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    Structured Review

    TaKaRa hek293 cells
    ADAM10 expression is repressed by the G-quadruplex motif. A , <t>HEK293</t> cells were transiently transfected with the indicated ADAM10 cDNA constructs, and lysates were analyzed by immunoblotting for V5-tagged ADAM10, endogenous APP, β-actin as loading control, and GFP as transfection control. Supernatants were analyzed for APPsα secretion using antibody 2D8. Cellular APP is present in low molecular weight immature forms ( im ) and high molecular weight mature form ( m ). ADAM10 is present as a mature ( m ) form and predominantly as an immature ( im ) form. B , quantification of ADAM10 protein ( black bars ) and mRNA levels ( white bars ) from cells transfected with ADAM10 cDNA constructs shown in A . ADAM10 protein levels were normalized to GFP and actin levels. The signal for ADAM10 with the wild-type G-quadruplex GQ-WT ADAM10 was set to 1. Results are expressed as the means ± S.D. from three experiments made in triplicate. ADAM10 mRNA was normalized to glycerolaldehyde-3-phosphate-dehydrogenase mRNA levels, and the signal for GQ-WT ADAM10 was set to 1. Results are expressed as the means ± S.D. from three experiments. C , quantification of secreted APPsα from cells transfected with the indicated ADAM10 variants were shown in A . The signal for APPsα from GQ-WT ADAM10 transfected cells was set to 100%. Results are expressed as the means ± S.D. from three experiments. Asterisks indicate statistical significance (one-way analysis of variance with Dunnett's post test) relative to GQ-WT ADAM10 transfected cells (*, p
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    Images

    1) Product Images from "Translational Repression of the Disintegrin and Metalloprotease ADAM10 by a Stable G-quadruplex Secondary Structure in Its 5?-Untranslated Region *"

    Article Title: Translational Repression of the Disintegrin and Metalloprotease ADAM10 by a Stable G-quadruplex Secondary Structure in Its 5?-Untranslated Region *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.296921

    ADAM10 expression is repressed by the G-quadruplex motif. A , HEK293 cells were transiently transfected with the indicated ADAM10 cDNA constructs, and lysates were analyzed by immunoblotting for V5-tagged ADAM10, endogenous APP, β-actin as loading control, and GFP as transfection control. Supernatants were analyzed for APPsα secretion using antibody 2D8. Cellular APP is present in low molecular weight immature forms ( im ) and high molecular weight mature form ( m ). ADAM10 is present as a mature ( m ) form and predominantly as an immature ( im ) form. B , quantification of ADAM10 protein ( black bars ) and mRNA levels ( white bars ) from cells transfected with ADAM10 cDNA constructs shown in A . ADAM10 protein levels were normalized to GFP and actin levels. The signal for ADAM10 with the wild-type G-quadruplex GQ-WT ADAM10 was set to 1. Results are expressed as the means ± S.D. from three experiments made in triplicate. ADAM10 mRNA was normalized to glycerolaldehyde-3-phosphate-dehydrogenase mRNA levels, and the signal for GQ-WT ADAM10 was set to 1. Results are expressed as the means ± S.D. from three experiments. C , quantification of secreted APPsα from cells transfected with the indicated ADAM10 variants were shown in A . The signal for APPsα from GQ-WT ADAM10 transfected cells was set to 100%. Results are expressed as the means ± S.D. from three experiments. Asterisks indicate statistical significance (one-way analysis of variance with Dunnett's post test) relative to GQ-WT ADAM10 transfected cells (*, p
    Figure Legend Snippet: ADAM10 expression is repressed by the G-quadruplex motif. A , HEK293 cells were transiently transfected with the indicated ADAM10 cDNA constructs, and lysates were analyzed by immunoblotting for V5-tagged ADAM10, endogenous APP, β-actin as loading control, and GFP as transfection control. Supernatants were analyzed for APPsα secretion using antibody 2D8. Cellular APP is present in low molecular weight immature forms ( im ) and high molecular weight mature form ( m ). ADAM10 is present as a mature ( m ) form and predominantly as an immature ( im ) form. B , quantification of ADAM10 protein ( black bars ) and mRNA levels ( white bars ) from cells transfected with ADAM10 cDNA constructs shown in A . ADAM10 protein levels were normalized to GFP and actin levels. The signal for ADAM10 with the wild-type G-quadruplex GQ-WT ADAM10 was set to 1. Results are expressed as the means ± S.D. from three experiments made in triplicate. ADAM10 mRNA was normalized to glycerolaldehyde-3-phosphate-dehydrogenase mRNA levels, and the signal for GQ-WT ADAM10 was set to 1. Results are expressed as the means ± S.D. from three experiments. C , quantification of secreted APPsα from cells transfected with the indicated ADAM10 variants were shown in A . The signal for APPsα from GQ-WT ADAM10 transfected cells was set to 100%. Results are expressed as the means ± S.D. from three experiments. Asterisks indicate statistical significance (one-way analysis of variance with Dunnett's post test) relative to GQ-WT ADAM10 transfected cells (*, p

    Techniques Used: Expressing, Transfection, Construct, Molecular Weight

    Translational repression of a luciferase reporter by the ADAM10 G-quadruplex motif. A, Schematic representation of the plasmids used for reporter gene assays. The wild-type G-quadruplex sequence (GQ-WT) of the ADAM10 5′-UTR or mutated variants thereof were cloned directly in front of the Renilla coding region. B, 24 h after transfection of the indicated plasmids in HEK293 cells dual-luciferase assays were performed and mRNA was isolated. Renilla luciferase activity was normalized to Firefly luciferase activity and the value for GQ-WT was set to 100%. C T values for Renilla and Firefly luciferase mRNA were determined by quantitative RT-PCR and the ratio of C T Renilla/C T ). Results are expressed as means ± S.D. of at least three independent experiments made in triplicates.
    Figure Legend Snippet: Translational repression of a luciferase reporter by the ADAM10 G-quadruplex motif. A, Schematic representation of the plasmids used for reporter gene assays. The wild-type G-quadruplex sequence (GQ-WT) of the ADAM10 5′-UTR or mutated variants thereof were cloned directly in front of the Renilla coding region. B, 24 h after transfection of the indicated plasmids in HEK293 cells dual-luciferase assays were performed and mRNA was isolated. Renilla luciferase activity was normalized to Firefly luciferase activity and the value for GQ-WT was set to 100%. C T values for Renilla and Firefly luciferase mRNA were determined by quantitative RT-PCR and the ratio of C T Renilla/C T ). Results are expressed as means ± S.D. of at least three independent experiments made in triplicates.

    Techniques Used: Luciferase, Sequencing, Clone Assay, Transfection, Isolation, Activity Assay, Quantitative RT-PCR

    2) Product Images from "Targeted deletion of the aquaglyceroporin AQP9 is protective in a mouse model of Parkinson’s disease"

    Article Title: Targeted deletion of the aquaglyceroporin AQP9 is protective in a mouse model of Parkinson’s disease

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0194896

    HEK293 cells expressing h AQP9 or h DAT reveal higher sensitivity to MPP + . A-B) Immunofluorescence images of HEK293 cells expressing EGFP- h AQP9 (A) or YFP- h DAT (B) grown on coverslips confirm plasma membrane localization of the respective constructs (identified by antibodies to AQP9 or DAT). The cells were counterstained with Hoechst to visualize nuclei. C-G) Native HEK293 cells and HEK293 cells expressing EGFP- h AQP9 or YFP- h DAT were grown in 96-well plates and exposed to different concentrations of MPP + (four wells for each concentration). Cell viability was assessed after 24 hours using the MTT assay. Data were collected from independent plates (n = 3 for each construct) and normalized to respective untreated cells. Native HEK293 cells show sensitivity to MPP + only at very high concentrations (~100 μM). Cells expressing h DAT become sensitive at 1 μM MPP + , compared with 0.1 μM for cells expressing h AQP9. Overlay of the dose/response curve for the three groups (D) and the individual curves for native HEK293 cells (E), YFP- h DAT expressing (F) and EGFP- h AQP9 expressing HEK293 cells (G) are shown. Bars are mean ± SEM. Asterisks: significantly different from untreated controls; *p
    Figure Legend Snippet: HEK293 cells expressing h AQP9 or h DAT reveal higher sensitivity to MPP + . A-B) Immunofluorescence images of HEK293 cells expressing EGFP- h AQP9 (A) or YFP- h DAT (B) grown on coverslips confirm plasma membrane localization of the respective constructs (identified by antibodies to AQP9 or DAT). The cells were counterstained with Hoechst to visualize nuclei. C-G) Native HEK293 cells and HEK293 cells expressing EGFP- h AQP9 or YFP- h DAT were grown in 96-well plates and exposed to different concentrations of MPP + (four wells for each concentration). Cell viability was assessed after 24 hours using the MTT assay. Data were collected from independent plates (n = 3 for each construct) and normalized to respective untreated cells. Native HEK293 cells show sensitivity to MPP + only at very high concentrations (~100 μM). Cells expressing h DAT become sensitive at 1 μM MPP + , compared with 0.1 μM for cells expressing h AQP9. Overlay of the dose/response curve for the three groups (D) and the individual curves for native HEK293 cells (E), YFP- h DAT expressing (F) and EGFP- h AQP9 expressing HEK293 cells (G) are shown. Bars are mean ± SEM. Asterisks: significantly different from untreated controls; *p

    Techniques Used: Expressing, Immunofluorescence, Construct, Concentration Assay, MTT Assay

    HEK293 cells expressing EGFP- h AQP9 are more sensitive to arsenite than HEK293 cells expressing YFP- h DAT. A-C) Native HEK293 cells and HEK293 cells expressing EGFP- h AQP9 or YFP- h DAT were grown in 96-well plates and exposed to different concentrations of arsenite (eight wells for each concentration). Cell viability was assessed after 24 hours using the MTT assay. Data were collected from independent plates (n = 3 for each construct) and normalized to respective untreated cells. Both EGFP- h AQP9 and YFP- h DAT expressing cells showed higher sensitivity to arsenite, than native HEK293 cells, with EGFP- h AQP9 cells being the most sensitive. At the arsenite concentration of 10 μM, stably transfected EGFP- h AQP9 were the only cells showing toxin sensitivity (A). The curve showing IC50 values for arsenite calculated by nonlinear regression, log(inhibitor) vs response (three parameters) is shown (B). For log transformed data, the concentration 0 was set to 1 nM. Comparison of the IC50 values shows a significantly lower IC50 value for the HEK293 cells expressing EGFP- h AQP9 compared to the native HEK293 cells or HEK293 cells expressing YFP- h DAT (C). Bars are mean ± SEM. Asterisks: significantly different from untreated controls; *p
    Figure Legend Snippet: HEK293 cells expressing EGFP- h AQP9 are more sensitive to arsenite than HEK293 cells expressing YFP- h DAT. A-C) Native HEK293 cells and HEK293 cells expressing EGFP- h AQP9 or YFP- h DAT were grown in 96-well plates and exposed to different concentrations of arsenite (eight wells for each concentration). Cell viability was assessed after 24 hours using the MTT assay. Data were collected from independent plates (n = 3 for each construct) and normalized to respective untreated cells. Both EGFP- h AQP9 and YFP- h DAT expressing cells showed higher sensitivity to arsenite, than native HEK293 cells, with EGFP- h AQP9 cells being the most sensitive. At the arsenite concentration of 10 μM, stably transfected EGFP- h AQP9 were the only cells showing toxin sensitivity (A). The curve showing IC50 values for arsenite calculated by nonlinear regression, log(inhibitor) vs response (three parameters) is shown (B). For log transformed data, the concentration 0 was set to 1 nM. Comparison of the IC50 values shows a significantly lower IC50 value for the HEK293 cells expressing EGFP- h AQP9 compared to the native HEK293 cells or HEK293 cells expressing YFP- h DAT (C). Bars are mean ± SEM. Asterisks: significantly different from untreated controls; *p

    Techniques Used: Expressing, Concentration Assay, MTT Assay, Construct, Stable Transfection, Transfection, Transformation Assay

    3) Product Images from "DDX5 resolves R-loops at DNA double-strand breaks to promote DNA repair and avoid chromosomal deletions"

    Article Title: DDX5 resolves R-loops at DNA double-strand breaks to promote DNA repair and avoid chromosomal deletions

    Journal: Nar Cancer

    doi: 10.1093/narcan/zcaa028

    The CRISPR–Cas LMNA HDR system accumulates R-loops in a DDX5-dependent manner. ( A ) Illustration of the Cas9-directed knock-in of the Clover in the LMNA coding sequence. The red and the blue arrows represent both arms used for HDR. Cells were transfected with plasmids for CRISPR–Cas LMNA HDR analysis. B, E and H denote the location of the Bsr GI, Eco RI and Hind III restriction sites. qPCR amplification region is shown at the top of the red homology arm of the LMNA used for the DRIP-qPCR. ( B ) HEK293 cells were transfected with pcDNA or RNAse H1 (RNH1) expressing vector along with the siRNAs. Cells were then transfected with the CRISPR–Cas LMNA HDR system and iRFP plasmids and subjected to DRIP-qPCR analysis at both LMNA homology arms and the EGR1 control locus. ( C ) HEK293 cells were transfected with Flag-DDX5 and subjected to ChIP-qPCR analysis. The bar graphs are the average and SEM from three independent experiments. Statistical significance was assessed using t -test: * P
    Figure Legend Snippet: The CRISPR–Cas LMNA HDR system accumulates R-loops in a DDX5-dependent manner. ( A ) Illustration of the Cas9-directed knock-in of the Clover in the LMNA coding sequence. The red and the blue arrows represent both arms used for HDR. Cells were transfected with plasmids for CRISPR–Cas LMNA HDR analysis. B, E and H denote the location of the Bsr GI, Eco RI and Hind III restriction sites. qPCR amplification region is shown at the top of the red homology arm of the LMNA used for the DRIP-qPCR. ( B ) HEK293 cells were transfected with pcDNA or RNAse H1 (RNH1) expressing vector along with the siRNAs. Cells were then transfected with the CRISPR–Cas LMNA HDR system and iRFP plasmids and subjected to DRIP-qPCR analysis at both LMNA homology arms and the EGR1 control locus. ( C ) HEK293 cells were transfected with Flag-DDX5 and subjected to ChIP-qPCR analysis. The bar graphs are the average and SEM from three independent experiments. Statistical significance was assessed using t -test: * P

    Techniques Used: CRISPR, Knock-In, Sequencing, Transfection, Real-time Polymerase Chain Reaction, Amplification, Expressing, Plasmid Preparation, Chromatin Immunoprecipitation

