LRRC25 and RFP-p65 for 24 h, and then stained with anti-HA-tag DyLight 650 antibody. DAPI (blue) was used for nuclear staining, Scale bar: 10 μm. ( G ) WT and SQSTM1 KO cells were transfected with a NF-κB-luc reporter plasmid, together with Flag-p65/RelA, an empty vector or HA-LRRC25 and analyzed for NF-κB luciferase activity. Values are means ± SEM of three independent experiments (*p

    Article Snippet: Cell culture and transfection THP-1 cells, HeLa cells, and Human embryonic kidney 293T (HEK293T) cells were purchased from American Type Culture Collection.

    Techniques: Expressing, Transfection, Co-Immunoprecipitation Assay, Immunoprecipitation, Staining, Plasmid Preparation, Luciferase, Activity Assay

    CRY1 accelerates ubiquitin-mediated FOXO1 degradation. ( a ) Mouse primary hepatocytes were adenovirally infected with Ad-MOCK or Ad-CRY1. The cells were treated with 20 μM MG132 or vehicle for 4 h. Total cell lysates were analysed by western blotting with indicated antibodies. ( b ) HEK293T cells were transfected with GFP-CRY1 and/or FOXO1-MYC expression vectors. Co-immunoprecipitation with an anti-MYC antibody and western blotting were performed with the indicated antibodies. IP, immunoprecipitation. ( c ) COS-1 cells were co-transfected with plasmids encoding FOXO1-MYC, GFP-CRY1, and Ubiquitin-HA. After transfection, the cells were treated with MG132 (20 μM) for 6 h and then the cell lysates were subjected to immunoprecipitation with an anti-MYC antibody followed by western blotting with indicated antibodies. IP, immunoprecipitation. ( d ) Mouse primary hepatocytes were infected with Ad-MOCK or Ad-CRY1. After infection, the cells were treated with MG132 (20 μM) for 4 h. Nuclear and cytosolic fractions were isolated and analysed by western blotting with indicated antibodies. ( e ) COS-1 cells were co-transfected with plasmids encoding nFOXO1-MYC, GFP-CRY1, and Ubiquitin-HA. After transfection, the cells were challenged with MG132 (20 μM) for 6 h. The cell lysates were subjected to immunoprecipitation with an anti-MYC antibody. IP, immunoprecipitation. See Supplementary Fig. 13 for original full immunoblot.

    Journal: Nature Communications

    Article Title: SREBP1c-CRY1 signalling represses hepatic glucose production by promoting FOXO1 degradation during refeeding

    doi: 10.1038/ncomms12180

    Figure Lengend Snippet: CRY1 accelerates ubiquitin-mediated FOXO1 degradation. ( a ) Mouse primary hepatocytes were adenovirally infected with Ad-MOCK or Ad-CRY1. The cells were treated with 20 μM MG132 or vehicle for 4 h. Total cell lysates were analysed by western blotting with indicated antibodies. ( b ) HEK293T cells were transfected with GFP-CRY1 and/or FOXO1-MYC expression vectors. Co-immunoprecipitation with an anti-MYC antibody and western blotting were performed with the indicated antibodies. IP, immunoprecipitation. ( c ) COS-1 cells were co-transfected with plasmids encoding FOXO1-MYC, GFP-CRY1, and Ubiquitin-HA. After transfection, the cells were treated with MG132 (20 μM) for 6 h and then the cell lysates were subjected to immunoprecipitation with an anti-MYC antibody followed by western blotting with indicated antibodies. IP, immunoprecipitation. ( d ) Mouse primary hepatocytes were infected with Ad-MOCK or Ad-CRY1. After infection, the cells were treated with MG132 (20 μM) for 4 h. Nuclear and cytosolic fractions were isolated and analysed by western blotting with indicated antibodies. ( e ) COS-1 cells were co-transfected with plasmids encoding nFOXO1-MYC, GFP-CRY1, and Ubiquitin-HA. After transfection, the cells were challenged with MG132 (20 μM) for 6 h. The cell lysates were subjected to immunoprecipitation with an anti-MYC antibody. IP, immunoprecipitation. See Supplementary Fig. 13 for original full immunoblot.

    Article Snippet: Transient transfection and luciferase assays HEK293T cells (ATCC, CRL3216) were transiently transfected with various DNA plasmids using the calcium-phosphate method described previously .

    Techniques: Infection, Western Blot, Transfection, Expressing, Immunoprecipitation, Isolation

    CRY1 is involved in MDM2-mediated FOXO1 ubiquitination. ( a ) Mouse primary hepatocytes were infected with Ad-MOCK or Ad-CRY1 and/or siCON or siMDM2 . Total cell lysates were analysed by western blotting with indicated antibodies. ( b ) HEK293T cells were transfected with FLAG-MDM2, nFOXO1-MYC, and GFP-CRY1 expression vectors. Total cell lysates were subjected to co-immunoprecipitation with an anti-MYC antibody followed by western blotting with indicated antibodies. IP, immunoprecipitation. ( c ) COS-1 cells were co-transfected with plasmids encoding nFOXO1-MYC, FLAG-MDM2, FLAG-CRY1, and Ubiquitin-HA. After transfection, the cells were challenged with MG132 (20 μM) for 6 h. Cell lysates were subjected to immunoprecipitation with an anti-MYC antibody. IP, immunoprecipitation ( d ) COS-1 cells were co-transfected with plasmids encoding nFOXO1-MYC, FLAG-MDM2, Ubiquitin-HA, and siCRY1 . Cells were treated with MG132 (20 μM) for 6 h. Cell lysates were subjected to immunoprecipitation with an anti-MYC antibody. IP, immunoprecipitation. See Supplementary Fig. 13 for original full immunoblot.

    Journal: Nature Communications

    Article Title: SREBP1c-CRY1 signalling represses hepatic glucose production by promoting FOXO1 degradation during refeeding

    doi: 10.1038/ncomms12180

    Figure Lengend Snippet: CRY1 is involved in MDM2-mediated FOXO1 ubiquitination. ( a ) Mouse primary hepatocytes were infected with Ad-MOCK or Ad-CRY1 and/or siCON or siMDM2 . Total cell lysates were analysed by western blotting with indicated antibodies. ( b ) HEK293T cells were transfected with FLAG-MDM2, nFOXO1-MYC, and GFP-CRY1 expression vectors. Total cell lysates were subjected to co-immunoprecipitation with an anti-MYC antibody followed by western blotting with indicated antibodies. IP, immunoprecipitation. ( c ) COS-1 cells were co-transfected with plasmids encoding nFOXO1-MYC, FLAG-MDM2, FLAG-CRY1, and Ubiquitin-HA. After transfection, the cells were challenged with MG132 (20 μM) for 6 h. Cell lysates were subjected to immunoprecipitation with an anti-MYC antibody. IP, immunoprecipitation ( d ) COS-1 cells were co-transfected with plasmids encoding nFOXO1-MYC, FLAG-MDM2, Ubiquitin-HA, and siCRY1 . Cells were treated with MG132 (20 μM) for 6 h. Cell lysates were subjected to immunoprecipitation with an anti-MYC antibody. IP, immunoprecipitation. See Supplementary Fig. 13 for original full immunoblot.

    Article Snippet: Transient transfection and luciferase assays HEK293T cells (ATCC, CRL3216) were transiently transfected with various DNA plasmids using the calcium-phosphate method described previously .