    Increased DNA end deletions in DDX5-deficient cells are associated with local gene transcription. ( A ) Illustration of a tetracycline-induced (tetO) reporter system for NHEJ repair analysis in HEK293. The reporter construct is similar to the one described in Figure 4A except that the reporter expression is controlled by a tet-on promoter. ( B ) RT-qPCR analysis of the expression of puromycin in the absence (−Dox) or presence (+Dox) of 1 μg/ml Dox. ( C ) The HEK293-tetO-puro-GFP reporter cells were transfected with indicated siLuc control siRNA (siCTL) and siDDX5 #3 (siDDX5), respectively, in the absence (−Dox) or presence (+Dox) of 1 μg/ml Dox. Forty to forty-four hours after the siRNA transfection, the cells were then transfected with I-SceI-expressing vector (pCAG-I-SceI). Seventy hours after the plasmid transfection, the cells were harvested and the genomic DNA was extracted for PCR analysis using the primers shown in ( A ). The PCR primers amplify a DNA fragment with a size of 733 bp if the two ends are accurately repaired. Under the PCR reaction condition, the DNA (3573–1581 equals 1992 bp) without cleavage cannot be amplified. A representative agarose gel was shown for the analysis of the PCR products. ( D ) The PCR products amplified with the primers as in ( A ) were subjected to qPCR analysis targeting different regions surrounding the I-SceI sites. The ratio of F1/F2 that was normalized to the one in the siCTL sample. ( E ) The graph shows the average and SEM from three independent experiments performed in triplicates. ( F , G ) The HEK293-tetO-puro-GFP reporter cells were co-transfected with Flag-DDX5 and I-SceI-expressing plasmids in the absence (−Dox) or presence (+Dox) of 1 μg/ml Dox. ChIP-qPCR was performed to determine DDX5 occupancy near the I-SceI-cleaved DNA breaks (P1 and P2). MDM2 promoter region was used as a positive control. The results were normalized to IgG control at each condition. The graph shows the average and SEM from four independent experiments. Statistical significance was assessed using Student’s t -test: * P
    Figure Legend Snippet: Increased DNA end deletions in DDX5-deficient cells are associated with local gene transcription. ( A ) Illustration of a tetracycline-induced (tetO) reporter system for NHEJ repair analysis in HEK293. The reporter construct is similar to the one described in Figure 4A except that the reporter expression is controlled by a tet-on promoter. ( B ) RT-qPCR analysis of the expression of puromycin in the absence (−Dox) or presence (+Dox) of 1 μg/ml Dox. ( C ) The HEK293-tetO-puro-GFP reporter cells were transfected with indicated siLuc control siRNA (siCTL) and siDDX5 #3 (siDDX5), respectively, in the absence (−Dox) or presence (+Dox) of 1 μg/ml Dox. Forty to forty-four hours after the siRNA transfection, the cells were then transfected with I-SceI-expressing vector (pCAG-I-SceI). Seventy hours after the plasmid transfection, the cells were harvested and the genomic DNA was extracted for PCR analysis using the primers shown in ( A ). The PCR primers amplify a DNA fragment with a size of 733 bp if the two ends are accurately repaired. Under the PCR reaction condition, the DNA (3573–1581 equals 1992 bp) without cleavage cannot be amplified. A representative agarose gel was shown for the analysis of the PCR products. ( D ) The PCR products amplified with the primers as in ( A ) were subjected to qPCR analysis targeting different regions surrounding the I-SceI sites. The ratio of F1/F2 that was normalized to the one in the siCTL sample. ( E ) The graph shows the average and SEM from three independent experiments performed in triplicates. ( F , G ) The HEK293-tetO-puro-GFP reporter cells were co-transfected with Flag-DDX5 and I-SceI-expressing plasmids in the absence (−Dox) or presence (+Dox) of 1 μg/ml Dox. ChIP-qPCR was performed to determine DDX5 occupancy near the I-SceI-cleaved DNA breaks (P1 and P2). MDM2 promoter region was used as a positive control. The results were normalized to IgG control at each condition. The graph shows the average and SEM from four independent experiments. Statistical significance was assessed using Student’s t -test: * P

    Techniques Used: Non-Homologous End Joining, Construct, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Positive Control

    4) Product Images from "Human sorting nexin 2 protein interacts with Influenza A virus PA protein and has a negative regulatory effect on the virus replication"

    Article Title: Human sorting nexin 2 protein interacts with Influenza A virus PA protein and has a negative regulatory effect on the virus replication

    Journal: Molecular Biology Reports

    doi: 10.1007/s11033-021-06906-9

    Rescue effect of SNX2 proteins on influenza A/DkPen PA inhibitory activity to host gene expression. The HEK293 cells were grown in a 24-well plate (5 × 10 4 cells/well) for 24 h in standard culture conditions and transfected with constant amounts of pCAGGS-PA(D) (1 ng/well) and pSEAP (20 ng/well) plasmids and indicated amounts of the SNX2 plasmids in the figure. After 24 h of transfection, the reporter SEAP activities in culture media was detected with a commercial kit
    Figure Legend Snippet: Rescue effect of SNX2 proteins on influenza A/DkPen PA inhibitory activity to host gene expression. The HEK293 cells were grown in a 24-well plate (5 × 10 4 cells/well) for 24 h in standard culture conditions and transfected with constant amounts of pCAGGS-PA(D) (1 ng/well) and pSEAP (20 ng/well) plasmids and indicated amounts of the SNX2 plasmids in the figure. After 24 h of transfection, the reporter SEAP activities in culture media was detected with a commercial kit

    Techniques Used: Activity Assay, Expressing, Transfection

    5) Product Images from "Binding of Guanylyl Cyclase Activating Protein 1 (GCAP1) to Retinal Guanylyl Cyclase (RetGC1)"

    Article Title: Binding of Guanylyl Cyclase Activating Protein 1 (GCAP1) to Retinal Guanylyl Cyclase (RetGC1)

    Journal:

    doi: 10.1074/jbc.M801899200

    Characteristic cellular localization of GCAP1-GFP co-expressed with RetGC1. A , fluorescence of GCAP1-GFP in HEK293 cells expressed in the absence ( a ) and presence ( b-d ) of RetGC1, nc , nuclei; ni, nucleoli; a and b, fluorescence of GCAP1-GFP; c , immunofluorescence
    Figure Legend Snippet: Characteristic cellular localization of GCAP1-GFP co-expressed with RetGC1. A , fluorescence of GCAP1-GFP in HEK293 cells expressed in the absence ( a ) and presence ( b-d ) of RetGC1, nc , nuclei; ni, nucleoli; a and b, fluorescence of GCAP1-GFP; c , immunofluorescence

    Techniques Used: Fluorescence, Immunofluorescence

    Properties of dsRed-RetGC1 and GCAP1-GFP. A , activation of non-tagged RetGC1 in HEK293 cell membranes by purified non-tagged (□) or GFP-tagged (○) GCAP1; B , Ca 2+ sensitivity of the non-tagged RetGC1 reconstituted with the non-tagged
    Figure Legend Snippet: Properties of dsRed-RetGC1 and GCAP1-GFP. A , activation of non-tagged RetGC1 in HEK293 cell membranes by purified non-tagged (□) or GFP-tagged (○) GCAP1; B , Ca 2+ sensitivity of the non-tagged RetGC1 reconstituted with the non-tagged

    Techniques Used: Activation Assay, Purification

    6) Product Images from "Human sorting nexin 2 protein interacts with Influenza A virus PA protein and has a negative regulatory effect on the virus replication"

    Article Title: Human sorting nexin 2 protein interacts with Influenza A virus PA protein and has a negative regulatory effect on the virus replication

    Journal: Molecular Biology Reports

    doi: 10.1007/s11033-021-06906-9

    Rescue effect of SNX2 proteins on influenza A/DkPen PA inhibitory activity to host gene expression. The HEK293 cells were grown in a 24-well plate (5 × 10 4 cells/well) for 24 h in standard culture conditions and transfected with constant amounts of pCAGGS-PA(D) (1 ng/well) and pSEAP (20 ng/well) plasmids and indicated amounts of the SNX2 plasmids in the figure. After 24 h of transfection, the reporter SEAP activities in culture media was detected with a commercial kit
    Figure Legend Snippet: Rescue effect of SNX2 proteins on influenza A/DkPen PA inhibitory activity to host gene expression. The HEK293 cells were grown in a 24-well plate (5 × 10 4 cells/well) for 24 h in standard culture conditions and transfected with constant amounts of pCAGGS-PA(D) (1 ng/well) and pSEAP (20 ng/well) plasmids and indicated amounts of the SNX2 plasmids in the figure. After 24 h of transfection, the reporter SEAP activities in culture media was detected with a commercial kit

    Techniques Used: Activity Assay, Expressing, Transfection

    7) Product Images from "Attenuated fusogenicity and pathogenicity of SARS-CoV-2 Omicron variant"

    Article Title: Attenuated fusogenicity and pathogenicity of SARS-CoV-2 Omicron variant

    Journal: Nature

    doi: 10.1038/s41586-022-04462-1

    Cell–cell fusion mediated by the SARS-CoV-2 S protein. a , SARS-CoV-2 S-based fusion assay. Effector cells (S-expressing cells) and target cells (Calu-3 cells, HEK293-ACE2 cells and HEK293-ACE2/TMPRSS2 cells) were prepared, and the fusion activity was measured as described in the Methods. Assays were performed in quadruplicate, and fusion activity (arbitrary units) is shown. b , Coculture of S-expressing cells with HEK293-ACE2/TMPRSS2 cells. Left, representative images of S-expressing cells (green) cocultured with HEK293 cells (red, top) or HEK293-ACE2/TMPRSS2 cells (red, bottom). Nuclei were stained with Hoechst33342 (blue). Scale bars, 50 μm. Right, the size distributions of syncytia (yellow) in the HEK293-ACE2/TMPRSS2 cultures cocultured with the cells expressing the parental D614G S (50 yellow syncytia), Delta S (54 yellow syncytia) or Omicron S (58 yellow syncytia). Data are mean ± s.d. In a , statistically significant differences versus B.1.1 and Delta through time points were determined by multiple regression. FWERs calculated using the Holm method are indicated in the figures. In b , each dot indicates the result from an individual yellow syncytium. Statistically significant differences (* P
    Figure Legend Snippet: Cell–cell fusion mediated by the SARS-CoV-2 S protein. a , SARS-CoV-2 S-based fusion assay. Effector cells (S-expressing cells) and target cells (Calu-3 cells, HEK293-ACE2 cells and HEK293-ACE2/TMPRSS2 cells) were prepared, and the fusion activity was measured as described in the Methods. Assays were performed in quadruplicate, and fusion activity (arbitrary units) is shown. b , Coculture of S-expressing cells with HEK293-ACE2/TMPRSS2 cells. Left, representative images of S-expressing cells (green) cocultured with HEK293 cells (red, top) or HEK293-ACE2/TMPRSS2 cells (red, bottom). Nuclei were stained with Hoechst33342 (blue). Scale bars, 50 μm. Right, the size distributions of syncytia (yellow) in the HEK293-ACE2/TMPRSS2 cultures cocultured with the cells expressing the parental D614G S (50 yellow syncytia), Delta S (54 yellow syncytia) or Omicron S (58 yellow syncytia). Data are mean ± s.d. In a , statistically significant differences versus B.1.1 and Delta through time points were determined by multiple regression. FWERs calculated using the Holm method are indicated in the figures. In b , each dot indicates the result from an individual yellow syncytium. Statistically significant differences (* P

    Techniques Used: Single Vesicle Fusion Assay, Expressing, Activity Assay, Staining

    8) Product Images from "Expression profiles of interferon-related genes in cells infected with influenza A viruses or transiently transfected with plasmids encoding viral RNA polymerase"

    Article Title: Expression profiles of interferon-related genes in cells infected with influenza A viruses or transiently transfected with plasmids encoding viral RNA polymerase

    Journal: Turkish Journal of Biology

    doi: 10.3906/biy-2005-73

    The effects of influenza A virus PA proteins on the expression of SEAP reporter in transiently transfected HEK293 cells. A. The cells expressing native PA proteins. B. The cells expressing chimeric PA proteins. C. The cells expressing influenza PA proteins with WSN PB2 and PB1. D. The cells expressing influenza PA proteins with DkPen, PB2 and PB1. Total plasmid DNA adjusted to 250 ng/well with pCAGGS plasmid DNA (Niwa et al., 1991).
    Figure Legend Snippet: The effects of influenza A virus PA proteins on the expression of SEAP reporter in transiently transfected HEK293 cells. A. The cells expressing native PA proteins. B. The cells expressing chimeric PA proteins. C. The cells expressing influenza PA proteins with WSN PB2 and PB1. D. The cells expressing influenza PA proteins with DkPen, PB2 and PB1. Total plasmid DNA adjusted to 250 ng/well with pCAGGS plasmid DNA (Niwa et al., 1991).

    Techniques Used: Expressing, Transfection, Plasmid Preparation

    The heatmaps and Venn diagram of the genes related to the interferon response in HEK293 cells infected with influenza A viruses (WSN or DkPen) and noninfected cells (HEK293).
    Figure Legend Snippet: The heatmaps and Venn diagram of the genes related to the interferon response in HEK293 cells infected with influenza A viruses (WSN or DkPen) and noninfected cells (HEK293).

    Techniques Used: Infection

    The heatmaps and Venn diagram of the interferon response genes in HEK293 cells transiently transected with plasmid DNAs expressing influenza A virus RdRP enzyme (3P).
    Figure Legend Snippet: The heatmaps and Venn diagram of the interferon response genes in HEK293 cells transiently transected with plasmid DNAs expressing influenza A virus RdRP enzyme (3P).

    Techniques Used: Plasmid Preparation, Expressing

    The heatmaps and Venn diagram of the interferon response genes in HEK293 cells transiently transected with plasmid DNAs expressing the PA subunit of influenza A virus RdRP.
    Figure Legend Snippet: The heatmaps and Venn diagram of the interferon response genes in HEK293 cells transiently transected with plasmid DNAs expressing the PA subunit of influenza A virus RdRP.

    Techniques Used: Plasmid Preparation, Expressing

    Western blot analysis of native actin beta and HA-actin proteins in transiently transfected HEK293 cells. The cells were cotransfected with pCHA-ACTB plasmid and a plasmid expressing deleted PA protein (pCAGGS-cPA/DkPen, pCAGGS-cPA/WSN, pCAGGS-nPA/DkPen or pCAGGS-nPA/WSN). Actin beta and viral PA proteins were separated on 10% polyacrylamide gel and immunoblotted with monoclonal mouse anti-HA (for HA-ACTB), monoclonal anti actin (for ACTB and HA-ACTB) and rabbit polyclonal anti-PA antibodies.
    Figure Legend Snippet: Western blot analysis of native actin beta and HA-actin proteins in transiently transfected HEK293 cells. The cells were cotransfected with pCHA-ACTB plasmid and a plasmid expressing deleted PA protein (pCAGGS-cPA/DkPen, pCAGGS-cPA/WSN, pCAGGS-nPA/DkPen or pCAGGS-nPA/WSN). Actin beta and viral PA proteins were separated on 10% polyacrylamide gel and immunoblotted with monoclonal mouse anti-HA (for HA-ACTB), monoclonal anti actin (for ACTB and HA-ACTB) and rabbit polyclonal anti-PA antibodies.