    Techniques: Infection, Western Blot, Transfection, Expressing, Immunoprecipitation

    Anti-C2Cat antibodies immunoprecipitate more cPKC upon activation with PMA. ( A ) HEK293T cells were transfected with either WTPKCβΙ or ΔNPSPKCβΙ and treated with 100 nM PMA for 15 minutes. Fixed cells were then incubated with anti-PKCβΙ V5 domain antibodies and subsequently with anti-rabbit antibodies labeled with Alexa 555. ( B ) To evaluate the amount of active (membrane bound) PKCβΙ in HEK293T transfected cells. Cells treated or non-treated with 100 nM PMA were fractionated and probed for PKCβΙ in the particulate fraction by Western blot with anti-PKCβΙ-V5. ( C ) Transfected HEK293T cells were treated with 100 nM PMA and control cells were immunoprecipated with anti-C2Cat antibodies and probed for PKCβΙ with anti-PKCβΙ-V5 (upper panel). Total lysates were probed with anti-PKCβΙ to evaluate transfection levels, and for α-tubulin to evaluate the total amount of protein loaded. A representative blot of n = 3 is shown for ( B , C ). Quantitative analysis of the average of three independent experiments is shown normalized to non-treated cells, which was set to 1. Statistical significance was determine by ANOVA-Bonferroni test where **p

    Journal: Scientific Reports

    Article Title: Rational design and validation of an anti-protein kinase C active-state specific antibody based on conformational changes

    doi: 10.1038/srep22114

    Figure Lengend Snippet: Anti-C2Cat antibodies immunoprecipitate more cPKC upon activation with PMA. ( A ) HEK293T cells were transfected with either WTPKCβΙ or ΔNPSPKCβΙ and treated with 100 nM PMA for 15 minutes. Fixed cells were then incubated with anti-PKCβΙ V5 domain antibodies and subsequently with anti-rabbit antibodies labeled with Alexa 555. ( B ) To evaluate the amount of active (membrane bound) PKCβΙ in HEK293T transfected cells. Cells treated or non-treated with 100 nM PMA were fractionated and probed for PKCβΙ in the particulate fraction by Western blot with anti-PKCβΙ-V5. ( C ) Transfected HEK293T cells were treated with 100 nM PMA and control cells were immunoprecipated with anti-C2Cat antibodies and probed for PKCβΙ with anti-PKCβΙ-V5 (upper panel). Total lysates were probed with anti-PKCβΙ to evaluate transfection levels, and for α-tubulin to evaluate the total amount of protein loaded. A representative blot of n = 3 is shown for ( B , C ). Quantitative analysis of the average of three independent experiments is shown normalized to non-treated cells, which was set to 1. Statistical significance was determine by ANOVA-Bonferroni test where **p

    Article Snippet: Cell Culture Human Embryonic Kidney (HEK293T) cells were obtained from Dr. Malnic B. (Universidade de São Paulo, Instituto de Química) and Neuroblastoma cells (SK-N-SH) were obtained from American Type Culture Collection (Manassas, VA, USA).

    Techniques: Activation Assay, Transfection, Incubation, Labeling, Western Blot

    N-glycosylation may enhance plasma membrane localization of Panx2 when co-expressed with Panx1. Confocal micrographs of ( A ) Panx2-FLAG or ( B ) N86Q-FLAG (magenta) ectopically co-expressed with mouse Panx1 (green) in HEK293T cells. 72 h post-transfection Panx2-FLAG partially colocalized with Panx1 at the cell membrane (see black arrows in Linescan, panel A ) with a subpopulation still in intracellular compartments. N86Q-FLAG formed intracellular aggregates and showed limited colocalization with Panx1 at the plasma membrane (see black arrows in Linescan, panel B ). Yellow arrowheads denote regions of colocalization of Panx1 and FLAG labeling also depicted with black arrows in the corresponding linescans. Insets: Linescans showing the overlapping (black arrows) between fluorescence peaks to denote colocalization. Nuclei (blue, Hoechst 33342). Scale bars = 20 µm.

    Journal: International Journal of Molecular Sciences

    Article Title: N-Glycosylation Regulates Pannexin 2 Localization but Is Not Required for Interacting with Pannexin 1

    doi: 10.3390/ijms19071837

    Figure Lengend Snippet: N-glycosylation may enhance plasma membrane localization of Panx2 when co-expressed with Panx1. Confocal micrographs of ( A ) Panx2-FLAG or ( B ) N86Q-FLAG (magenta) ectopically co-expressed with mouse Panx1 (green) in HEK293T cells. 72 h post-transfection Panx2-FLAG partially colocalized with Panx1 at the cell membrane (see black arrows in Linescan, panel A ) with a subpopulation still in intracellular compartments. N86Q-FLAG formed intracellular aggregates and showed limited colocalization with Panx1 at the plasma membrane (see black arrows in Linescan, panel B ). Yellow arrowheads denote regions of colocalization of Panx1 and FLAG labeling also depicted with black arrows in the corresponding linescans. Insets: Linescans showing the overlapping (black arrows) between fluorescence peaks to denote colocalization. Nuclei (blue, Hoechst 33342). Scale bars = 20 µm.

    Article Snippet: Normal rat kidney (NRK) (ATCC® CRL-6509™) and human embryonic kidney cells (HEK293T) (ATCC® CRL-3216™) were obtained from ATCC (Manassas, VA, USA).

    Techniques: Transfection, Labeling, Fluorescence

    Panx2 glycosylation is not required for the interaction with Panx1 by immunoprecipitation. ( A ) Reciprocal co-immunoprecipitation (co-IP) experiments showed that both Panx2 and N86Q co-IP in a complex with overexpressed Panx1 in HEK293T cells. Colored arrowheads denote bands of co-IP proteins detected in WB. ( B , C ) of co-IP shows that the interaction of N86Q with Panx1 is not significantly different ( p > 0.05, N = 4) than with Panx2, and in both cases the complexes only involved the lower glycosylated species of Panx1 (Gly-0 and Gly-1). Beads Ctrl denote control IPs done in parallel without antibodies. Protein sizes in kDa.

    Journal: International Journal of Molecular Sciences

    Article Title: N-Glycosylation Regulates Pannexin 2 Localization but Is Not Required for Interacting with Pannexin 1

    doi: 10.3390/ijms19071837

    Figure Lengend Snippet: Panx2 glycosylation is not required for the interaction with Panx1 by immunoprecipitation. ( A ) Reciprocal co-immunoprecipitation (co-IP) experiments showed that both Panx2 and N86Q co-IP in a complex with overexpressed Panx1 in HEK293T cells. Colored arrowheads denote bands of co-IP proteins detected in WB. ( B , C ) of co-IP shows that the interaction of N86Q with Panx1 is not significantly different ( p > 0.05, N = 4) than with Panx2, and in both cases the complexes only involved the lower glycosylated species of Panx1 (Gly-0 and Gly-1). Beads Ctrl denote control IPs done in parallel without antibodies. Protein sizes in kDa.

    Article Snippet: Normal rat kidney (NRK) (ATCC® CRL-6509™) and human embryonic kidney cells (HEK293T) (ATCC® CRL-3216™) were obtained from ATCC (Manassas, VA, USA).

    Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot

    Asn86 is the N-glycosylation site of Panx2. ( A ) Based on sequence analysis, Panx2 (Uniprot ID: Q6IMP4-1) is predicted to contain four transmembrane domains, one intracellular (IL) and two extracellular loops (EL). The predicted N-glycosylation site is located at Asn86 in the first extracellular loop (EL1) (red residue). ( B ) Western blot (WB) comparing wildtype Panx2 and mutant N86Q, the latter shows a faster migrating band than the wildtype counterpart, indicative of decreased molecular weight. ( C ) Cell lysates of HEK293T transiently expressing Panx2 and N86Q mutant were subjected to enzymatic digestions with PNGase F and EndoH N-glycosidases. WB analysis confirmed that N86 is the only glycosylation site for Panx2 since only the wildtype protein exhibited a band shift after treatment with both glycosidases, and the de-glycosylated Panx2 band ran to the same position as the N86Q mutant. GAPDH was used as loading control. Molecular weights are noted in kDa.