    Techniques Used: Western Blot, Transfection, Plasmid Preparation, Expressing

    The expression profiles of interferon response genes in HEK293 cells infected with influenza A viruses (WSN or DkPen) and noninfected cells (HEK293).
    Figure Legend Snippet: The expression profiles of interferon response genes in HEK293 cells infected with influenza A viruses (WSN or DkPen) and noninfected cells (HEK293).

    Techniques Used: Expressing, Infection

    9) Product Images from "Expression profiles of interferon-related genes in cells infected with influenza A viruses or transiently transfected with plasmids encoding viral RNA polymerase"

    Article Title: Expression profiles of interferon-related genes in cells infected with influenza A viruses or transiently transfected with plasmids encoding viral RNA polymerase

    Journal: Turkish Journal of Biology

    doi: 10.3906/biy-2005-73

    The effects of influenza A virus PA proteins on the expression of SEAP reporter in transiently transfected HEK293 cells. A. The cells expressing native PA proteins. B. The cells expressing chimeric PA proteins. C. The cells expressing influenza PA proteins with WSN PB2 and PB1. D. The cells expressing influenza PA proteins with DkPen, PB2 and PB1. Total plasmid DNA adjusted to 250 ng/well with pCAGGS plasmid DNA (Niwa et al., 1991).
    Figure Legend Snippet: The effects of influenza A virus PA proteins on the expression of SEAP reporter in transiently transfected HEK293 cells. A. The cells expressing native PA proteins. B. The cells expressing chimeric PA proteins. C. The cells expressing influenza PA proteins with WSN PB2 and PB1. D. The cells expressing influenza PA proteins with DkPen, PB2 and PB1. Total plasmid DNA adjusted to 250 ng/well with pCAGGS plasmid DNA (Niwa et al., 1991).

    Techniques Used: Expressing, Transfection, Plasmid Preparation

    The heatmaps and Venn diagram of the genes related to the interferon response in HEK293 cells infected with influenza A viruses (WSN or DkPen) and noninfected cells (HEK293).
    Figure Legend Snippet: The heatmaps and Venn diagram of the genes related to the interferon response in HEK293 cells infected with influenza A viruses (WSN or DkPen) and noninfected cells (HEK293).

    Techniques Used: Infection

    The heatmaps and Venn diagram of the interferon response genes in HEK293 cells transiently transected with plasmid DNAs expressing influenza A virus RdRP enzyme (3P).
    Figure Legend Snippet: The heatmaps and Venn diagram of the interferon response genes in HEK293 cells transiently transected with plasmid DNAs expressing influenza A virus RdRP enzyme (3P).

    Techniques Used: Plasmid Preparation, Expressing

    The heatmaps and Venn diagram of the interferon response genes in HEK293 cells transiently transected with plasmid DNAs expressing the PA subunit of influenza A virus RdRP.
    Figure Legend Snippet: The heatmaps and Venn diagram of the interferon response genes in HEK293 cells transiently transected with plasmid DNAs expressing the PA subunit of influenza A virus RdRP.

    Techniques Used: Plasmid Preparation, Expressing

    Western blot analysis of native actin beta and HA-actin proteins in transiently transfected HEK293 cells. The cells were cotransfected with pCHA-ACTB plasmid and a plasmid expressing deleted PA protein (pCAGGS-cPA/DkPen, pCAGGS-cPA/WSN, pCAGGS-nPA/DkPen or pCAGGS-nPA/WSN). Actin beta and viral PA proteins were separated on 10% polyacrylamide gel and immunoblotted with monoclonal mouse anti-HA (for HA-ACTB), monoclonal anti actin (for ACTB and HA-ACTB) and rabbit polyclonal anti-PA antibodies.
    Figure Legend Snippet: Western blot analysis of native actin beta and HA-actin proteins in transiently transfected HEK293 cells. The cells were cotransfected with pCHA-ACTB plasmid and a plasmid expressing deleted PA protein (pCAGGS-cPA/DkPen, pCAGGS-cPA/WSN, pCAGGS-nPA/DkPen or pCAGGS-nPA/WSN). Actin beta and viral PA proteins were separated on 10% polyacrylamide gel and immunoblotted with monoclonal mouse anti-HA (for HA-ACTB), monoclonal anti actin (for ACTB and HA-ACTB) and rabbit polyclonal anti-PA antibodies.

    Techniques Used: Western Blot, Transfection, Plasmid Preparation, Expressing

    The expression profiles of interferon response genes in HEK293 cells infected with influenza A viruses (WSN or DkPen) and noninfected cells (HEK293).
    Figure Legend Snippet: The expression profiles of interferon response genes in HEK293 cells infected with influenza A viruses (WSN or DkPen) and noninfected cells (HEK293).

    Techniques Used: Expressing, Infection

    10) Product Images from "Cell-to-Cell Transmission Can Overcome Multiple Donor and Target Cell Barriers Imposed on Cell-Free HIV"

    Article Title: Cell-to-Cell Transmission Can Overcome Multiple Donor and Target Cell Barriers Imposed on Cell-Free HIV

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0053138

    Co-culturing cells leads to a larger proportion of infected target cells and a higher proviral content. ( A ) Infection by cell-free or by co-cultures with MT4, Jurkat or primary CD4+ T cells were repeated as in Figure 5B using HEK293 donor cells producing HIV IRES-GFP . Target cells were identified based on their expression of CD2 (MT4 cells) or CD3 (Jurkat and primary CD4 T cells). GFP fluorescence was gated based on the background fluorescence from a corresponding sample treated with 1 µM of efavirenz (EFV). Numbers represent the average percent of GFP-positive cells +/− the standard deviation of 2 (efavirenz control) or 6 (no drug) inoculations. MFI values correspond to the average mean fluorescence +/− the standard deviation of 2 (efavirenz control) or 6 (no drug) inoculations. ( B ) Comparison of the GFP fluorescence intensity of cells infected by cell-free virus or by co-culture from the gated population in panel (A). Histograms represent the combination of 3 measurements. Fluorescence was normalized to the fluorescence from the corresponding efavirenz-controls to account for fluorescence shifts due to sample variability. The grey dashed line represents cell-free infection and solid black line represent co-culture infection. The black vertical dashed line represents the limit of the gates shown in (A). ( C ) An experiment as in (A) was repeated with wild-type HIV NL4-3 and target cells were stained with CFSE. CFSE-positive cells expressing HIV Gag above the efavirenz-treated control (see Figure S5 ) were sorted and the number of HIV integration events was analyzed by Alu -PCR. Control samples were treated with 1 µM efavirenz (+EFV). Error bars represent the standard deviation of 3 measurements of integration.
    Figure Legend Snippet: Co-culturing cells leads to a larger proportion of infected target cells and a higher proviral content. ( A ) Infection by cell-free or by co-cultures with MT4, Jurkat or primary CD4+ T cells were repeated as in Figure 5B using HEK293 donor cells producing HIV IRES-GFP . Target cells were identified based on their expression of CD2 (MT4 cells) or CD3 (Jurkat and primary CD4 T cells). GFP fluorescence was gated based on the background fluorescence from a corresponding sample treated with 1 µM of efavirenz (EFV). Numbers represent the average percent of GFP-positive cells +/− the standard deviation of 2 (efavirenz control) or 6 (no drug) inoculations. MFI values correspond to the average mean fluorescence +/− the standard deviation of 2 (efavirenz control) or 6 (no drug) inoculations. ( B ) Comparison of the GFP fluorescence intensity of cells infected by cell-free virus or by co-culture from the gated population in panel (A). Histograms represent the combination of 3 measurements. Fluorescence was normalized to the fluorescence from the corresponding efavirenz-controls to account for fluorescence shifts due to sample variability. The grey dashed line represents cell-free infection and solid black line represent co-culture infection. The black vertical dashed line represents the limit of the gates shown in (A). ( C ) An experiment as in (A) was repeated with wild-type HIV NL4-3 and target cells were stained with CFSE. CFSE-positive cells expressing HIV Gag above the efavirenz-treated control (see Figure S5 ) were sorted and the number of HIV integration events was analyzed by Alu -PCR. Control samples were treated with 1 µM efavirenz (+EFV). Error bars represent the standard deviation of 3 measurements of integration.

    Techniques Used: Infection, Expressing, Fluorescence, Standard Deviation, Co-Culture Assay, Staining, Polymerase Chain Reaction

    Sensitivity of HIV transmission by cell-free virus or by transmission in co-cultures to neutralizing antibodies. ( A ) A barrier was placed into the cell-free path of HIV at the extracellular step using neutralizing antibodies. ( B ) The infectivity of cell-free HIV NL4-3-GLuc on MT4 cells was measured in the absence and presence of increasing amounts of neutralizing antibodies (NAb) 4E10, 2G12, and 2F5 (µg/ml). Infectivity was normalized to 1 using mock-treated control. ( C ) Scheme depicts the experimental approach to test the sensitivity of HIV spreading in co-cultures to neutralizing antibodies. At 6 h post-transfection, donor and target cells were co-cultured and cell-cell contacts were allowed to form for 2 h. Transmission events occurring during these 2 h were measured by terminating transmission with saquinavir (SQV) and allowing all previous infection events to proceed for 36 h and result in luciferase expression. These base level infections were set to 1. In parallel, the ability of neutralizing antibodies (NAb) to interfere with the spreading of infection to indicated cell types during a window of 4 h was determined. At the end of the 4 h incubation transmissions were terminated using SQV and the produced luciferase was determined 32 h later. ( D–F ) Dose response for indicated neutralizing antibodies 4E10, 2G12, 2F5 in co-cultures of HEK293 HIV-producer cells with the indicated target cell lines as described in (C). ( G ) The data point at 2.5 µg/ml from figures D–F. Infectivity is normalized to the baseline infection levels measured during the first 2 h of co-culture. Error bars represent the standard error of the mean from 3 experiments.
    Figure Legend Snippet: Sensitivity of HIV transmission by cell-free virus or by transmission in co-cultures to neutralizing antibodies. ( A ) A barrier was placed into the cell-free path of HIV at the extracellular step using neutralizing antibodies. ( B ) The infectivity of cell-free HIV NL4-3-GLuc on MT4 cells was measured in the absence and presence of increasing amounts of neutralizing antibodies (NAb) 4E10, 2G12, and 2F5 (µg/ml). Infectivity was normalized to 1 using mock-treated control. ( C ) Scheme depicts the experimental approach to test the sensitivity of HIV spreading in co-cultures to neutralizing antibodies. At 6 h post-transfection, donor and target cells were co-cultured and cell-cell contacts were allowed to form for 2 h. Transmission events occurring during these 2 h were measured by terminating transmission with saquinavir (SQV) and allowing all previous infection events to proceed for 36 h and result in luciferase expression. These base level infections were set to 1. In parallel, the ability of neutralizing antibodies (NAb) to interfere with the spreading of infection to indicated cell types during a window of 4 h was determined. At the end of the 4 h incubation transmissions were terminated using SQV and the produced luciferase was determined 32 h later. ( D–F ) Dose response for indicated neutralizing antibodies 4E10, 2G12, 2F5 in co-cultures of HEK293 HIV-producer cells with the indicated target cell lines as described in (C). ( G ) The data point at 2.5 µg/ml from figures D–F. Infectivity is normalized to the baseline infection levels measured during the first 2 h of co-culture. Error bars represent the standard error of the mean from 3 experiments.

    Techniques Used: Transmission Assay, Infection, Transfection, Cell Culture, Luciferase, Expressing, Incubation, Produced, Co-Culture Assay

    The relative contribution of cell-free to co-culture mediated transmission is affected by the donor cell type. Different donor cells (HEK293, HeLa, Jurkat) were co-cultured with HeLa cells expressing CD4/CXCR4, MT4 cells and Jurkat cells. The efficiency of virus transmission in the cell-free mode and in the co-culture-dependent mode was compared as described in Figure 1A . Data for HEK293 donor cells are as Figure 5B . Error bars represent the standard error of the mean from 2 experiments.
    Figure Legend Snippet: The relative contribution of cell-free to co-culture mediated transmission is affected by the donor cell type. Different donor cells (HEK293, HeLa, Jurkat) were co-cultured with HeLa cells expressing CD4/CXCR4, MT4 cells and Jurkat cells. The efficiency of virus transmission in the cell-free mode and in the co-culture-dependent mode was compared as described in Figure 1A . Data for HEK293 donor cells are as Figure 5B . Error bars represent the standard error of the mean from 2 experiments.

    Techniques Used: Co-Culture Assay, Transmission Assay, Cell Culture, Expressing

    Concentration of HIV-Gag to sites of cell-cell contacts. ( A–C ) HEK293 cells were co-transfected with HIV NL4-3 and fluorescently tagged HIV NL4-3-GFP (green) or HIV NL4-3-RFP (red) and co-cultured with the dye-labeled target T cells MT4 cells (CFSE, green)(A), Jurkat cells (CMTPX, red))(B), and SupT1 cells (CFSE, green)(C) and imaged by confocal fluorescence microscopy. Size bars represent 10 µm.
    Figure Legend Snippet: Concentration of HIV-Gag to sites of cell-cell contacts. ( A–C ) HEK293 cells were co-transfected with HIV NL4-3 and fluorescently tagged HIV NL4-3-GFP (green) or HIV NL4-3-RFP (red) and co-cultured with the dye-labeled target T cells MT4 cells (CFSE, green)(A), Jurkat cells (CMTPX, red))(B), and SupT1 cells (CFSE, green)(C) and imaged by confocal fluorescence microscopy. Size bars represent 10 µm.

    Techniques Used: Concentration Assay, Transfection, Cell Culture, Labeling, Fluorescence, Microscopy

    Co-culture can overcome an entry barrier into poorly susceptible T cells. ( A ) The existence of potential entry barriers in the cell-free path of HIV transmission was explored by varying target cells. ( B ) An experiment as in ( Figure 1B ) was performed to compare cell-free HIV NL4-3-GLuc infection and spreading infections in co-cultures of HEK293 donor cells with indicated target cells. For comparison, the data for permissive HeLa and MT4 from Figure 1B are shown to the left. Error bars represent the standard error of the mean from 7–8 experiments. ( C ) The binding capability of cell-free HIV NL4-3-GLuc on indicated cell types was tested at 37°C for 2 h (∼9 ng/ml of p24). The cells were then washed to remove unbound particles. Cells were then lysed and analyzed by α-p24-ELISA. “ND” for “non-detectable” by ELISA in > 600,000 infected cells. Error bars represent the standard error of the mean from 2 experiments. ( D ) Relative virus binding was measured as in (C) for indicated cell types following spinoculation with concentrated HIV NL4-3-GLuc virus (∼600 ng/ml). ( E ) Late reverse transcription was measured following incubation at 37°C for 36 h. Data shown in D–E were determined in parallel within one experiment and 3 experiments were combined. Error bars represent the standard error of the mean.
    Figure Legend Snippet: Co-culture can overcome an entry barrier into poorly susceptible T cells. ( A ) The existence of potential entry barriers in the cell-free path of HIV transmission was explored by varying target cells. ( B ) An experiment as in ( Figure 1B ) was performed to compare cell-free HIV NL4-3-GLuc infection and spreading infections in co-cultures of HEK293 donor cells with indicated target cells. For comparison, the data for permissive HeLa and MT4 from Figure 1B are shown to the left. Error bars represent the standard error of the mean from 7–8 experiments. ( C ) The binding capability of cell-free HIV NL4-3-GLuc on indicated cell types was tested at 37°C for 2 h (∼9 ng/ml of p24). The cells were then washed to remove unbound particles. Cells were then lysed and analyzed by α-p24-ELISA. “ND” for “non-detectable” by ELISA in > 600,000 infected cells. Error bars represent the standard error of the mean from 2 experiments. ( D ) Relative virus binding was measured as in (C) for indicated cell types following spinoculation with concentrated HIV NL4-3-GLuc virus (∼600 ng/ml). ( E ) Late reverse transcription was measured following incubation at 37°C for 36 h. Data shown in D–E were determined in parallel within one experiment and 3 experiments were combined. Error bars represent the standard error of the mean.

    Techniques Used: Co-Culture Assay, Transmission Assay, Infection, Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation

    Experimental approach for comparing HIV transmission by cell-free virus or by transmission in co-cultures. ( A ) Left panel: Schematic illustration of cell-free and cell-to-cell transmission. Viruses should be able to spread by cell-free transmission if individual steps of the viral life cycle are efficient (1 = viral gene expression, 2 = virus release, 3 = stability of extracellular virus, 4 = virus entry into target cell). Right panel: Schematic illustration for the experimental comparison of HIV transmission by cell-free virus or by transmission in co-cultures using the same pool of transfected virus-producer cells. See text for details. ( B ) Transmission of HIV NL4-3-GLuc by cell-free or in co-cultures were compared between highly permissive donor HEK293 and target cells (HEK293 and HeLa cells expressing CD4 and CXCR4 receptors, MT4 T cells). Infectivity was measured as relative light units of luciferase (RLU). Error bars represent the standard error of the mean from 2–8 experiments.
    Figure Legend Snippet: Experimental approach for comparing HIV transmission by cell-free virus or by transmission in co-cultures. ( A ) Left panel: Schematic illustration of cell-free and cell-to-cell transmission. Viruses should be able to spread by cell-free transmission if individual steps of the viral life cycle are efficient (1 = viral gene expression, 2 = virus release, 3 = stability of extracellular virus, 4 = virus entry into target cell). Right panel: Schematic illustration for the experimental comparison of HIV transmission by cell-free virus or by transmission in co-cultures using the same pool of transfected virus-producer cells. See text for details. ( B ) Transmission of HIV NL4-3-GLuc by cell-free or in co-cultures were compared between highly permissive donor HEK293 and target cells (HEK293 and HeLa cells expressing CD4 and CXCR4 receptors, MT4 T cells). Infectivity was measured as relative light units of luciferase (RLU). Error bars represent the standard error of the mean from 2–8 experiments.

    Techniques Used: Transmission Assay, Expressing, Transfection, Infection, Luciferase

    The reduction in release of cell-free HIV Δvpu by tetherin is overcome in co-cultures. ( A ) A barrier was placed into the cell-free path of HIV at the step of virus release by expressing tetherin. ( B ) HEK293 cells producing HIV LAI-Δvpu and increasing amounts of tetherin (ng) were analyzed for viral gene expression in cells and virus release into the culture supernatant by Western blotting using the α-p24 antibody. The expression of HA-tagged tetherin was confirmed using α-HA antibodies. ( C ) Relative HIV LAI-Δvpu-GLuc infectivity released by HEK293 producer cells expressing increasing amounts of tetherin (red) or transmitted from producer cells to indicated target cell types in co-cultures. The ratio of infectivity to non-tetherin expressing cells was calculated. ( D ) Fold of rescue of co-culture over cell-free for the data shown in (C). Fold-rescue was determined by calculating the ratio of the infectivity in co-cultures over the infectivity of cell-free virus at the corresponding tetherin plasmid dose. Error bars represent the standard error of the mean from 3 experiments. The effects of tetherin expression on wild-type HIV LAI are presented in Figure S2 .
    Figure Legend Snippet: The reduction in release of cell-free HIV Δvpu by tetherin is overcome in co-cultures. ( A ) A barrier was placed into the cell-free path of HIV at the step of virus release by expressing tetherin. ( B ) HEK293 cells producing HIV LAI-Δvpu and increasing amounts of tetherin (ng) were analyzed for viral gene expression in cells and virus release into the culture supernatant by Western blotting using the α-p24 antibody. The expression of HA-tagged tetherin was confirmed using α-HA antibodies. ( C ) Relative HIV LAI-Δvpu-GLuc infectivity released by HEK293 producer cells expressing increasing amounts of tetherin (red) or transmitted from producer cells to indicated target cell types in co-cultures. The ratio of infectivity to non-tetherin expressing cells was calculated. ( D ) Fold of rescue of co-culture over cell-free for the data shown in (C). Fold-rescue was determined by calculating the ratio of the infectivity in co-cultures over the infectivity of cell-free virus at the corresponding tetherin plasmid dose. Error bars represent the standard error of the mean from 3 experiments. The effects of tetherin expression on wild-type HIV LAI are presented in Figure S2 .

    Techniques Used: Expressing, Western Blot, Infection, Co-Culture Assay, Plasmid Preparation

    Co-culture can overcome low viral gene expression. ( A ) A barrier was placed into the cell-free path of HIV by lowering viral gene expression. ( B ) HEK293 producer cells transfected in 12-well plates with decreasing amounts of viral plasmid DNA (µg) were incubated for 30 h and cell lysates and viral supernatant were analyzed by Western blot using α-p24 antibodies. ( C ) Relative HIV NL4-3-GLuc infectivity released by HEK293 producer cells (red) or transmitted from producer cells to indicated target cell types in co-cultures. Infectivity measured for the highest amount of transfected DNA was set to 1. ( D ) Fold of rescue of co-culture over cell-free from (C). Fold-rescue was determined by calculating the ratio of the infectivity in co-cultures over the infectivity of cell-free virus at the corresponding plasmid dose. Error bars represent the standard error of the mean from 3 experiments.
    Figure Legend Snippet: Co-culture can overcome low viral gene expression. ( A ) A barrier was placed into the cell-free path of HIV by lowering viral gene expression. ( B ) HEK293 producer cells transfected in 12-well plates with decreasing amounts of viral plasmid DNA (µg) were incubated for 30 h and cell lysates and viral supernatant were analyzed by Western blot using α-p24 antibodies. ( C ) Relative HIV NL4-3-GLuc infectivity released by HEK293 producer cells (red) or transmitted from producer cells to indicated target cell types in co-cultures. Infectivity measured for the highest amount of transfected DNA was set to 1. ( D ) Fold of rescue of co-culture over cell-free from (C). Fold-rescue was determined by calculating the ratio of the infectivity in co-cultures over the infectivity of cell-free virus at the corresponding plasmid dose. Error bars represent the standard error of the mean from 3 experiments.

    Techniques Used: Co-Culture Assay, Expressing, Transfection, Plasmid Preparation, Incubation, Western Blot, Infection

    11) Product Images from "The Exon Junction Complex Component Y14 Modulates the Activity of the Methylosome in Biogenesis of Spliceosomal Small Nuclear Ribonucleoproteins *"

    Article Title: The Exon Junction Complex Component Y14 Modulates the Activity of the Methylosome in Biogenesis of Spliceosomal Small Nuclear Ribonucleoproteins *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.190587

    Y14 promotes formation of larger PRMT5-containing complex. A , cytoplasmic extracts prepared from mock, FLAG-Y14-, or DDX3-expressing stable HEK293 cells were fractionated on a 5–20% sucrose gradient. Odd-numbered fractions were analyzed by immunoblotting
    Figure Legend Snippet: Y14 promotes formation of larger PRMT5-containing complex. A , cytoplasmic extracts prepared from mock, FLAG-Y14-, or DDX3-expressing stable HEK293 cells were fractionated on a 5–20% sucrose gradient. Odd-numbered fractions were analyzed by immunoblotting

    Techniques Used: Expressing

    Y14 modulates methylation activity of PRMT5 in vivo . Total cell lysates were prepared from the following cells: mock or FLAG-Y14-expressing HEK293 cells ( A ), HEK293 cells that were mock-transfected or transfected with luciferase ( Luc ), Y14-targeting siRNAs,
    Figure Legend Snippet: Y14 modulates methylation activity of PRMT5 in vivo . Total cell lysates were prepared from the following cells: mock or FLAG-Y14-expressing HEK293 cells ( A ), HEK293 cells that were mock-transfected or transfected with luciferase ( Luc ), Y14-targeting siRNAs,

    Techniques Used: Methylation, Activity Assay, In Vivo, Expressing, Transfection, Luciferase

    Y14, but not other EJC factors, interacts with the methylosome. In all panels, HEK293 cells were transiently transfected with the empty vector (−) or the expression vector for each FLAG-tagged protein as indicated above the gels. Cell lysates
    Figure Legend Snippet: Y14, but not other EJC factors, interacts with the methylosome. In all panels, HEK293 cells were transiently transfected with the empty vector (−) or the expression vector for each FLAG-tagged protein as indicated above the gels. Cell lysates

    Techniques Used: Transfection, Plasmid Preparation, Expressing

    Y14 associates with snRNP assembly complex. HEK293 cells were mock-transfected or transiently transfected with the expression vector FLAG-Y14 alone ( B , D , and F ) or together with the vector of HA-SmB ( A ). Anti-FLAG immunoprecipitates ( IP ) were subjected
    Figure Legend Snippet: Y14 associates with snRNP assembly complex. HEK293 cells were mock-transfected or transiently transfected with the expression vector FLAG-Y14 alone ( B , D , and F ) or together with the vector of HA-SmB ( A ). Anti-FLAG immunoprecipitates ( IP ) were subjected

    Techniques Used: Transfection, Expressing, Plasmid Preparation

    12) Product Images from "High molecular weight fibroblast growth factor 2 induces apoptosis by interacting with complement component 1 Q subcomponent–binding protein in vitro. High molecular weight fibroblast growth factor 2 induces apoptosis by interacting with complement component 1 Q subcomponent–binding protein in vitro"

    Article Title: High molecular weight fibroblast growth factor 2 induces apoptosis by interacting with complement component 1 Q subcomponent–binding protein in vitro. High molecular weight fibroblast growth factor 2 induces apoptosis by interacting with complement component 1 Q subcomponent–binding protein in vitro

    Journal: Journal of Cellular Biochemistry

    doi: 10.1002/jcb.27131

    C1QBP is crucial for the mitochondrial localization of hi‐FGF2. VDAC (mitochondrial marker) and β‐actin (cytosolic marker) levels were used as controls. HEK293 cells were transiently transfected with si‐ C1QBP for 3 days prior to Western blot analysis by using the indicated antibodies. CON, control nontransfected cells; C1QBP, complement component 1 Q subcomponent–binding protein; hi‐FGF2, hi‐FGF2–overexpressing cells; si‐ C1QBP , C1QBP knockdown cells; si‐ C1QBP + hi‐FGF2, cells transfected with si‐ C1QBP and hi‐FGF2–expressing vector; VDAC, voltage dependent anion channel
    Figure Legend Snippet: C1QBP is crucial for the mitochondrial localization of hi‐FGF2. VDAC (mitochondrial marker) and β‐actin (cytosolic marker) levels were used as controls. HEK293 cells were transiently transfected with si‐ C1QBP for 3 days prior to Western blot analysis by using the indicated antibodies. CON, control nontransfected cells; C1QBP, complement component 1 Q subcomponent–binding protein; hi‐FGF2, hi‐FGF2–overexpressing cells; si‐ C1QBP , C1QBP knockdown cells; si‐ C1QBP + hi‐FGF2, cells transfected with si‐ C1QBP and hi‐FGF2–expressing vector; VDAC, voltage dependent anion channel

    Techniques Used: Marker, Transfection, Western Blot, Binding Assay, Expressing, Plasmid Preparation

    C1QBP expression is necessary for hi‐FGF2–induced apoptosis. (A) Flow cytometry analysis for determining mitochondrial membrane depolarization in HEK293 cells transfected with empty pDsRed vector, hi‐FGF2–expressing vector, si‐Scr–expressing, si‐ C1QBP –expressing, or si‐ C1QBP + hi‐FGF2–expressing vector. Right, percentage of apoptotic cells in different samples. Data are presented as the mean ± SEM (n = 3); ** P
    Figure Legend Snippet: C1QBP expression is necessary for hi‐FGF2–induced apoptosis. (A) Flow cytometry analysis for determining mitochondrial membrane depolarization in HEK293 cells transfected with empty pDsRed vector, hi‐FGF2–expressing vector, si‐Scr–expressing, si‐ C1QBP –expressing, or si‐ C1QBP + hi‐FGF2–expressing vector. Right, percentage of apoptotic cells in different samples. Data are presented as the mean ± SEM (n = 3); ** P

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

    Hi‐FGF2 overexpression upregulates C1QBP expression. (A) C1QBP protein levels in hi‐FGF2–overexpressing and control HEK293 cells. Left, representative immunoblots showing C1QBP expression; β‐actin was used for normalization. Right, quantification of results. ** P
    Figure Legend Snippet: Hi‐FGF2 overexpression upregulates C1QBP expression. (A) C1QBP protein levels in hi‐FGF2–overexpressing and control HEK293 cells. Left, representative immunoblots showing C1QBP expression; β‐actin was used for normalization. Right, quantification of results. ** P

    Techniques Used: Over Expression, Expressing, Western Blot

    Hi‐FGF2 interacts with C1QBP in HEK293 cells. (A) CoIP of hi‐FGF2 and C1QBP. Western blot analysis was performed using anti‐C1QBP and anti‐RFP antibodies. CoIP was performed using 10% cell lysate as a loading control. Input control (lanes 1 to 3), rabbit IgG (lanes 4 to 6), and immunoprecipitated FGF2 (lanes 10 to 12). (B) CoIP of C1QBP and hi‐FGF2. Cell extracts were immunoprecipitated using the anti‐FGF2 antibody. Western blot analysis was performed using anti‐C1QBP and anti‐RFP antibodies. CoIP was performed using 10% cell lysate as a loading control. Input control (lanes 1 to 3), rabbit IgG (lanes 4 to 6), and immunoprecipitated C1QBP (lanes 10 to 12). CON, control; IgG, immunoglobulin G
    Figure Legend Snippet: Hi‐FGF2 interacts with C1QBP in HEK293 cells. (A) CoIP of hi‐FGF2 and C1QBP. Western blot analysis was performed using anti‐C1QBP and anti‐RFP antibodies. CoIP was performed using 10% cell lysate as a loading control. Input control (lanes 1 to 3), rabbit IgG (lanes 4 to 6), and immunoprecipitated FGF2 (lanes 10 to 12). (B) CoIP of C1QBP and hi‐FGF2. Cell extracts were immunoprecipitated using the anti‐FGF2 antibody. Western blot analysis was performed using anti‐C1QBP and anti‐RFP antibodies. CoIP was performed using 10% cell lysate as a loading control. Input control (lanes 1 to 3), rabbit IgG (lanes 4 to 6), and immunoprecipitated C1QBP (lanes 10 to 12). CON, control; IgG, immunoglobulin G