    Journal: International Journal of Molecular Sciences

    Article Title: N-Glycosylation Regulates Pannexin 2 Localization but Is Not Required for Interacting with Pannexin 1

    doi: 10.3390/ijms19071837

    Figure Lengend Snippet: Asn86 is the N-glycosylation site of Panx2. ( A ) Based on sequence analysis, Panx2 (Uniprot ID: Q6IMP4-1) is predicted to contain four transmembrane domains, one intracellular (IL) and two extracellular loops (EL). The predicted N-glycosylation site is located at Asn86 in the first extracellular loop (EL1) (red residue). ( B ) Western blot (WB) comparing wildtype Panx2 and mutant N86Q, the latter shows a faster migrating band than the wildtype counterpart, indicative of decreased molecular weight. ( C ) Cell lysates of HEK293T transiently expressing Panx2 and N86Q mutant were subjected to enzymatic digestions with PNGase F and EndoH N-glycosidases. WB analysis confirmed that N86 is the only glycosylation site for Panx2 since only the wildtype protein exhibited a band shift after treatment with both glycosidases, and the de-glycosylated Panx2 band ran to the same position as the N86Q mutant. GAPDH was used as loading control. Molecular weights are noted in kDa.

    Article Snippet: Normal rat kidney (NRK) (ATCC® CRL-6509™) and human embryonic kidney cells (HEK293T) (ATCC® CRL-3216™) were obtained from ATCC (Manassas, VA, USA).

    Techniques: Sequencing, Western Blot, Mutagenesis, Molecular Weight, Expressing, Electrophoretic Mobility Shift Assay

    Analysis of Panx2 and N86Q localization in AD293 and HEK293T cells expressing endogenous PANX1. ( A ) Immunolabeling of Panx2 and N86Q mutant (green) showed that both localized mostly intracellularly, but the N86Q mutant aggregated intracellularly in AD293 cells. Scale bars = 20 μm. ( B ) A subpopulation of AD293 cells displayed Panx2 localization at the cell surface, less evident with the N86Q mutant (indicated with white arrows) Scale bars = 5 μm. Nuclei (blue). ( C ) Cell Surface Biotinylation Assays on AD293 cells showed a weak detection of the Panx2 wildtype but not N86Q mutant in surface-labeled fractions. GAPDH was used as a control for biotin internalization. ( D ) Immunoblots of AD293 and HEK293T cells confirmed that both cell lines express endogenous PANX1. Overexpressed human PANX1 served as positive control (Ctrl +) and endogenous α-tubulin was used as loading control. Line dividing upper panel of PANX1 WB indicates differences in exposure of the same blot to show a better detection of endogenous PANX1 compared to the overexpressed positive control. ( E ) Cell surface biotinylation experiments performed on HEK293T cells showed that overexpressed Panx2 and the mutant N86Q are detectable at the cell surface. Protein disulfide-isomerase (PDI) was used as a control for biotin internalization. ( F ) Densitometric analysis and quantification of cell surface biotinylation experiments performed in HEK293T cells revealed a significant reduction of N86Q cell surface detection compared to Panx2. Cell surface detection was calculated relative to the total protein in input lanes. Statistical significance was considered when p

    Journal: International Journal of Molecular Sciences

    Article Title: N-Glycosylation Regulates Pannexin 2 Localization but Is Not Required for Interacting with Pannexin 1

    doi: 10.3390/ijms19071837

    Figure Lengend Snippet: Analysis of Panx2 and N86Q localization in AD293 and HEK293T cells expressing endogenous PANX1. ( A ) Immunolabeling of Panx2 and N86Q mutant (green) showed that both localized mostly intracellularly, but the N86Q mutant aggregated intracellularly in AD293 cells. Scale bars = 20 μm. ( B ) A subpopulation of AD293 cells displayed Panx2 localization at the cell surface, less evident with the N86Q mutant (indicated with white arrows) Scale bars = 5 μm. Nuclei (blue). ( C ) Cell Surface Biotinylation Assays on AD293 cells showed a weak detection of the Panx2 wildtype but not N86Q mutant in surface-labeled fractions. GAPDH was used as a control for biotin internalization. ( D ) Immunoblots of AD293 and HEK293T cells confirmed that both cell lines express endogenous PANX1. Overexpressed human PANX1 served as positive control (Ctrl +) and endogenous α-tubulin was used as loading control. Line dividing upper panel of PANX1 WB indicates differences in exposure of the same blot to show a better detection of endogenous PANX1 compared to the overexpressed positive control. ( E ) Cell surface biotinylation experiments performed on HEK293T cells showed that overexpressed Panx2 and the mutant N86Q are detectable at the cell surface. Protein disulfide-isomerase (PDI) was used as a control for biotin internalization. ( F ) Densitometric analysis and quantification of cell surface biotinylation experiments performed in HEK293T cells revealed a significant reduction of N86Q cell surface detection compared to Panx2. Cell surface detection was calculated relative to the total protein in input lanes. Statistical significance was considered when p

    Article Snippet: Normal rat kidney (NRK) (ATCC® CRL-6509™) and human embryonic kidney cells (HEK293T) (ATCC® CRL-3216™) were obtained from ATCC (Manassas, VA, USA).

    Techniques: Expressing, Immunolabeling, Mutagenesis, Labeling, Western Blot, Positive Control

    FIN mediates the COX2/PGE 2 pathway by increasing the activity of COX2 promoter. The plasmid harboring a COX2 promoter fragment and a firefly luciferase (FL) gene (5ng/well separately) was transfected into HEK293T cells in a 24-well-plate and a CMV-Renilla luciferase (RL) plasmid (10ng/well) served as a transfection control. FIN (50μM) was treated for 6 h before harvesting the cells for dual-luciferase assay. Transfected cells treated with DMSO were set as negative control. Results are shown in mean ± SEM and representative of 3 independent experiments. **p

    Journal: PLoS ONE

    Article Title: Finasteride Enhances the Generation of Human Myeloid-Derived Suppressor Cells by Up-Regulating the COX2/PGE2 Pathway

    doi: 10.1371/journal.pone.0156549

    Figure Lengend Snippet: FIN mediates the COX2/PGE 2 pathway by increasing the activity of COX2 promoter. The plasmid harboring a COX2 promoter fragment and a firefly luciferase (FL) gene (5ng/well separately) was transfected into HEK293T cells in a 24-well-plate and a CMV-Renilla luciferase (RL) plasmid (10ng/well) served as a transfection control. FIN (50μM) was treated for 6 h before harvesting the cells for dual-luciferase assay. Transfected cells treated with DMSO were set as negative control. Results are shown in mean ± SEM and representative of 3 independent experiments. **p

    Article Snippet: Cell culture HEK293T cells (Cat. No. CRL-1573)were purchased from ATCC (American Type Culture Collection) and maintained in the conditioned Dulbecco’s Modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA) with 10% fetal calf serum (Gibco), 100 units/ml penicillin and 100 μg/ml streptomycin (Gibco) at 37°C and 5% CO2.