    Techniques Used: Co-Immunoprecipitation Assay, Western Blot, Immunoprecipitation

    13) Product Images from "Neddylation of Coro1a determines the fate of multivesicular bodies and biogenesis of extracellular vesicles). Neddylation of Coro1a determines the fate of multivesicular bodies and biogenesis of extracellular vesicles"

    Article Title: Neddylation of Coro1a determines the fate of multivesicular bodies and biogenesis of extracellular vesicles). Neddylation of Coro1a determines the fate of multivesicular bodies and biogenesis of extracellular vesicles

    Journal: Journal of Extracellular Vesicles

    doi: 10.1002/jev2.12153

    Lysine 233 is the main neddylation site in Coro1a. (a, b) WB analysis of NEDD8‐Coro1a and Coro1a in the lysates of HEK293 cells overexpressing mCherry‐Coro1a‐Flag or mCherry‐Coro1a‐K233R‐Flag and NEDD8‐HA (a), or mCherry‐Coro1a‐Flag or mCherry‐Coro1a‐K233R‐Flag (b) with or without 100 nM MLN4924 treatment after IP with anti‐Flag. (c) WB analysis of the in vitro neddylation of Coro1a in the presence of purified NEDD8, NAE1 and UBA3 (E1); UBE2F (E2); mCherry‐TRIM4‐His (E3); and mCherry‐Coro1a‐Flag or mCherry‐Coro1a‐K233R‐Flag proteins. Red arrow indicated NEDD8‐Coro1a. (d) Left, confocal microscopy analysis of the MVB marker CD63 in Coro1a‐Flag or Coro1a‐K233R‐Flag overexpressing HeLa cells treated with DMSO or 20 nM Baf A1 for 12 h. Scale bar, 10 μm. Right, quantification of CD63 + spots per cell. Each dot indicates the number of CD63 + spots per cell. (e, f) Flow cytometric ratio of CD9 + EVs in the supernatants of Coro1a‐Flag or Coro1a‐K233R‐Flag overexpressing HEK293 cells treated with DMSO, 20 nM Baf A1 (e) or 100 nM MLN4924 (f) for 12 h. Representative results from three independent experiments are shown. n , sample number; ns, not significant; * P
    Figure Legend Snippet: Lysine 233 is the main neddylation site in Coro1a. (a, b) WB analysis of NEDD8‐Coro1a and Coro1a in the lysates of HEK293 cells overexpressing mCherry‐Coro1a‐Flag or mCherry‐Coro1a‐K233R‐Flag and NEDD8‐HA (a), or mCherry‐Coro1a‐Flag or mCherry‐Coro1a‐K233R‐Flag (b) with or without 100 nM MLN4924 treatment after IP with anti‐Flag. (c) WB analysis of the in vitro neddylation of Coro1a in the presence of purified NEDD8, NAE1 and UBA3 (E1); UBE2F (E2); mCherry‐TRIM4‐His (E3); and mCherry‐Coro1a‐Flag or mCherry‐Coro1a‐K233R‐Flag proteins. Red arrow indicated NEDD8‐Coro1a. (d) Left, confocal microscopy analysis of the MVB marker CD63 in Coro1a‐Flag or Coro1a‐K233R‐Flag overexpressing HeLa cells treated with DMSO or 20 nM Baf A1 for 12 h. Scale bar, 10 μm. Right, quantification of CD63 + spots per cell. Each dot indicates the number of CD63 + spots per cell. (e, f) Flow cytometric ratio of CD9 + EVs in the supernatants of Coro1a‐Flag or Coro1a‐K233R‐Flag overexpressing HEK293 cells treated with DMSO, 20 nM Baf A1 (e) or 100 nM MLN4924 (f) for 12 h. Representative results from three independent experiments are shown. n , sample number; ns, not significant; * P

    Techniques Used: Western Blot, In Vitro, Purification, Confocal Microscopy, Marker

    UBE2F and TRIM4 are the E2 and E3 for Coro1a neddylation. (a) WB analysis of NEDD8‐Coro1a and Coro1a in the lysates of Ube2f fl/fl BMDCs and Ube2f −/‐ BMDMs or Ube2m fl/fl BMDCs and Ube2m −/‐ BMDMs treated with or without 100 nM MLN4924 for 12 h after IP with anti‐Coro1a. (b) Flow cytometric ratio of CD9 + EVs in the supernatants of Ube2f fl/fl BMDCs and Ube2f −/‐ BMDMs or Ube2m fl/fl BMDCs and Ube2m −/‐ BMDMs. (c, d) WB analysis of Coro1a and TRIM4 (c) or Coro1a and TRIM21 (d) in the lysates of HEK293 cells overexpressing mCherry‐Coro1a‐Flag and mCherry‐TRIM4‐His (c) or mCherry‐Coro1a‐Flag and TRIM21‐Myc (d) after IP with anti‐His (c) or anti‐Myc (d). (e) WB analysis of TRIM4, UBE2M and UBE2F in the lysates of mCherry‐TRIM4‐His‐overexpressing HEK293 cells with UBE2F‐Myc‐His or UBE2M overexpression after IP with anti‐Myc. (f) WB analysis of NEDD8‐Coro1a and Coro1a in the lysates of HEK293 cells transfected with NC siRNA or Trim4 siRNA and the mCherry‐Coro1a‐Flag expression vector with or without 100 nM MLN4924 treatment for 12 h after IP with anti‐Flag. (g) Flow cytometric ratio of CD9 + EVs in the supernatants of HEK293 cells transfected with NC or Trim4 siRNA. (h) WB analysis of the in vitro neddylation of Coro1a in the presence of the purified NEDD8, NAE1 and UBA3 (E1); UBE2F or UBE2M (E2); mCherry‐TRIM4‐His (E3); and mCherry‐Coro1a‐Flag proteins. Red arrow indicated NEDD8‐Coro1a. Representative results from three independent experiments are shown. n , sample number; ns, not significant; *** P
    Figure Legend Snippet: UBE2F and TRIM4 are the E2 and E3 for Coro1a neddylation. (a) WB analysis of NEDD8‐Coro1a and Coro1a in the lysates of Ube2f fl/fl BMDCs and Ube2f −/‐ BMDMs or Ube2m fl/fl BMDCs and Ube2m −/‐ BMDMs treated with or without 100 nM MLN4924 for 12 h after IP with anti‐Coro1a. (b) Flow cytometric ratio of CD9 + EVs in the supernatants of Ube2f fl/fl BMDCs and Ube2f −/‐ BMDMs or Ube2m fl/fl BMDCs and Ube2m −/‐ BMDMs. (c, d) WB analysis of Coro1a and TRIM4 (c) or Coro1a and TRIM21 (d) in the lysates of HEK293 cells overexpressing mCherry‐Coro1a‐Flag and mCherry‐TRIM4‐His (c) or mCherry‐Coro1a‐Flag and TRIM21‐Myc (d) after IP with anti‐His (c) or anti‐Myc (d). (e) WB analysis of TRIM4, UBE2M and UBE2F in the lysates of mCherry‐TRIM4‐His‐overexpressing HEK293 cells with UBE2F‐Myc‐His or UBE2M overexpression after IP with anti‐Myc. (f) WB analysis of NEDD8‐Coro1a and Coro1a in the lysates of HEK293 cells transfected with NC siRNA or Trim4 siRNA and the mCherry‐Coro1a‐Flag expression vector with or without 100 nM MLN4924 treatment for 12 h after IP with anti‐Flag. (g) Flow cytometric ratio of CD9 + EVs in the supernatants of HEK293 cells transfected with NC or Trim4 siRNA. (h) WB analysis of the in vitro neddylation of Coro1a in the presence of the purified NEDD8, NAE1 and UBA3 (E1); UBE2F or UBE2M (E2); mCherry‐TRIM4‐His (E3); and mCherry‐Coro1a‐Flag proteins. Red arrow indicated NEDD8‐Coro1a. Representative results from three independent experiments are shown. n , sample number; ns, not significant; *** P

    Techniques Used: Western Blot, Over Expression, Transfection, Expressing, Plasmid Preparation, In Vitro, Purification

    Neddylation‐mediated inhibition of EV secretion is Coro1a dependent. (a, b), WB analysis of NEDD8 and Coro1a in the lysates of HEK293 cells without (a) or with (b) mCherry‐Coro1a‐Flag overexpression followed by 100 nM MLN4924 treatment for 12 h after IP with anti‐Coro1a (a) or anti‐Flag (b). (c), Flow cytometric ratio of CD9 + EVs in the supernatants of HEK293 cells transfected with NC or Coro1a siRNA. (d), Left, representative TIRF microscopic images of the CD63 + endosome distribution in HeLa cells transfected with NC or Coro1a siRNA. Scale bar, 10 μm. Right panel, quantification of CD63 + vesicles in the subplasmalemmal region per cell. Each dot indicates the number of CD63 + vesicles per cell. (e) Flow cytometric ratio of CD9 + EVs in the supernatants of HEK293 cells with or without Coro1a overexpression. (f) Flow cytometric ratio of CD9 + EVs in the supernatants of HEK293 and HeLa cells transfected with NC or Coro1a siRNA and treated with or without 100 nM MLN4924 for 12 h. (g) Flow cytometric ratio of CD9 + EVs in the supernatants of BMDMs differentiated from the bone marrow cells of Coro1a fl/fl or Lysm Cre Coro1a fl/fl mice. (h) ELISA analysis of F4/80 + EVs in the sera of Coro1a fl/fl or Lysm Cre Coro1a fl/fl mice that intraperitoneally injected with 30 mg kg −1 MLN4924 for 72 h. (i) Left, representative TIRF microscopic images of the CD63 + endosome distribution in Coro1a‐silenced HeLa cells treated with DMSO or 100 nM MLN4924 for 12 h. Scale bar, 10 μm. Right panel, quantification of CD63 + vesicles in the subplasmalemmal region per cell. Each dot indicates the number of CD63 + vesicles per cell. Representative results from three independent experiments are shown. n , sample number; ns, not significant; * P
    Figure Legend Snippet: Neddylation‐mediated inhibition of EV secretion is Coro1a dependent. (a, b), WB analysis of NEDD8 and Coro1a in the lysates of HEK293 cells without (a) or with (b) mCherry‐Coro1a‐Flag overexpression followed by 100 nM MLN4924 treatment for 12 h after IP with anti‐Coro1a (a) or anti‐Flag (b). (c), Flow cytometric ratio of CD9 + EVs in the supernatants of HEK293 cells transfected with NC or Coro1a siRNA. (d), Left, representative TIRF microscopic images of the CD63 + endosome distribution in HeLa cells transfected with NC or Coro1a siRNA. Scale bar, 10 μm. Right panel, quantification of CD63 + vesicles in the subplasmalemmal region per cell. Each dot indicates the number of CD63 + vesicles per cell. (e) Flow cytometric ratio of CD9 + EVs in the supernatants of HEK293 cells with or without Coro1a overexpression. (f) Flow cytometric ratio of CD9 + EVs in the supernatants of HEK293 and HeLa cells transfected with NC or Coro1a siRNA and treated with or without 100 nM MLN4924 for 12 h. (g) Flow cytometric ratio of CD9 + EVs in the supernatants of BMDMs differentiated from the bone marrow cells of Coro1a fl/fl or Lysm Cre Coro1a fl/fl mice. (h) ELISA analysis of F4/80 + EVs in the sera of Coro1a fl/fl or Lysm Cre Coro1a fl/fl mice that intraperitoneally injected with 30 mg kg −1 MLN4924 for 72 h. (i) Left, representative TIRF microscopic images of the CD63 + endosome distribution in Coro1a‐silenced HeLa cells treated with DMSO or 100 nM MLN4924 for 12 h. Scale bar, 10 μm. Right panel, quantification of CD63 + vesicles in the subplasmalemmal region per cell. Each dot indicates the number of CD63 + vesicles per cell. Representative results from three independent experiments are shown. n , sample number; ns, not significant; * P

    Techniques Used: Inhibition, Western Blot, Over Expression, Transfection, Mouse Assay, Enzyme-linked Immunosorbent Assay, Injection

    Neddylation promotes the lysosomal degradation of MVBs. (a, b) Left, confocal microscopy analysis of the MVB marker CD63 in HeLa cells treated with DMSO or 100 nM MLN4924 for 12 h (a) or HeLa cells transfected with NC or Uba3 siRNA (B). Scale bar, 10 μm. Right panel, quantification of CD63 + spots and average particle size per cell. Each dot indicates the number of CD63 + spots and the average particle size per cell. (c) Left, EM images of MVBs (blue arrows) in HEK293 cells treated with DMSO or MLN4924 for 12 h. Scale bar, 500 nm. Yellow arrows indicate MVB and lysosome hybrids. Right, quantification of MVBs per cell per 100 μm 2 and ILVs per MVB. Each dot indicates the number of MVBs per section and ILVs per MVB per cell. (d) Left, confocal microscopy analysis of CD63 and LAMP1 colocalization in HeLa cells treated with DMSO or 100 nM MLN4924 for 12 h. Scale bar, 10 μm. Right panel, each dot indicates the percentage of the colocalized area among the total LAMP1 area per cell. (e) Left, confocal microscopy analysis of mCherry‐GFP‐CD63 endosomes in HeLa cells treated with DMSO or 100 nM MLN4924 for 12 h. Scale bar, 10 μm. Right, the percentage of mCherry + GFP − spots among the total mCherry + spots per cell. Each dot indicates the proportion of red CD63 puncta per cell. (f, g) Flow cytometric ratio of CD9 + EVs in the supernatants of HEK293 and HeLa cells treated with 100 nM MLN4924 for 12 h (f) or overexpressing NEDD8 (g) in the presence of 20 nM Baf A1. (H) WB analysis of HRS in HEK293 cells treated with DMSO or 100 nM MLN4924 for 12 h. (i) Left, Representative TIRF microscopic images of the CD63 + MVB distribution in HeLa cells treated with DMSO or 100 nM MLN4924 for 12 h. Scale bar, 10 μm. Right, quantification of CD63 + spots in the subplasmalemmal region per cell. Each dot indicates the number of CD63 + spots per cell. Representative results from three independent experiments are shown. n , sample number; ns, not significant; * P
    Figure Legend Snippet: Neddylation promotes the lysosomal degradation of MVBs. (a, b) Left, confocal microscopy analysis of the MVB marker CD63 in HeLa cells treated with DMSO or 100 nM MLN4924 for 12 h (a) or HeLa cells transfected with NC or Uba3 siRNA (B). Scale bar, 10 μm. Right panel, quantification of CD63 + spots and average particle size per cell. Each dot indicates the number of CD63 + spots and the average particle size per cell. (c) Left, EM images of MVBs (blue arrows) in HEK293 cells treated with DMSO or MLN4924 for 12 h. Scale bar, 500 nm. Yellow arrows indicate MVB and lysosome hybrids. Right, quantification of MVBs per cell per 100 μm 2 and ILVs per MVB. Each dot indicates the number of MVBs per section and ILVs per MVB per cell. (d) Left, confocal microscopy analysis of CD63 and LAMP1 colocalization in HeLa cells treated with DMSO or 100 nM MLN4924 for 12 h. Scale bar, 10 μm. Right panel, each dot indicates the percentage of the colocalized area among the total LAMP1 area per cell. (e) Left, confocal microscopy analysis of mCherry‐GFP‐CD63 endosomes in HeLa cells treated with DMSO or 100 nM MLN4924 for 12 h. Scale bar, 10 μm. Right, the percentage of mCherry + GFP − spots among the total mCherry + spots per cell. Each dot indicates the proportion of red CD63 puncta per cell. (f, g) Flow cytometric ratio of CD9 + EVs in the supernatants of HEK293 and HeLa cells treated with 100 nM MLN4924 for 12 h (f) or overexpressing NEDD8 (g) in the presence of 20 nM Baf A1. (H) WB analysis of HRS in HEK293 cells treated with DMSO or 100 nM MLN4924 for 12 h. (i) Left, Representative TIRF microscopic images of the CD63 + MVB distribution in HeLa cells treated with DMSO or 100 nM MLN4924 for 12 h. Scale bar, 10 μm. Right, quantification of CD63 + spots in the subplasmalemmal region per cell. Each dot indicates the number of CD63 + spots per cell. Representative results from three independent experiments are shown. n , sample number; ns, not significant; * P

    Techniques Used: Confocal Microscopy, Marker, Transfection, Western Blot

    Neddylation inhibits EV secretion. (a) Flow cytometric analysis of EVs in the supernatants of HEK293 cells treated with DMSO or 100 nM MLN4924 for 12 h. Left, representative dot plots showing CD9 and CD81 staining of EVs captured with anti‑CD63‑coated beads after incubation with cell culture supernatants. Right, the ratio of CD9 + and CD81 + EVs. (b–d) EVs were purified from equal numbers of HEK293 cells treated with DMSO or 100 nM MLN4924 for 12 h. The BCA assay was used to determine the amount of EV protein (b). NTA to determine the EV concentration (c). WB analysis to detect the indicated EV markers (d). (e) WB analysis to detect the indicated proteins in HEK293 cells overexpressing NEDD8. (f) Flow cytometric ratio of CD9 + EVs in the supernatants of HEK293 cells overexpressing NEDD8. (g) ELISA analysis of CD9 + and CD81 + EVs in sera of mice intraperitoneally injected with MLN4924 at the indicated dose for 72 h. Representative results from three independent experiments are shown. n , sample number; * P
    Figure Legend Snippet: Neddylation inhibits EV secretion. (a) Flow cytometric analysis of EVs in the supernatants of HEK293 cells treated with DMSO or 100 nM MLN4924 for 12 h. Left, representative dot plots showing CD9 and CD81 staining of EVs captured with anti‑CD63‑coated beads after incubation with cell culture supernatants. Right, the ratio of CD9 + and CD81 + EVs. (b–d) EVs were purified from equal numbers of HEK293 cells treated with DMSO or 100 nM MLN4924 for 12 h. The BCA assay was used to determine the amount of EV protein (b). NTA to determine the EV concentration (c). WB analysis to detect the indicated EV markers (d). (e) WB analysis to detect the indicated proteins in HEK293 cells overexpressing NEDD8. (f) Flow cytometric ratio of CD9 + EVs in the supernatants of HEK293 cells overexpressing NEDD8. (g) ELISA analysis of CD9 + and CD81 + EVs in sera of mice intraperitoneally injected with MLN4924 at the indicated dose for 72 h. Representative results from three independent experiments are shown. n , sample number; * P

    Techniques Used: Staining, Incubation, Cell Culture, Purification, BIA-KA, Concentration Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Mouse Assay, Injection

    NEDD8‐Coro1a mediates the recruitment of Rab7 to MVBs. (a) Left, confocal microscopy analysis of CD63 in HeLa cells transfected with the Rab7a T22N dominant‐negative mutant. Scale bar, 10 μm. Right, quantification of CD63 + spots per cell. Each dot indicates the number of CD63 + spots per cell. (b) Flow cytometric ratio of CD9 + EVs in the supernatants of HEK293 cells transfected with the Rab7a T22N . (c) Left, confocal microscopy analysis of CD63 and Rab7a T22N colocalization in HeLa cells overexpressing mCherry‐Rab7a T22N and Coro1a‐Flag or Coro1a‐K233R‐Flag. Scale bar, 10 μm. Right, quantification of spots showing colocalization per cell. Each dot indicates the percentage of spots per cell showing colocalization. (d) 3D‐SIM analysis of Rab7a T22N on CD63 + MVBs of HeLa cells overexpressing mCherry‐Rab7a T22N and Coro1a‐Flag or Coro1a‐K233R‐Flag and the corresponding reconstructed renderings by Imaris 9.5 (Rightmost). Yellow indicated contact surface. Scale bar, 3 μm. Right, quantification of the contact area of Rab7a T22N with per MVBs. Each dot represents the mean contact area in each MVB from individual cell. (e) Left, electron micrographs of HEK293 cells overexpressing Coro1a‐Flag or Coro1a‐K233R‐Flag with anti‐Rab7 staining (conjugated with 18 nm gold). Representative mRab7 on MVBs (blue arrows) and ILVs (red arrows) are shown. Scale bars, 500 nm. Right panel, quantification of mRab7 gold particles on MVBs and ILVs per 1 μm 2 . Each dot indicates the number of mRab7 gold particles per section per cell. (f) Left, confocal microscopy analysis of PLA + spots showing the interaction between Coro1a and Rab7 in HeLa cells treated with DMSO or MLN4924 at the indicated dose for 12 h. Scale bar, 10 μm. Right, quantification of PLA + spots per cell. (g) WB analysis of Rab7a and Coro1a in the lysates of HEK293 cells transfected with vectors for mCherry‐Coro1a‐Flag and mCherry‐Rab7a‐His expression and NC or Trim4 siRNA with or without 100 nM MLN4924 treatment for 12 h after IP with anti‐Flag. (h) WB analysis of the in vitro interaction between the purified mCherry‐Rab7a‐His protein and mCherry‐Coro1a‐Flag protein from HEK293 cells with mock vector transfection or the mCherry‐Coro1a‐Flag protein from HEK293 cells with NEDD8 vector transfection [mCherry‐Coro1a‐Flag (NEDD8)] after IP with anti‐Flag. (i) WB analysis of Coro1a and Rab7a in the lysates of HEK293 cells overexpressing mCherry‐Rab7a‐His and mCherry‐Coro1a‐Flag or mCherry‐Coro1a‐K233R‐Flag after IP with anti‐His. (j) Left, confocal microscopy analysis of PLA + spots showing the interaction between Rab7a and GDI2 in HeLa cells overexpressing Coro1a‐Flag or Coro1a‐K233R‐Flag with or without Mon1a and Mon1b silencing. Scale bar, 10 μm. Right, quantification of PLA + spots per cell. (k) The images of Coro1a in Rab5 Q79L endosomes of HeLa cells treated with DMSO or 100 nM MLN4924 for 12 h analysed by 3D‐SIM and the corresponding reconstructed renderings by Imaris 9.5 (Rightmost). Yellow indicated contact surface. Scale bar, 3 μm. Right, quantification of the contact area of Coro1a with per Rab5 Q79L endosome. Each dot represents the total contact area of Coro1a + spots per Rab5 Q79L endosome. (l) Left, electron micrographs of HEK293 cells treated with DMSO or 100 nM MLN4924 for 12 h followed by anti‐Coro1a staining (conjugated with 18 nm gold). Representative mCoro1a on MVBs (blue arrows) and ILVs (red arrows) are shown. Scale bars, 1 μm. Right, quantification of Coro1a gold particles per MVB per 1 μm 2 . Each dot indicates the number of Coro1a gold particles per section per cell. Representative results from three independent experiments are shown. n , sample number; ns, not significant; * P
    Figure Legend Snippet: NEDD8‐Coro1a mediates the recruitment of Rab7 to MVBs. (a) Left, confocal microscopy analysis of CD63 in HeLa cells transfected with the Rab7a T22N dominant‐negative mutant. Scale bar, 10 μm. Right, quantification of CD63 + spots per cell. Each dot indicates the number of CD63 + spots per cell. (b) Flow cytometric ratio of CD9 + EVs in the supernatants of HEK293 cells transfected with the Rab7a T22N . (c) Left, confocal microscopy analysis of CD63 and Rab7a T22N colocalization in HeLa cells overexpressing mCherry‐Rab7a T22N and Coro1a‐Flag or Coro1a‐K233R‐Flag. Scale bar, 10 μm. Right, quantification of spots showing colocalization per cell. Each dot indicates the percentage of spots per cell showing colocalization. (d) 3D‐SIM analysis of Rab7a T22N on CD63 + MVBs of HeLa cells overexpressing mCherry‐Rab7a T22N and Coro1a‐Flag or Coro1a‐K233R‐Flag and the corresponding reconstructed renderings by Imaris 9.5 (Rightmost). Yellow indicated contact surface. Scale bar, 3 μm. Right, quantification of the contact area of Rab7a T22N with per MVBs. Each dot represents the mean contact area in each MVB from individual cell. (e) Left, electron micrographs of HEK293 cells overexpressing Coro1a‐Flag or Coro1a‐K233R‐Flag with anti‐Rab7 staining (conjugated with 18 nm gold). Representative mRab7 on MVBs (blue arrows) and ILVs (red arrows) are shown. Scale bars, 500 nm. Right panel, quantification of mRab7 gold particles on MVBs and ILVs per 1 μm 2 . Each dot indicates the number of mRab7 gold particles per section per cell. (f) Left, confocal microscopy analysis of PLA + spots showing the interaction between Coro1a and Rab7 in HeLa cells treated with DMSO or MLN4924 at the indicated dose for 12 h. Scale bar, 10 μm. Right, quantification of PLA + spots per cell. (g) WB analysis of Rab7a and Coro1a in the lysates of HEK293 cells transfected with vectors for mCherry‐Coro1a‐Flag and mCherry‐Rab7a‐His expression and NC or Trim4 siRNA with or without 100 nM MLN4924 treatment for 12 h after IP with anti‐Flag. (h) WB analysis of the in vitro interaction between the purified mCherry‐Rab7a‐His protein and mCherry‐Coro1a‐Flag protein from HEK293 cells with mock vector transfection or the mCherry‐Coro1a‐Flag protein from HEK293 cells with NEDD8 vector transfection [mCherry‐Coro1a‐Flag (NEDD8)] after IP with anti‐Flag. (i) WB analysis of Coro1a and Rab7a in the lysates of HEK293 cells overexpressing mCherry‐Rab7a‐His and mCherry‐Coro1a‐Flag or mCherry‐Coro1a‐K233R‐Flag after IP with anti‐His. (j) Left, confocal microscopy analysis of PLA + spots showing the interaction between Rab7a and GDI2 in HeLa cells overexpressing Coro1a‐Flag or Coro1a‐K233R‐Flag with or without Mon1a and Mon1b silencing. Scale bar, 10 μm. Right, quantification of PLA + spots per cell. (k) The images of Coro1a in Rab5 Q79L endosomes of HeLa cells treated with DMSO or 100 nM MLN4924 for 12 h analysed by 3D‐SIM and the corresponding reconstructed renderings by Imaris 9.5 (Rightmost). Yellow indicated contact surface. Scale bar, 3 μm. Right, quantification of the contact area of Coro1a with per Rab5 Q79L endosome. Each dot represents the total contact area of Coro1a + spots per Rab5 Q79L endosome. (l) Left, electron micrographs of HEK293 cells treated with DMSO or 100 nM MLN4924 for 12 h followed by anti‐Coro1a staining (conjugated with 18 nm gold). Representative mCoro1a on MVBs (blue arrows) and ILVs (red arrows) are shown. Scale bars, 1 μm. Right, quantification of Coro1a gold particles per MVB per 1 μm 2 . Each dot indicates the number of Coro1a gold particles per section per cell. Representative results from three independent experiments are shown. n , sample number; ns, not significant; * P

    Techniques Used: Confocal Microscopy, Transfection, Dominant Negative Mutation, Staining, Proximity Ligation Assay, Western Blot, Expressing, In Vitro, Purification, Plasmid Preparation

    14) Product Images from "Diabetes and Pancreatic Exocrine Dysfunction Due to Mutations in the Carboxyl Ester Lipase Gene-Maturity Onset Diabetes of the Young (CEL-MODY): A PROTEIN MISFOLDING DISEASE*"

    Article Title: Diabetes and Pancreatic Exocrine Dysfunction Due to Mutations in the Carboxyl Ester Lipase Gene-Maturity Onset Diabetes of the Young (CEL-MODY): A PROTEIN MISFOLDING DISEASE*

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.222679

    Prolonged time course for the secretion of CEL. CEL-MUT protein appears to be more stable than the CEL-WT. HEK293 cells were metabolically labeled and chased for the indicated time periods, and the proteins in cell extract and growth medium were immunoprecipitated
    Figure Legend Snippet: Prolonged time course for the secretion of CEL. CEL-MUT protein appears to be more stable than the CEL-WT. HEK293 cells were metabolically labeled and chased for the indicated time periods, and the proteins in cell extract and growth medium were immunoprecipitated

    Techniques Used: Metabolic Labelling, Labeling, Immunoprecipitation

    UPR in HEK293 cells expressing different CEL proteins. Detergent-soluble proteins in lysates of cells expressing CEL-WT, CEL-MUT, and EV were analyzed by immunoblotting using specific antibodies (see under “Experimental Procedures”). A
    Figure Legend Snippet: UPR in HEK293 cells expressing different CEL proteins. Detergent-soluble proteins in lysates of cells expressing CEL-WT, CEL-MUT, and EV were analyzed by immunoblotting using specific antibodies (see under “Experimental Procedures”). A

    Techniques Used: Expressing

    CEL-MUT protein forms higher oligomeric forms that are SDS-resistant and Triton X-100-insoluble. A , metabolically labeling of HEK293 cells, stably expressing CEL-WT ( left panel ) and CEL-MUT ( right panel ). Subsequently, the cells were lysed, and the CEL
    Figure Legend Snippet: CEL-MUT protein forms higher oligomeric forms that are SDS-resistant and Triton X-100-insoluble. A , metabolically labeling of HEK293 cells, stably expressing CEL-WT ( left panel ) and CEL-MUT ( right panel ). Subsequently, the cells were lysed, and the CEL

    Techniques Used: Metabolic Labelling, Labeling, Stable Transfection, Expressing

    CEL-MUT forms extra- and intracellular aggregates in HEK293 cells. HEK293 cells stably transfected with CEL-MUT and CEL-WT were processed for immunoperoxidase electron microscopy as described under “Experimental Procedures.” A , extracellular
    Figure Legend Snippet: CEL-MUT forms extra- and intracellular aggregates in HEK293 cells. HEK293 cells stably transfected with CEL-MUT and CEL-WT were processed for immunoperoxidase electron microscopy as described under “Experimental Procedures.” A , extracellular