    Techniques: Activity Assay, Plasmid Preparation, Luciferase, Transfection, Negative Control

    The USP25 dimer stabilizes endogenous tankyrase in HEK293T cells. a Flag-USP25FL, Flag-USP25FL ΔIL, Flag-USP25FL C178A, Flag-USP25FL ΔIL C178A, Flag-USP25NCD, and Flag-USP25NCD ΔIL were transfected in HEK293T cells and the levels of endogenous tankyrases1/2 were analyzed by western blot (WB). GFP was transfected as a control. Plot of the quantification of tankyrase1/2 levels, corrected with tubulin and relative to GFP. Data values are mean ± s.d. and n ≥ 3 technical replicates. Significance was measured by a two-tailed unpaired t test for all lanes relative to GFP and between USP25FL and USP25FL ΔIL. * P

    Journal: Nature Communications

    Article Title: A quaternary tetramer assembly inhibits the deubiquitinating activity of USP25

    doi: 10.1038/s41467-018-07510-5

    Figure Lengend Snippet: The USP25 dimer stabilizes endogenous tankyrase in HEK293T cells. a Flag-USP25FL, Flag-USP25FL ΔIL, Flag-USP25FL C178A, Flag-USP25FL ΔIL C178A, Flag-USP25NCD, and Flag-USP25NCD ΔIL were transfected in HEK293T cells and the levels of endogenous tankyrases1/2 were analyzed by western blot (WB). GFP was transfected as a control. Plot of the quantification of tankyrase1/2 levels, corrected with tubulin and relative to GFP. Data values are mean ± s.d. and n ≥ 3 technical replicates. Significance was measured by a two-tailed unpaired t test for all lanes relative to GFP and between USP25FL and USP25FL ΔIL. * P

    Article Snippet: Cell culture analysis The HEK293T cell line (CRL-1573; ATCC) was used for ectopic expression of Flag-USP25 and its mutants.

    Techniques: Transfection, Western Blot, Two Tailed Test

    Stimulation of poly-ubiquitination of c-Myc by Rabring7. A and B . HEK293T cells were co-transfected with FLAG-wild-type-c-Myc together with two doses of HA-wild-type Rabring7 (A) or HA-C229S-Rabring7 (B). Forty-four hrs after transfection, 25 μM MG132 was added to the culture medium, and the cells were cultured for an additional 4 hrs. Proteins in cells were then immunoprecipitated with an anti-FLAG antibody, and the precipitates were analyzed by Western blotting with an anti-multi-ubiquitin antibody. C . HEK293T cells were co-transfected with FLAG-T58A-c-Myc together with two doses of HA-wild-type Rabring7 and subjected to ubiquitination assays as described in the legends of Figures 6A and 6B . D. HEK293T cells were co-transfected with FLAG-MM-1α, T7-Rabring7 and HA-ubiquitin. Forty-eight hrs after transfection, total cell lysates were prepared and the cytoplasm and nucleus were fractionated as described in Experimental procedures. Proteins extracted from them were immunoprecipitated with an anti-FLAG antibody and analyzed by Western blotting with anti-HA, anti-FLAG and anti-T7 antibodies. Fractions of the cytoplasm and nucleus were also blotted with anti-HSP60 (SC-6216, Santa Cruz biotechnology, Santa Cruz, CA) and anti-Lamin B (SC-13115, Santa Cruz biotechnology) antibodies. E. HEK293T cells were co-transfected with EGFP-c-Myc, FLAG-MM-1α, T7-Rabring7 and HA-ubiquitin. Proteins were analyzed as described in the legend of Figure 6D .

    Journal: PLoS ONE

    Article Title: Rabring7 Degrades c-Myc through Complex Formation with MM-1

    doi: 10.1371/journal.pone.0041891

    Figure Lengend Snippet: Stimulation of poly-ubiquitination of c-Myc by Rabring7. A and B . HEK293T cells were co-transfected with FLAG-wild-type-c-Myc together with two doses of HA-wild-type Rabring7 (A) or HA-C229S-Rabring7 (B). Forty-four hrs after transfection, 25 μM MG132 was added to the culture medium, and the cells were cultured for an additional 4 hrs. Proteins in cells were then immunoprecipitated with an anti-FLAG antibody, and the precipitates were analyzed by Western blotting with an anti-multi-ubiquitin antibody. C . HEK293T cells were co-transfected with FLAG-T58A-c-Myc together with two doses of HA-wild-type Rabring7 and subjected to ubiquitination assays as described in the legends of Figures 6A and 6B . D. HEK293T cells were co-transfected with FLAG-MM-1α, T7-Rabring7 and HA-ubiquitin. Forty-eight hrs after transfection, total cell lysates were prepared and the cytoplasm and nucleus were fractionated as described in Experimental procedures. Proteins extracted from them were immunoprecipitated with an anti-FLAG antibody and analyzed by Western blotting with anti-HA, anti-FLAG and anti-T7 antibodies. Fractions of the cytoplasm and nucleus were also blotted with anti-HSP60 (SC-6216, Santa Cruz biotechnology, Santa Cruz, CA) and anti-Lamin B (SC-13115, Santa Cruz biotechnology) antibodies. E. HEK293T cells were co-transfected with EGFP-c-Myc, FLAG-MM-1α, T7-Rabring7 and HA-ubiquitin. Proteins were analyzed as described in the legend of Figure 6D .

    Article Snippet: To examine endogenous interaction of c-Myc with Rabring7, proteins extracted from HEK293T cells were immunoprecipitated with an agarose-conjugated chicken anti-c-Myc antibody (Abcam, Cambridge, UK) or with an agarose-conjugated chicken IgY (Acris Antibodies, San Diego, CA) under the same conditions as those for the in vitro binding assay, and the precipitates were subjected to Western blotting with anti-c-Myc (1∶1000, N262, Santa Cruz, California, CA) and anti-Rabring7 (1∶1000, ab80432, Abcam) antibodies.

    Techniques: Transfection, Cell Culture, Immunoprecipitation, Western Blot

    Binding of Rabring7 to c-Myc. A . MBP-Rabring7 or MBP expressed in and purified from E. coli was reacted with 35 S-labeled c-Myc, transthyretin (TTR) and MM-1α, and pull-down assays were carried out as described in Experimental procedures. B . HEK293 cells were transfected with various combinations of expression vectors for HA-Rabring7 and FLAG-c-Myc. At 48 hrs after transfection, proteins prepared from cells were immunoprecipitated with an anti-FLAG antibody, and the precipitates were analyzed by Western blotting with anti-HA and anti-FLAG antibodies. C . Proteins prepared from HEK293T cells were immunoprecipitated with an anti-c-Myc antibody or with IgY, and the precipitates were analyzed by Western blotting with anti-Rabring7 and anti-c-Myc antibodies. D . H1299 cells were co-transfected with expression vectors for T7-Rabring7, HA-MM-1α and FLAG-c-Myc. At 48 hrs after transfection, proteins extracted from cells were immunoprecipitated with an agarose-conjugated anti-FLAG antibody and the proteins bound to agarose bead were eluted with FLAG peptide. The eluted proteins were then immunoprecipitated with an anti-HA antibody and precipitates were analyzed by Western blotting with anti-T7, anti-HA and anti-FLAG antibodies as described in Experimental procedures.

    Journal: PLoS ONE

    Article Title: Rabring7 Degrades c-Myc through Complex Formation with MM-1

    doi: 10.1371/journal.pone.0041891

    Figure Lengend Snippet: Binding of Rabring7 to c-Myc. A . MBP-Rabring7 or MBP expressed in and purified from E. coli was reacted with 35 S-labeled c-Myc, transthyretin (TTR) and MM-1α, and pull-down assays were carried out as described in Experimental procedures. B . HEK293 cells were transfected with various combinations of expression vectors for HA-Rabring7 and FLAG-c-Myc. At 48 hrs after transfection, proteins prepared from cells were immunoprecipitated with an anti-FLAG antibody, and the precipitates were analyzed by Western blotting with anti-HA and anti-FLAG antibodies. C . Proteins prepared from HEK293T cells were immunoprecipitated with an anti-c-Myc antibody or with IgY, and the precipitates were analyzed by Western blotting with anti-Rabring7 and anti-c-Myc antibodies. D . H1299 cells were co-transfected with expression vectors for T7-Rabring7, HA-MM-1α and FLAG-c-Myc. At 48 hrs after transfection, proteins extracted from cells were immunoprecipitated with an agarose-conjugated anti-FLAG antibody and the proteins bound to agarose bead were eluted with FLAG peptide. The eluted proteins were then immunoprecipitated with an anti-HA antibody and precipitates were analyzed by Western blotting with anti-T7, anti-HA and anti-FLAG antibodies as described in Experimental procedures.

    Article Snippet: To examine endogenous interaction of c-Myc with Rabring7, proteins extracted from HEK293T cells were immunoprecipitated with an agarose-conjugated chicken anti-c-Myc antibody (Abcam, Cambridge, UK) or with an agarose-conjugated chicken IgY (Acris Antibodies, San Diego, CA) under the same conditions as those for the in vitro binding assay, and the precipitates were subjected to Western blotting with anti-c-Myc (1∶1000, N262, Santa Cruz, California, CA) and anti-Rabring7 (1∶1000, ab80432, Abcam) antibodies.

    Techniques: Binding Assay, Purification, Labeling, Transfection, Expressing, Immunoprecipitation, Western Blot

    Identification of Rabring7 as an MM-1-binding protein. A . Proteins prepared from H1299 cells and F-MM-1-expressing H1299 cells were immunoprecipitated with an anti-FLAG antibody, and the precipitates were subjected to TOF-MS analyses as described in Experimental procedures. B . H1299 cells (upper panel) and HEK293 cells (lower panel) were transfected with various combinations of expression vectors for HA-Rabring7 and FLAG-MM-1α. At 48 hrs after transfection, proteins prepared from cells were immunoprecipitated with an anti-FLAG antibody or with IgG, and the precipitates were analyzed by Western blotting with anti-HA and anti-FLAG antibodies. C . Proteins prepared from HEK293T cells were immunoprecipitated with an anti-MM-1 antibody or with IgG, and the precipitates were analyzed by Western blotting with anti-Rabring7 and anti-MM-1 antibodies. D . FLAG-MM-1 expressed in and purified from E. coli was reacted with 35 S-labeled Rabring7 that had been synthesized by using reticulocyte lysates. Proteins in a reaction mixture were then immunoprecipitated with an anti-FLAG antibody or with IgG, and the precipitates were separated on a gel followed by fluorography.

    Journal: PLoS ONE

    Article Title: Rabring7 Degrades c-Myc through Complex Formation with MM-1

    doi: 10.1371/journal.pone.0041891

    Figure Lengend Snippet: Identification of Rabring7 as an MM-1-binding protein. A . Proteins prepared from H1299 cells and F-MM-1-expressing H1299 cells were immunoprecipitated with an anti-FLAG antibody, and the precipitates were subjected to TOF-MS analyses as described in Experimental procedures. B . H1299 cells (upper panel) and HEK293 cells (lower panel) were transfected with various combinations of expression vectors for HA-Rabring7 and FLAG-MM-1α. At 48 hrs after transfection, proteins prepared from cells were immunoprecipitated with an anti-FLAG antibody or with IgG, and the precipitates were analyzed by Western blotting with anti-HA and anti-FLAG antibodies. C . Proteins prepared from HEK293T cells were immunoprecipitated with an anti-MM-1 antibody or with IgG, and the precipitates were analyzed by Western blotting with anti-Rabring7 and anti-MM-1 antibodies. D . FLAG-MM-1 expressed in and purified from E. coli was reacted with 35 S-labeled Rabring7 that had been synthesized by using reticulocyte lysates. Proteins in a reaction mixture were then immunoprecipitated with an anti-FLAG antibody or with IgG, and the precipitates were separated on a gel followed by fluorography.

    Article Snippet: To examine endogenous interaction of c-Myc with Rabring7, proteins extracted from HEK293T cells were immunoprecipitated with an agarose-conjugated chicken anti-c-Myc antibody (Abcam, Cambridge, UK) or with an agarose-conjugated chicken IgY (Acris Antibodies, San Diego, CA) under the same conditions as those for the in vitro binding assay, and the precipitates were subjected to Western blotting with anti-c-Myc (1∶1000, N262, Santa Cruz, California, CA) and anti-Rabring7 (1∶1000, ab80432, Abcam) antibodies.

    Techniques: Binding Assay, Expressing, Immunoprecipitation, Mass Spectrometry, Transfection, Western Blot, Purification, Labeling, Synthesized

    Effect of Rabring7 on stability of c-Myc. A . HEK293T cells were co-transfected with a constant amount of FLAG-MM-1α and with various doses of T7-Rabring7, and proteins in cells were analyzed by Western blotting with anti-T7, anti-FLAG and anti-actin antibodies. B . HEK293T cells were co-transfected with constant amounts of FLAG-MM-1α and FLAG-c-Myc together with various doses of T7-Rabring7, and proteins in cells were analyzed by Western blotting with anti-T7, anti-FLAG and anti-actin antibodies. C . Expression levels of MM-1α, Rabring7 and actin in vector- and MM-1-expressing H1299 cells were analyzed by Western blotting with anti-MM-1, anti-Rabring7 and anti-actin antibodies. D . Vector- and MM-1-expressing H1299 cells were transfected with a constant amount of FLAG-c-Myc and with various amounts of T7-Rabring7. At 48 hrs after transfection, protein in the cells were analyzed by Western blotting with anti-T7, anti-FLAG and anti-actin antibodies. E . The intensity of bands corresponding to FLAG-c-Myc and actin in Fig. 6D was quantified, and relative intensity of FLAG-c-Myc to that of actin is shown. Values are means ± S.D. n = 3 experiments. Significance: ** p

    Journal: PLoS ONE

    Article Title: Rabring7 Degrades c-Myc through Complex Formation with MM-1

    doi: 10.1371/journal.pone.0041891

    Figure Lengend Snippet: Effect of Rabring7 on stability of c-Myc. A . HEK293T cells were co-transfected with a constant amount of FLAG-MM-1α and with various doses of T7-Rabring7, and proteins in cells were analyzed by Western blotting with anti-T7, anti-FLAG and anti-actin antibodies. B . HEK293T cells were co-transfected with constant amounts of FLAG-MM-1α and FLAG-c-Myc together with various doses of T7-Rabring7, and proteins in cells were analyzed by Western blotting with anti-T7, anti-FLAG and anti-actin antibodies. C . Expression levels of MM-1α, Rabring7 and actin in vector- and MM-1-expressing H1299 cells were analyzed by Western blotting with anti-MM-1, anti-Rabring7 and anti-actin antibodies. D . Vector- and MM-1-expressing H1299 cells were transfected with a constant amount of FLAG-c-Myc and with various amounts of T7-Rabring7. At 48 hrs after transfection, protein in the cells were analyzed by Western blotting with anti-T7, anti-FLAG and anti-actin antibodies. E . The intensity of bands corresponding to FLAG-c-Myc and actin in Fig. 6D was quantified, and relative intensity of FLAG-c-Myc to that of actin is shown. Values are means ± S.D. n = 3 experiments. Significance: ** p

    Article Snippet: To examine endogenous interaction of c-Myc with Rabring7, proteins extracted from HEK293T cells were immunoprecipitated with an agarose-conjugated chicken anti-c-Myc antibody (Abcam, Cambridge, UK) or with an agarose-conjugated chicken IgY (Acris Antibodies, San Diego, CA) under the same conditions as those for the in vitro binding assay, and the precipitates were subjected to Western blotting with anti-c-Myc (1∶1000, N262, Santa Cruz, California, CA) and anti-Rabring7 (1∶1000, ab80432, Abcam) antibodies.