    Techniques Used: Stable Transfection, Transfection, Electron Microscopy

    In vitro N - and O -linked deglycosylation of CEL proteins variants. Acetone-precipitated proteins from the cell-free growth medium of HEK293 cells, stably expressing CEL-WT and CEL-MUT, were subjected to N -linked ( N -glycosidase F) and/or O -linked ( O -glycosidase
    Figure Legend Snippet: In vitro N - and O -linked deglycosylation of CEL proteins variants. Acetone-precipitated proteins from the cell-free growth medium of HEK293 cells, stably expressing CEL-WT and CEL-MUT, were subjected to N -linked ( N -glycosidase F) and/or O -linked ( O -glycosidase

    Techniques Used: In Vitro, Stable Transfection, Expressing

    Time course for the secretion of CEL-WT and CEL-MUT by stably transfected HEK293 cells. A , pulse-chase analysis of HEK293 stably expressing CEL-WT ( left panel ) and CEL-MUT ( right panel ). The cells were metabolically labeled with [ 35 S]Met/Cys for 5 min,
    Figure Legend Snippet: Time course for the secretion of CEL-WT and CEL-MUT by stably transfected HEK293 cells. A , pulse-chase analysis of HEK293 stably expressing CEL-WT ( left panel ) and CEL-MUT ( right panel ). The cells were metabolically labeled with [ 35 S]Met/Cys for 5 min,

    Techniques Used: Stable Transfection, Transfection, Pulse Chase, Expressing, Metabolic Labelling, Labeling

    15) Product Images from "Ehrlichia chaffeensis Induces Monocyte Inflammatory Responses through MyD88, ERK, and NF-?B but Not through TRIF, Interleukin-1 Receptor 1 (IL-1R1)/IL-18R1, or Toll-Like Receptors ▿"

    Article Title: Ehrlichia chaffeensis Induces Monocyte Inflammatory Responses through MyD88, ERK, and NF-?B but Not through TRIF, Interleukin-1 Receptor 1 (IL-1R1)/IL-18R1, or Toll-Like Receptors ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.05640-11

    IL-8 induction in HEK293 cells by E. chaffeensis is mediated by ERK. (A) Effects of inhibitors on IL-8 promoter activation in HEK293 cells in response to E. chaffeensis Wakulla added at 40 IFU/cell and incubated at 37°C for 2 h. All inhibitors
    Figure Legend Snippet: IL-8 induction in HEK293 cells by E. chaffeensis is mediated by ERK. (A) Effects of inhibitors on IL-8 promoter activation in HEK293 cells in response to E. chaffeensis Wakulla added at 40 IFU/cell and incubated at 37°C for 2 h. All inhibitors

    Techniques Used: Activation Assay, Incubation

    E. chaffeensis induces cytokines and activates IL-8 and NF-κB promoters in HEK293 cells. (A) HEK293 cells infected with E. chaffeensis . Host-free E. chaffeensis was inoculated and incubated with HEK293 cells for 6 days. The cells were stained
    Figure Legend Snippet: E. chaffeensis induces cytokines and activates IL-8 and NF-κB promoters in HEK293 cells. (A) HEK293 cells infected with E. chaffeensis . Host-free E. chaffeensis was inoculated and incubated with HEK293 cells for 6 days. The cells were stained

    Techniques Used: Infection, Incubation, Staining

    16) Product Images from "The C-Terminal 42 Residues of the Tula Virus Gn Protein Regulate Interferon Induction ▿"

    Article Title: The C-Terminal 42 Residues of the Tula Virus Gn Protein Regulate Interferon Induction ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01945-10

    TULV Gn-T inhibition of TBK1- and TRAF2-directed NF-κB activation. HEK293 cells transfected with an NF-κB promoter-luciferase reporter construct in the presence or absence of activating TBK1 (A) or TRAF2 (B) expression vectors and plasmids
    Figure Legend Snippet: TULV Gn-T inhibition of TBK1- and TRAF2-directed NF-κB activation. HEK293 cells transfected with an NF-κB promoter-luciferase reporter construct in the presence or absence of activating TBK1 (A) or TRAF2 (B) expression vectors and plasmids

    Techniques Used: Inhibition, Activation Assay, Transfection, Luciferase, Construct, Expressing

    TULV Gn-T regulates RIG-I- and TBK1-directed ISRE and IFN-β transcription. HEK293 cells were transfected with an ISRE-driven luciferase reporter construct (A, B, and D) or an IFN-β-luciferase reporter construct (C) in the presence or absence
    Figure Legend Snippet: TULV Gn-T regulates RIG-I- and TBK1-directed ISRE and IFN-β transcription. HEK293 cells were transfected with an ISRE-driven luciferase reporter construct (A, B, and D) or an IFN-β-luciferase reporter construct (C) in the presence or absence

    Techniques Used: Transfection, Luciferase, Construct

    TULV C42 inhibits TBK1-directed ISRE and IFN-β activation. (A) HEK293 cells were transfected with an ISRE-driven luciferase reporter construct in the presence or absence of an activating TBK1 expression vector. Cells were cotransfected with plasmids
    Figure Legend Snippet: TULV C42 inhibits TBK1-directed ISRE and IFN-β activation. (A) HEK293 cells were transfected with an ISRE-driven luciferase reporter construct in the presence or absence of an activating TBK1 expression vector. Cells were cotransfected with plasmids

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

    17) Product Images from "Gq-coupled receptors as mechanosensors mediating myogenic vasoconstriction"

    Article Title: Gq-coupled receptors as mechanosensors mediating myogenic vasoconstriction

    Journal: The EMBO Journal

    doi: 10.1038/emboj.2008.233

    Inverse agonists and antagonists prevent mechanical activation of GPCRs. Whole-cell measurements of H 1 R ( A – C , E ) or M 5 R 100 s ( D , F ) with stimulation by positive pipette pressure (A), with vertical stretch (B) or with hypotonicity (C–F) in the presence or absence of 100 μM diphenhydramine, ‘DPH' (E) or 1 μM atropine (F). Agonist stimulation was performed with 100 μM histamine, ‘His' or with 100 μM carbachol ‘CCh'. Direct TRPC6 stimulation was performed with 100 μM OAG. Current time courses at ±60 mV, zero current levels (stippled lines), scale bars: 500 pA (A), 200 pA (B–E) or 1 nA (F) and 50 s (A, C–F) or 100 s (B). ( G ) Comparison of TRPC6 current suppression by atropine (1 μM), pirenzepine (1 μM), diphenhydramine (100 μM), losartan (1 μM) and candesartan, ‘Cand', (100 nM) with hypotonic stimulation of HEK293 cells co-expressing TRPC6 and the respective receptors. ( H ) Analysis of NPo values in cell-attached patches. (H) (top) Representative current traces before, during the first (left and right) and the fifths (right) second after application of −10 cm H 2 O pipette pressure. The closed state is indicated with ‘c' (right), scale bars (5 pA, 20 ms).
    Figure Legend Snippet: Inverse agonists and antagonists prevent mechanical activation of GPCRs. Whole-cell measurements of H 1 R ( A – C , E ) or M 5 R 100 s ( D , F ) with stimulation by positive pipette pressure (A), with vertical stretch (B) or with hypotonicity (C–F) in the presence or absence of 100 μM diphenhydramine, ‘DPH' (E) or 1 μM atropine (F). Agonist stimulation was performed with 100 μM histamine, ‘His' or with 100 μM carbachol ‘CCh'. Direct TRPC6 stimulation was performed with 100 μM OAG. Current time courses at ±60 mV, zero current levels (stippled lines), scale bars: 500 pA (A), 200 pA (B–E) or 1 nA (F) and 50 s (A, C–F) or 100 s (B). ( G ) Comparison of TRPC6 current suppression by atropine (1 μM), pirenzepine (1 μM), diphenhydramine (100 μM), losartan (1 μM) and candesartan, ‘Cand', (100 nM) with hypotonic stimulation of HEK293 cells co-expressing TRPC6 and the respective receptors. ( H ) Analysis of NPo values in cell-attached patches. (H) (top) Representative current traces before, during the first (left and right) and the fifths (right) second after application of −10 cm H 2 O pipette pressure. The closed state is indicated with ‘c' (right), scale bars (5 pA, 20 ms).

    Techniques Used: Activation Assay, Transferring, Expressing, Mass Spectrometry

    Indirect TRPC6 activation by membrane stretch through AT 1 R. Whole-cell recordings from transiently AT 1 R ( A , B ) or AT 1 R-Venus ( C , D ) and TRPC6 co-expressing HEK293 cells. (A–D) IV relationships before ‘Basal' and during hypotonicity (A, B) and direct membrane stretch (C, D) with and without 1 μM losartan, ‘Losa', are displayed; current time courses at ±60 mV, zero current levels (stippled lines), time scale bars: 50 s (A, B), 100 s (C, D) and current scale bars: 500 pA (A, B), 2 nA (C, D).
    Figure Legend Snippet: Indirect TRPC6 activation by membrane stretch through AT 1 R. Whole-cell recordings from transiently AT 1 R ( A , B ) or AT 1 R-Venus ( C , D ) and TRPC6 co-expressing HEK293 cells. (A–D) IV relationships before ‘Basal' and during hypotonicity (A, B) and direct membrane stretch (C, D) with and without 1 μM losartan, ‘Losa', are displayed; current time courses at ±60 mV, zero current levels (stippled lines), time scale bars: 50 s (A, B), 100 s (C, D) and current scale bars: 500 pA (A, B), 2 nA (C, D).

    Techniques Used: Activation Assay, Expressing

    Osmotically induced membrane stretch activates phospholipase C and G proteins. ( A – D ) Whole-cell recordings from HEK293 cells co-expressing TRPC6 and H 1 R. Recording with 10 μM U73122 (A) or with 10 μM U73343 (B) in the pipette solution. Recordings of cells pretreated with 100 ng ml −1 PTX for 16 h (C) or with 2 mM GDP-β-S in the pipette solution (D). IV relationships before ‘Basal', during hypotonic stimulation, ‘Hypo' and receptor stimulation with 100 μM histamine, ‘His' are shown. (A–D) Insets show current time courses at ±60 mV, hypotonic and histamine stimulations, zero current levels (stippled lines); scale bars: 500 pA, 50 s.
    Figure Legend Snippet: Osmotically induced membrane stretch activates phospholipase C and G proteins. ( A – D ) Whole-cell recordings from HEK293 cells co-expressing TRPC6 and H 1 R. Recording with 10 μM U73122 (A) or with 10 μM U73343 (B) in the pipette solution. Recordings of cells pretreated with 100 ng ml −1 PTX for 16 h (C) or with 2 mM GDP-β-S in the pipette solution (D). IV relationships before ‘Basal', during hypotonic stimulation, ‘Hypo' and receptor stimulation with 100 μM histamine, ‘His' are shown. (A–D) Insets show current time courses at ±60 mV, hypotonic and histamine stimulations, zero current levels (stippled lines); scale bars: 500 pA, 50 s.

    Techniques Used: Expressing, Transferring

    Expression of AT 1 R leads to mechanosensitivity of aortic SMCs. Whole-cell recordings from non-transfected ( A ) and A7r5 cells over-expressing AT 1 R ( B – D ). (A, B) IV relationships before ‘Basal', during hypotonic stimulation, ‘Hypo', and receptor stimulation with 1 μM vasopressin ‘VP' (left) and current density analyses at ±60 mV (right) are displayed. (C, D) Current time courses at ±60 mV with zero current levels (stippled lines); time scale bars 60 s and 100 pA (left) and current densities in the presence of neutralizing antibody, ‘nAb' (right) are displayed. ( E ) [Ca 2+ ] i increases by hypotonic stimulation in HEK293 cells transfected with different amounts of AT 1 R-Venus cDNAs. Venus fluorescence was correlated with receptor densities. Numbers indicate the number of summarized cells and of independent transfections. ( F ) Concentration–response curves to AII of fura-2-loaded HEK293 cells stably expressing AT 1 R-Venus in isotonic and in hypotonic (273 mOsm kg −1 ) solutions.
    Figure Legend Snippet: Expression of AT 1 R leads to mechanosensitivity of aortic SMCs. Whole-cell recordings from non-transfected ( A ) and A7r5 cells over-expressing AT 1 R ( B – D ). (A, B) IV relationships before ‘Basal', during hypotonic stimulation, ‘Hypo', and receptor stimulation with 1 μM vasopressin ‘VP' (left) and current density analyses at ±60 mV (right) are displayed. (C, D) Current time courses at ±60 mV with zero current levels (stippled lines); time scale bars 60 s and 100 pA (left) and current densities in the presence of neutralizing antibody, ‘nAb' (right) are displayed. ( E ) [Ca 2+ ] i increases by hypotonic stimulation in HEK293 cells transfected with different amounts of AT 1 R-Venus cDNAs. Venus fluorescence was correlated with receptor densities. Numbers indicate the number of summarized cells and of independent transfections. ( F ) Concentration–response curves to AII of fura-2-loaded HEK293 cells stably expressing AT 1 R-Venus in isotonic and in hypotonic (273 mOsm kg −1 ) solutions.

    Techniques Used: Expressing, Transfection, Fluorescence, Concentration Assay, Stable Transfection

    TRPC6 per se is not mechanosensitive. ( A ) Whole-cell recordings from HEK293 cells expressing TRPC6; application of hypotonic stimulus (250 mOsm kg −1 ), ‘Hypo', and of 100 μM OAG are indicated. Current time courses at ±60 mV, zero current levels (stippled lines), time scale bar (50 s) and current scale bar (200 pA). ( B – E ) Single-channel recordings in inside-out patches with negative pipette pressure and subsequent SAG bath application. (B) Original current trace, scale bar (2 pA, 10 s) (top); 13 consecutive traces on an expanded time-scale at the indicated time points, scale bar (5 pA, 20 ms) (middle); the respective graph of consecutive open probabilities (NPo) in 1-s steps (bottom) is displayed. (C, D) Analysis of mean NPo values. (E) Representative expanded current traces around the time point of pressure application, scale bar (5 pA, 10 ms).
    Figure Legend Snippet: TRPC6 per se is not mechanosensitive. ( A ) Whole-cell recordings from HEK293 cells expressing TRPC6; application of hypotonic stimulus (250 mOsm kg −1 ), ‘Hypo', and of 100 μM OAG are indicated. Current time courses at ±60 mV, zero current levels (stippled lines), time scale bar (50 s) and current scale bar (200 pA). ( B – E ) Single-channel recordings in inside-out patches with negative pipette pressure and subsequent SAG bath application. (B) Original current trace, scale bar (2 pA, 10 s) (top); 13 consecutive traces on an expanded time-scale at the indicated time points, scale bar (5 pA, 20 ms) (middle); the respective graph of consecutive open probabilities (NPo) in 1-s steps (bottom) is displayed. (C, D) Analysis of mean NPo values. (E) Representative expanded current traces around the time point of pressure application, scale bar (5 pA, 10 ms).