    Techniques: Transfection, Western Blot, Expressing, Plasmid Preparation

    Mitochondrial t 6 A37 formation is sensitive to intracellular bicarbonate concentration. a Kinetic analyses of mitochondrial t 6 A37 formation mediated by YRDC and OSGEPL1. Initial velocities of t 6 A37 formation were measured against variable concentrations of mt-tRNA Thr , L -Thr, ATP, and bicarbonate. Km values for each substrate are indicated. b Hypomodification of t 6 A37 in mt-tRNAs in HEK293T cells cultured in non-bicarbonate medium. XICs generated by integration of multiply-charged negative ions of A37-containing fragments of mt-tRNA Ser(AGY) (top panels), mt-tRNA Asn (second panels), mt-tRNA Thr (third panels), mt-tRNA Lys with s 2 U34 (fourth panels) and ct-tRNA Ile (bottom panels) bearing A37 (blue) and t 6 A37 (black) (Supplementary Table 1 ) isolated from HEK293T cells cultured with normal DMEM medium (44 mM NaHCO 3 ) in 5% CO 2 (left panels) and non-bicarbonate medium in air (right panels). mt-tRNA Ser(AGY) and other tRNAs were isolated from the cells cultured for 6 and 3 days, respectively. t 6 A frequencies (%) are described as mean values ± s.d. of technical triplicate

    Journal: Nature Communications

    Article Title: CO2-sensitive tRNA modification associated with human mitochondrial disease

    doi: 10.1038/s41467-018-04250-4

    Figure Lengend Snippet: Mitochondrial t 6 A37 formation is sensitive to intracellular bicarbonate concentration. a Kinetic analyses of mitochondrial t 6 A37 formation mediated by YRDC and OSGEPL1. Initial velocities of t 6 A37 formation were measured against variable concentrations of mt-tRNA Thr , L -Thr, ATP, and bicarbonate. Km values for each substrate are indicated. b Hypomodification of t 6 A37 in mt-tRNAs in HEK293T cells cultured in non-bicarbonate medium. XICs generated by integration of multiply-charged negative ions of A37-containing fragments of mt-tRNA Ser(AGY) (top panels), mt-tRNA Asn (second panels), mt-tRNA Thr (third panels), mt-tRNA Lys with s 2 U34 (fourth panels) and ct-tRNA Ile (bottom panels) bearing A37 (blue) and t 6 A37 (black) (Supplementary Table 1 ) isolated from HEK293T cells cultured with normal DMEM medium (44 mM NaHCO 3 ) in 5% CO 2 (left panels) and non-bicarbonate medium in air (right panels). mt-tRNA Ser(AGY) and other tRNAs were isolated from the cells cultured for 6 and 3 days, respectively. t 6 A frequencies (%) are described as mean values ± s.d. of technical triplicate

    Article Snippet: HEK293T cells were transfected with a pX330 vector bearing the sgRNA sequence and with pEGFP-N1 (Clontech) and pLL3.7 vectors containing the puromycin resistance gene; transfections were performed using FuGENE HD (Promega).

    Techniques: Concentration Assay, Cell Culture, Generated, Isolation

    YRDC is responsible for t 6 A37 formation in mt-tRNAs. a Subcellular localization of wild-type (WT) and mutant YRDC (S17F, A15F/S17F) in HeLa cells immunostained with an anti-FLAG antibody (Green). Nuclei and mitochondria were stained with DAPI (blue) and MitoTracker (Red), respectively. All images were superimposed to generate the merged panel. Scale bars: 20 μm. b Mitochondrial localization of YRDC. Whole-cell lysates (W.L.) and mitochondrial fractions (Mito.) from HEK293T cells transfected with WT, variant with N-terminal truncation (a.a. Δ2–15), and mutant (A15F/S17F) YRDC constructs were subjected to western blotting with anti-FLAG antibody to detect YRDC variants, anti-CO1 (mitochondrial marker), and anti-GAPDH (cytoplasmic marker). Uncut gel images are provided in Supplementary Fig. 15 . c Determination of the cleavage site in the MTS of YRDC. Schematic depiction of YRDC with a predicted MTS (blue) and a conserved region homologous to that of E. coli YrdC (green). Multiple cleavage sites in the long and the short isoforms of YRDC expressed in HeLa cells are indicated by white and black arrowheads, respectively. The CID spectrum represents the sequence of the N-terminal tryptic peptide of the short isoform with the cleavage site at position 52. The precursor ion for CID is m/z 565.28. Product ions are assigned on the peptide sequence. d Top: schematic of human YRDC gene and the site of insertion introduced with the CRISPR–Cas9 system. Shaded boxes indicate coding regions; open boxes indicate untranslated regions of exons; lines indicate introns. Inset: exon 1 of WT YRDC. The target sequence of the single guide RNA (sgRNA) is underlined; the protospacer adjacent motif (PAM) sequence is outlined. Bottom: sequence of the frameshift cell line (FS#1); the inserted C is indicated in red. e Extracted ion chromatograms (XICs) generated by integration of multiply-charged negative ions of the A37-containing fragments of human mt-tRNA Ile with A37 (top) or t 6 A37 (bottom) (Supplementary Table 1 ) isolated from WT (left) and FS#1 (right) cell lines. t 6 A frequencies (%) are described as mean values ± s.d. of technical triplicate. f XICs generated by integration of multiply-charged negative ions of A37-containing fragments of human ct-tRNA Ile(IAU) with A37 (top) or t 6 A37 (bottom) (Supplementary Table 1 ) isolated from WT (left) and FS#1 (right) cell lines

    Journal: Nature Communications

    Article Title: CO2-sensitive tRNA modification associated with human mitochondrial disease

    doi: 10.1038/s41467-018-04250-4

    Figure Lengend Snippet: YRDC is responsible for t 6 A37 formation in mt-tRNAs. a Subcellular localization of wild-type (WT) and mutant YRDC (S17F, A15F/S17F) in HeLa cells immunostained with an anti-FLAG antibody (Green). Nuclei and mitochondria were stained with DAPI (blue) and MitoTracker (Red), respectively. All images were superimposed to generate the merged panel. Scale bars: 20 μm. b Mitochondrial localization of YRDC. Whole-cell lysates (W.L.) and mitochondrial fractions (Mito.) from HEK293T cells transfected with WT, variant with N-terminal truncation (a.a. Δ2–15), and mutant (A15F/S17F) YRDC constructs were subjected to western blotting with anti-FLAG antibody to detect YRDC variants, anti-CO1 (mitochondrial marker), and anti-GAPDH (cytoplasmic marker). Uncut gel images are provided in Supplementary Fig. 15 . c Determination of the cleavage site in the MTS of YRDC. Schematic depiction of YRDC with a predicted MTS (blue) and a conserved region homologous to that of E. coli YrdC (green). Multiple cleavage sites in the long and the short isoforms of YRDC expressed in HeLa cells are indicated by white and black arrowheads, respectively. The CID spectrum represents the sequence of the N-terminal tryptic peptide of the short isoform with the cleavage site at position 52. The precursor ion for CID is m/z 565.28. Product ions are assigned on the peptide sequence. d Top: schematic of human YRDC gene and the site of insertion introduced with the CRISPR–Cas9 system. Shaded boxes indicate coding regions; open boxes indicate untranslated regions of exons; lines indicate introns. Inset: exon 1 of WT YRDC. The target sequence of the single guide RNA (sgRNA) is underlined; the protospacer adjacent motif (PAM) sequence is outlined. Bottom: sequence of the frameshift cell line (FS#1); the inserted C is indicated in red. e Extracted ion chromatograms (XICs) generated by integration of multiply-charged negative ions of the A37-containing fragments of human mt-tRNA Ile with A37 (top) or t 6 A37 (bottom) (Supplementary Table 1 ) isolated from WT (left) and FS#1 (right) cell lines. t 6 A frequencies (%) are described as mean values ± s.d. of technical triplicate. f XICs generated by integration of multiply-charged negative ions of A37-containing fragments of human ct-tRNA Ile(IAU) with A37 (top) or t 6 A37 (bottom) (Supplementary Table 1 ) isolated from WT (left) and FS#1 (right) cell lines

    Article Snippet: HEK293T cells were transfected with a pX330 vector bearing the sgRNA sequence and with pEGFP-N1 (Clontech) and pLL3.7 vectors containing the puromycin resistance gene; transfections were performed using FuGENE HD (Promega).

    Techniques: Mutagenesis, Staining, Transfection, Variant Assay, Construct, Western Blot, Marker, Sequencing, CRISPR, Generated, Isolation

    Mitochondrial dysfunction in OSGE P L1 KO cells. a Growth curves for WT HEK293T, OSGE P L1 KO#1, and OSGE P L1 KO#2 cells cultured in the presence of glucose (left) or galactose (right) as the primary carbon source. Mean values ± s.e.m. of four independent cultures are plotted. b Oxygen consumption rates of WT, OSGE P L1 KO#1, and OSGE P L1 KO#2 cells measured using an XFp extracellular flux analyzer. Mean values ± s.d. of biological triplicates are compared. * P

    Journal: Nature Communications

    Article Title: CO2-sensitive tRNA modification associated with human mitochondrial disease

    doi: 10.1038/s41467-018-04250-4

    Figure Lengend Snippet: Mitochondrial dysfunction in OSGE P L1 KO cells. a Growth curves for WT HEK293T, OSGE P L1 KO#1, and OSGE P L1 KO#2 cells cultured in the presence of glucose (left) or galactose (right) as the primary carbon source. Mean values ± s.e.m. of four independent cultures are plotted. b Oxygen consumption rates of WT, OSGE P L1 KO#1, and OSGE P L1 KO#2 cells measured using an XFp extracellular flux analyzer. Mean values ± s.d. of biological triplicates are compared. * P

    Article Snippet: HEK293T cells were transfected with a pX330 vector bearing the sgRNA sequence and with pEGFP-N1 (Clontech) and pLL3.7 vectors containing the puromycin resistance gene; transfections were performed using FuGENE HD (Promega).

    Techniques: Cell Culture

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gks257

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

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

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

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gks257

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

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

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

    p38 promotes mTORC1 activation and stress granule formation when PI3K is inactive. (A) p38 mediates mTORC1 activation when PI3K is inactive. MCF-7 cells were serum-starved and treated with arsenite in the presence of carrier (DMSO) or wortmannin (100 nM, PI3K inhibitor). In addition, the cells were treated with carrier (DMSO) versus LY2228820 (1 μM, p38 inhibitor). MK2-pT334, Akt-pT308, Akt-pS473, p70-S6K-pT389, and eIF2α-pS51 were monitored by immunoblot. Data represent five biological replicates. (B) Quantification of data shown in (A) when PI3K is active. Akt-pS473 and p70-S6K-pT389 were compared between carrier (DMSO) and LY2228820-treated cells using a two-way ANOVA followed by a Bonferroni multiple comparison test across five biological replicates. Data represent the mean ± SEM. The P -values for the Bonferroni multiple comparison tests are shown. *** P ≤ 0.001. (C) Quantification of data shown in (A) when PI3K is inactive. Akt-pS473 and p70-S6K-pT389 were compared between wortmannin- and wortmannin + LY2228820–treated cells using a two-way ANOVA followed by a Bonferroni multiple comparison test across five biological replicates. Data represent the mean ± SEM. The P -values for the Bonferroni multiple comparison tests are shown. * P ≤ 0.05; *** P ≤ 0.001. (D) p38 drives mTORC1 activity when PI3K is inactive. Quantification of data shown in Fig S14A . 4E-BP1-pT37/46 relative intensity was normalized separately for conditions without or with wortmannin. Significance of 4E-BP1-pT37/46 inhibition by LY2228820 was tested using a two-tailed t test across five biological replicates. Data represent the mean ± SEM. * P ≤ 0.05. (E) Prediction on the extent of mTORC1 inhibition upon LY2228820 treatment when PI3K is active or inactive. Prediction was performed with model V. The red lines depict the time points measured experimentally ( Fig 5A–C ). (F) When PI3K activity declines, p38 drives mTORC1 activity. Quantification of data shown in Fig S13 L. MCF-7 cells were serum-starved and treated with arsenite for 60 min in the presence of different concentrations of wortmannin (as indicated, PI3K inhibitor) in carrier (DMSO) versus LY2228820 (1 μM, p38 inhibitor)-treated cells. p70-S6K-pT389 relative intensity was normalized separately for each wortmannin concentration. Significance of p70-S6K-pT389 inhibition by LY2228820 was tested using a two-tailed t test across five biological replicates. Data represent the mean ± SEM. * P ≤ 0.05. (G) p38 drives mTORC1 activity in several cell lines, as PI3K activity declines. Quantification of data shown in Fig S14D–G . MCF-7, CAL51, LN18, HEK293T, and HeLa cells were serum-starved and exposed to arsenite for 60 min in combination with wortmannin (100 nM, PI3K inhibitor) and/or LY2228820 (1 mM, p38 inhibitor). Data represent 3–4 biological replicates (see Fig S14D–G ). 4E-BP1-pT37/46 relative intensity was normalized separately for conditions without or with wortmannin. Significance of 4E-BP1-pT37/46 inhibition by LY2228820 was tested using a two-tailed t test across three biological replicates. Data represent the mean ± SEM. * P ≤ 0.05; ** P ≤ 0.01. (H) Stress granule numbers upon PI3K and p38 inhibition. MCF-7 cells were serum-starved and treated with arsenite for 30 min in the presence of carrier (DMSO), wortmannin (100 nM, PI3K inhibitor), LY2228820 (1 μM, p38 inhibitor), or wortmannin + LY2228820. Stress granules were visualized by immunofluorescence staining of G3BP1. Nuclei were visualized with Hoechst 33342. Data represent four biological replicates. White square indicates region of insert and blue arrow highlights stress granules; scale bar 10 μm. (I) Quantification of data shown in (H). The number of stress granules (SGs) per cell (normalized to the arsenite condition) across four biological replicates. Stress granule formation between carrier and LY2228820 as well as wortmannin- and wortmannin + LY2228820–treated cells was compared using a two-tailed t test across four biological replicates. Data represent the mean ± SEM. * P ≤ 0.01. ns, not significant.

    Journal: Life Science Alliance

    Article Title: The PI3K and MAPK/p38 pathways control stress granule assembly in a hierarchical manner

    doi: 10.26508/lsa.201800257

    Figure Lengend Snippet: p38 promotes mTORC1 activation and stress granule formation when PI3K is inactive. (A) p38 mediates mTORC1 activation when PI3K is inactive. MCF-7 cells were serum-starved and treated with arsenite in the presence of carrier (DMSO) or wortmannin (100 nM, PI3K inhibitor). In addition, the cells were treated with carrier (DMSO) versus LY2228820 (1 μM, p38 inhibitor). MK2-pT334, Akt-pT308, Akt-pS473, p70-S6K-pT389, and eIF2α-pS51 were monitored by immunoblot. Data represent five biological replicates. (B) Quantification of data shown in (A) when PI3K is active. Akt-pS473 and p70-S6K-pT389 were compared between carrier (DMSO) and LY2228820-treated cells using a two-way ANOVA followed by a Bonferroni multiple comparison test across five biological replicates. Data represent the mean ± SEM. The P -values for the Bonferroni multiple comparison tests are shown. *** P ≤ 0.001. (C) Quantification of data shown in (A) when PI3K is inactive. Akt-pS473 and p70-S6K-pT389 were compared between wortmannin- and wortmannin + LY2228820–treated cells using a two-way ANOVA followed by a Bonferroni multiple comparison test across five biological replicates. Data represent the mean ± SEM. The P -values for the Bonferroni multiple comparison tests are shown. * P ≤ 0.05; *** P ≤ 0.001. (D) p38 drives mTORC1 activity when PI3K is inactive. Quantification of data shown in Fig S14A . 4E-BP1-pT37/46 relative intensity was normalized separately for conditions without or with wortmannin. Significance of 4E-BP1-pT37/46 inhibition by LY2228820 was tested using a two-tailed t test across five biological replicates. Data represent the mean ± SEM. * P ≤ 0.05. (E) Prediction on the extent of mTORC1 inhibition upon LY2228820 treatment when PI3K is active or inactive. Prediction was performed with model V. The red lines depict the time points measured experimentally ( Fig 5A–C ). (F) When PI3K activity declines, p38 drives mTORC1 activity. Quantification of data shown in Fig S13 L. MCF-7 cells were serum-starved and treated with arsenite for 60 min in the presence of different concentrations of wortmannin (as indicated, PI3K inhibitor) in carrier (DMSO) versus LY2228820 (1 μM, p38 inhibitor)-treated cells. p70-S6K-pT389 relative intensity was normalized separately for each wortmannin concentration. Significance of p70-S6K-pT389 inhibition by LY2228820 was tested using a two-tailed t test across five biological replicates. Data represent the mean ± SEM. * P ≤ 0.05. (G) p38 drives mTORC1 activity in several cell lines, as PI3K activity declines. Quantification of data shown in Fig S14D–G . MCF-7, CAL51, LN18, HEK293T, and HeLa cells were serum-starved and exposed to arsenite for 60 min in combination with wortmannin (100 nM, PI3K inhibitor) and/or LY2228820 (1 mM, p38 inhibitor). Data represent 3–4 biological replicates (see Fig S14D–G ). 4E-BP1-pT37/46 relative intensity was normalized separately for conditions without or with wortmannin. Significance of 4E-BP1-pT37/46 inhibition by LY2228820 was tested using a two-tailed t test across three biological replicates. Data represent the mean ± SEM. * P ≤ 0.05; ** P ≤ 0.01. (H) Stress granule numbers upon PI3K and p38 inhibition. MCF-7 cells were serum-starved and treated with arsenite for 30 min in the presence of carrier (DMSO), wortmannin (100 nM, PI3K inhibitor), LY2228820 (1 μM, p38 inhibitor), or wortmannin + LY2228820. Stress granules were visualized by immunofluorescence staining of G3BP1. Nuclei were visualized with Hoechst 33342. Data represent four biological replicates. White square indicates region of insert and blue arrow highlights stress granules; scale bar 10 μm. (I) Quantification of data shown in (H). The number of stress granules (SGs) per cell (normalized to the arsenite condition) across four biological replicates. Stress granule formation between carrier and LY2228820 as well as wortmannin- and wortmannin + LY2228820–treated cells was compared using a two-tailed t test across four biological replicates. Data represent the mean ± SEM. * P ≤ 0.01. ns, not significant.

    Article Snippet: HeLa, MCF-7, and HEK293T cells were validated by DSMZ for their origin using short tandem repeat analysis.

    Techniques: Activation Assay, Activity Assay, Inhibition, Two Tailed Test, Concentration Assay, Immunofluorescence, Staining

    PI3K and p38 control mTORC1 in a hierarchical manner in several cell lines. (A) When PI3K activity declines, p38 drives 4E-BP1-pT37/46 in MCF-7 cells. MCF-7 cells were serum-starved and treated with arsenite for 60 min upon wortmannin (100 nM, PI3K inhibitor) and/or LY2228820 (1 mM, p38 inhibitor). Data represent five biological replicates. (B) Comparison of the expression of mTORC1 targets. MCF-7, CAL51, LN18, HEK293T, and HeLa cells were serum-starved. p70-S6K and 4E-BP1 were monitored by immunoblot. Data represent three to four biological replicates. (C) Quantification of data shown in (B). p70-S6K and 4E-BP1 levels were compared between cell lines using a two-tailed t test across three to four biological replicates. Data represent the mean ± SEM. (D) When PI3K activity declines, p38 drives 4E-BP1-pT37/46 in CAL51 cells. MCF-7 and CAL51 cells were serum-starved and treated with arsenite for 60 min. For CAL51, the treatment was combined with wortmannin (100 nM, PI3K inhibitor) and/or LY2228820 (1 mM, p38 inhibitor). Data represent four biological replicates. (E) When PI3K activity declines, p38 drives 4E-BP1-pT37/46 in LN18 cells. MCF-7 and LN18 cells were treated as described in (D). Data represent four biological replicates. (F) When PI3K activity declines, p38 drives 4E-BP1-pT37/46 in HEK293T cells. MCF-7 and HEK293T cells were treated as described in (D). Data represent three biological replicates. (G) p38 drives 4E-BP1-pT37/46 in HeLa cells. MCF-7 and HeLa cells were serum-starved and exposed to arsenite for 60 min. For HeLa, the treatment was combined with wortmannin (100 nM, PI3K inhibitor) or LY2228820 (1 mM, p38 inhibitor). Note that in HeLa cells, Akt-pT308 is not arsenite inducible, indicative of low PI3K activity. Data represent three biological replicates.

    Journal: Life Science Alliance

    Article Title: The PI3K and MAPK/p38 pathways control stress granule assembly in a hierarchical manner

    doi: 10.26508/lsa.201800257

    Figure Lengend Snippet: PI3K and p38 control mTORC1 in a hierarchical manner in several cell lines. (A) When PI3K activity declines, p38 drives 4E-BP1-pT37/46 in MCF-7 cells. MCF-7 cells were serum-starved and treated with arsenite for 60 min upon wortmannin (100 nM, PI3K inhibitor) and/or LY2228820 (1 mM, p38 inhibitor). Data represent five biological replicates. (B) Comparison of the expression of mTORC1 targets. MCF-7, CAL51, LN18, HEK293T, and HeLa cells were serum-starved. p70-S6K and 4E-BP1 were monitored by immunoblot. Data represent three to four biological replicates. (C) Quantification of data shown in (B). p70-S6K and 4E-BP1 levels were compared between cell lines using a two-tailed t test across three to four biological replicates. Data represent the mean ± SEM. (D) When PI3K activity declines, p38 drives 4E-BP1-pT37/46 in CAL51 cells. MCF-7 and CAL51 cells were serum-starved and treated with arsenite for 60 min. For CAL51, the treatment was combined with wortmannin (100 nM, PI3K inhibitor) and/or LY2228820 (1 mM, p38 inhibitor). Data represent four biological replicates. (E) When PI3K activity declines, p38 drives 4E-BP1-pT37/46 in LN18 cells. MCF-7 and LN18 cells were treated as described in (D). Data represent four biological replicates. (F) When PI3K activity declines, p38 drives 4E-BP1-pT37/46 in HEK293T cells. MCF-7 and HEK293T cells were treated as described in (D). Data represent three biological replicates. (G) p38 drives 4E-BP1-pT37/46 in HeLa cells. MCF-7 and HeLa cells were serum-starved and exposed to arsenite for 60 min. For HeLa, the treatment was combined with wortmannin (100 nM, PI3K inhibitor) or LY2228820 (1 mM, p38 inhibitor). Note that in HeLa cells, Akt-pT308 is not arsenite inducible, indicative of low PI3K activity. Data represent three biological replicates.

    Article Snippet: HeLa, MCF-7, and HEK293T cells were validated by DSMZ for their origin using short tandem repeat analysis.

    Techniques: Activity Assay, Expressing, Two Tailed Test