    Techniques Used: Expressing, Transferring, Mass Spectrometry

    18) Product Images from "Thiostrepton interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates"

    Article Title: Thiostrepton interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.12602

    Thiostrepton induces accumulation of a labile reporter in human cells. (A) Schematic representation of a fluorescence-based DIAP1-sensor, represented by HEK293 cells stably cotransfected with DIAP1ΔR-YFP fusion and Rpr-HA. IAP-antagonist Rpr binds to DIAP1 and triggers its ubiquitination and degradation. (B) Fluorescence micrographs of DIAP1-sensor cells, showing DIAP1ΔR-YFP fluorescence response following DMSO, MG-132, Comp-3 (synthetic IAP-antagonist) and Thsp treatment for 18 hrs. (C) Chemical structure of Thsp with highlighted reactive dehydroalanine residues (DHA 3 /DHA 16 /DHA 17 ) and the dehydrobutyrine residue (DHB 8 ). (D) Diagram showing the determination of EC 50 for MG-132 and Thsp in DIAP1-sensor cells. (E) Thsp triggers accumulation of polyubiquitinated DIAP1 species. DIAP1ΔR-YFP was immunoprecipitated from IAP-sensor cells treated with the specified compounds, and the ubiquitination status and DIAP1 levels were determined by WB with anti-ubiquitin and anti-DIAP1 antibodies.
    Figure Legend Snippet: Thiostrepton induces accumulation of a labile reporter in human cells. (A) Schematic representation of a fluorescence-based DIAP1-sensor, represented by HEK293 cells stably cotransfected with DIAP1ΔR-YFP fusion and Rpr-HA. IAP-antagonist Rpr binds to DIAP1 and triggers its ubiquitination and degradation. (B) Fluorescence micrographs of DIAP1-sensor cells, showing DIAP1ΔR-YFP fluorescence response following DMSO, MG-132, Comp-3 (synthetic IAP-antagonist) and Thsp treatment for 18 hrs. (C) Chemical structure of Thsp with highlighted reactive dehydroalanine residues (DHA 3 /DHA 16 /DHA 17 ) and the dehydrobutyrine residue (DHB 8 ). (D) Diagram showing the determination of EC 50 for MG-132 and Thsp in DIAP1-sensor cells. (E) Thsp triggers accumulation of polyubiquitinated DIAP1 species. DIAP1ΔR-YFP was immunoprecipitated from IAP-sensor cells treated with the specified compounds, and the ubiquitination status and DIAP1 levels were determined by WB with anti-ubiquitin and anti-DIAP1 antibodies.

    Techniques Used: Fluorescence, Stable Transfection, Immunoprecipitation, Western Blot

    Proteasome inhibition is dependent on Thiostrepton ability to bind covalently to proteins. (A) Time-dependent Rpr–Thsp adducts formation is prevented by L-Cysteine supplementation. Rpr was incubated in vitro with Thsp or Thsp/L-Cysteine combination and the formation of Rpr adducts was monitored by WB with Rpr-specific antibody. This is consistent with Thsp binding covalently to the Cysteine residue of Rpr (Cys49), a process that is blocked by addition of excess L-Cysteine. (B) Microscopic images showing the fluorescence response of DIAP1 sensor cells and proteasome sensors cells following treatment with Thsp or combination Thsp/L-Cysteine. Excess L-Cysteine blocks Thsp-induced accumulation in sensors’ fluorescence (or blocks Thsp-induced proteasome inhibition), which is consistent with a model where Thsp covalent attachment to Cysteine residues is determinant for its proteasome inhibitory activity. (C) Thsp is a slow inhibitor of the proteasome activity in the cell when compared to chymotrypsin activity inhibitor MG-132, and the proteasome inhibition by Thsp correlates with formation of Thsp–p53 adducts in the cell. HEK293 IL-1R cells were treated with MG-132 or Thsp followed by stimulation with IL-1 (10 ng/ml) for 15 min., to trigger proteasomal degradation of IκBα (NF-κB activation). Persistence of IκBα in samples pre-treated with MG-132 or Thsp and subsequently with IL-1, is an indication of proteasome inhibition. IκBα stabilization in Thsp treated samples correlates with accumulation of p53 multimeric bands (an indicator of Thsp–protein adducts formation). (D) WB detection of polyubiquitination and Mcl1 accumulation in extracts of Raji cells. The cells were treated with CHX, CHX/MG-132 and CHX/Thsp for 4 hrs, washed and supplemented with fresh media and then harvested at different time-points (1–6 hrs). GAPDH was used as a loading control. The extent of polyubiquitination and Mcl-1 levels, 4 hrs after washing off MG-132 are largely reversed to non-treated state. For comparison, these effects remained steady or increased 4 hrs after Thsp wash off, which is consistent with a covalent-binding proteasome inhibition model.
    Figure Legend Snippet: Proteasome inhibition is dependent on Thiostrepton ability to bind covalently to proteins. (A) Time-dependent Rpr–Thsp adducts formation is prevented by L-Cysteine supplementation. Rpr was incubated in vitro with Thsp or Thsp/L-Cysteine combination and the formation of Rpr adducts was monitored by WB with Rpr-specific antibody. This is consistent with Thsp binding covalently to the Cysteine residue of Rpr (Cys49), a process that is blocked by addition of excess L-Cysteine. (B) Microscopic images showing the fluorescence response of DIAP1 sensor cells and proteasome sensors cells following treatment with Thsp or combination Thsp/L-Cysteine. Excess L-Cysteine blocks Thsp-induced accumulation in sensors’ fluorescence (or blocks Thsp-induced proteasome inhibition), which is consistent with a model where Thsp covalent attachment to Cysteine residues is determinant for its proteasome inhibitory activity. (C) Thsp is a slow inhibitor of the proteasome activity in the cell when compared to chymotrypsin activity inhibitor MG-132, and the proteasome inhibition by Thsp correlates with formation of Thsp–p53 adducts in the cell. HEK293 IL-1R cells were treated with MG-132 or Thsp followed by stimulation with IL-1 (10 ng/ml) for 15 min., to trigger proteasomal degradation of IκBα (NF-κB activation). Persistence of IκBα in samples pre-treated with MG-132 or Thsp and subsequently with IL-1, is an indication of proteasome inhibition. IκBα stabilization in Thsp treated samples correlates with accumulation of p53 multimeric bands (an indicator of Thsp–protein adducts formation). (D) WB detection of polyubiquitination and Mcl1 accumulation in extracts of Raji cells. The cells were treated with CHX, CHX/MG-132 and CHX/Thsp for 4 hrs, washed and supplemented with fresh media and then harvested at different time-points (1–6 hrs). GAPDH was used as a loading control. The extent of polyubiquitination and Mcl-1 levels, 4 hrs after washing off MG-132 are largely reversed to non-treated state. For comparison, these effects remained steady or increased 4 hrs after Thsp wash off, which is consistent with a covalent-binding proteasome inhibition model.

    Techniques Used: Inhibition, Incubation, In Vitro, Western Blot, Binding Assay, Fluorescence, Activity Assay, Activation Assay

    Thiostrepton interacts covalently with proteins in human cells. (A) WB detection of Usp24 and p53 in HEK293 cell extracts incubated with different concentrations of Thsp, for different times. Note the formation of p53 and Usp24 dimers/multimers, an effect that is dependent on Thsp concentration and incubation time. (B) Time-dependent formation of p53 multimers in HEK293 cells treated with Thsp (10 μM), as detected by WB. (C) WB detection of DIAP1ΔR-YFP in DIAP1-sensor cells treated with DMSO, MG-132, IAP-antagonist (Comp-3) and Thsp for 21 hrs. Besides DIAP1ΔR-YFP accumulation, Thsp also triggers formation of DIAP1 dimers and multimers. (D) WB detection of Rpr-HA and p21 in DIAP1-sensor cells treated with DMSO, MG-132 and Thsp for 21 hrs. Thiostrepton -dependent formation of Rpr dimers/multimers becomes apparent. (E) Left, Coomassie-stained SDS-PAGE gel showing purified Rpr protein incubated at RT for different times. Right, cartoon of monomeric Rpr with known structural elements, as well as cartoon of Rpr dimer. (F) MALDI–ToF analysis of purified Rpr protein, which confirms the MW of Rpr and Rpr dimer. (G) Left, Coomassie-stained SDS-PAGE gel showing the apparent formation of Rpr–Thsp adducts following incubation of Rpr with Thsp, in a time-dependent fashion. Right, schematic model of Rpr–Thsp adduct and of Thsp bridging between two Rpr molecules, (Rpr) 2 -Thsp. (H) MALDI–ToF confirmation of Rpr–Thsp and (Rpr) 2 –Thsp adducts formation.
    Figure Legend Snippet: Thiostrepton interacts covalently with proteins in human cells. (A) WB detection of Usp24 and p53 in HEK293 cell extracts incubated with different concentrations of Thsp, for different times. Note the formation of p53 and Usp24 dimers/multimers, an effect that is dependent on Thsp concentration and incubation time. (B) Time-dependent formation of p53 multimers in HEK293 cells treated with Thsp (10 μM), as detected by WB. (C) WB detection of DIAP1ΔR-YFP in DIAP1-sensor cells treated with DMSO, MG-132, IAP-antagonist (Comp-3) and Thsp for 21 hrs. Besides DIAP1ΔR-YFP accumulation, Thsp also triggers formation of DIAP1 dimers and multimers. (D) WB detection of Rpr-HA and p21 in DIAP1-sensor cells treated with DMSO, MG-132 and Thsp for 21 hrs. Thiostrepton -dependent formation of Rpr dimers/multimers becomes apparent. (E) Left, Coomassie-stained SDS-PAGE gel showing purified Rpr protein incubated at RT for different times. Right, cartoon of monomeric Rpr with known structural elements, as well as cartoon of Rpr dimer. (F) MALDI–ToF analysis of purified Rpr protein, which confirms the MW of Rpr and Rpr dimer. (G) Left, Coomassie-stained SDS-PAGE gel showing the apparent formation of Rpr–Thsp adducts following incubation of Rpr with Thsp, in a time-dependent fashion. Right, schematic model of Rpr–Thsp adduct and of Thsp bridging between two Rpr molecules, (Rpr) 2 -Thsp. (H) MALDI–ToF confirmation of Rpr–Thsp and (Rpr) 2 –Thsp adducts formation.

    Techniques Used: Western Blot, Incubation, Concentration Assay, Staining, SDS Page, Purification

    Effect of Thiostrepton on proteasome activity in human cells and in vitro . (A) Left, schematic representation of the ZsGreen-MODC sensor cell line. Human HEK293 cells were stably transfected with a construct that entails Zoanthus sp . GFP gene fused to mouse ornithine decarboxylase degron (MODC). Right, microscopy images showing the fluorescence of the labile GFP-MODC reporter following DMSO, MG-132, Comp-3 and Thsp treatment for 21 hrs. (B) WB detection of polyubiquitin species and p21 accumulation in extracts of MIA-PaCa-2 cells treated with DMSO, MG-132 and Thsp for 20 hrs. (C) Effect of Thsp on catalytic activities (chymotrypsin-, trypsin- and caspase-like) of the purified bovine liver 20S proteasome. (D) Effect of Thsp and MG-132 on the activity of purified bovine 26S proteasome.
    Figure Legend Snippet: Effect of Thiostrepton on proteasome activity in human cells and in vitro . (A) Left, schematic representation of the ZsGreen-MODC sensor cell line. Human HEK293 cells were stably transfected with a construct that entails Zoanthus sp . GFP gene fused to mouse ornithine decarboxylase degron (MODC). Right, microscopy images showing the fluorescence of the labile GFP-MODC reporter following DMSO, MG-132, Comp-3 and Thsp treatment for 21 hrs. (B) WB detection of polyubiquitin species and p21 accumulation in extracts of MIA-PaCa-2 cells treated with DMSO, MG-132 and Thsp for 20 hrs. (C) Effect of Thsp on catalytic activities (chymotrypsin-, trypsin- and caspase-like) of the purified bovine liver 20S proteasome. (D) Effect of Thsp and MG-132 on the activity of purified bovine 26S proteasome.

    Techniques Used: Activity Assay, In Vitro, Stable Transfection, Transfection, Construct, Microscopy, Fluorescence, Western Blot, Purification

    19) Product Images from "Expression profiles of interferon-related genes in cells infected with influenza A viruses or transiently transfected with plasmids encoding viral RNA polymerase"

    Article Title: Expression profiles of interferon-related genes in cells infected with influenza A viruses or transiently transfected with plasmids encoding viral RNA polymerase

    Journal: Turkish Journal of Biology

    doi: 10.3906/biy-2005-73

    The effects of influenza A virus PA proteins on the expression of SEAP reporter in transiently transfected HEK293 cells. A. The cells expressing native PA proteins. B. The cells expressing chimeric PA proteins. C. The cells expressing influenza PA proteins with WSN PB2 and PB1. D. The cells expressing influenza PA proteins with DkPen, PB2 and PB1. Total plasmid DNA adjusted to 250 ng/well with pCAGGS plasmid DNA (Niwa et al., 1991).
    Figure Legend Snippet: The effects of influenza A virus PA proteins on the expression of SEAP reporter in transiently transfected HEK293 cells. A. The cells expressing native PA proteins. B. The cells expressing chimeric PA proteins. C. The cells expressing influenza PA proteins with WSN PB2 and PB1. D. The cells expressing influenza PA proteins with DkPen, PB2 and PB1. Total plasmid DNA adjusted to 250 ng/well with pCAGGS plasmid DNA (Niwa et al., 1991).

    Techniques Used: Expressing, Transfection, Plasmid Preparation

    The heatmaps and Venn diagram of the genes related to the interferon response in HEK293 cells infected with influenza A viruses (WSN or DkPen) and noninfected cells (HEK293).
    Figure Legend Snippet: The heatmaps and Venn diagram of the genes related to the interferon response in HEK293 cells infected with influenza A viruses (WSN or DkPen) and noninfected cells (HEK293).

    Techniques Used: Infection

    The heatmaps and Venn diagram of the interferon response genes in HEK293 cells transiently transected with plasmid DNAs expressing influenza A virus RdRP enzyme (3P).
    Figure Legend Snippet: The heatmaps and Venn diagram of the interferon response genes in HEK293 cells transiently transected with plasmid DNAs expressing influenza A virus RdRP enzyme (3P).

    Techniques Used: Plasmid Preparation, Expressing

    The heatmaps and Venn diagram of the interferon response genes in HEK293 cells transiently transected with plasmid DNAs expressing the PA subunit of influenza A virus RdRP.
    Figure Legend Snippet: The heatmaps and Venn diagram of the interferon response genes in HEK293 cells transiently transected with plasmid DNAs expressing the PA subunit of influenza A virus RdRP.

    Techniques Used: Plasmid Preparation, Expressing

    Western blot analysis of native actin beta and HA-actin proteins in transiently transfected HEK293 cells. The cells were cotransfected with pCHA-ACTB plasmid and a plasmid expressing deleted PA protein (pCAGGS-cPA/DkPen, pCAGGS-cPA/WSN, pCAGGS-nPA/DkPen or pCAGGS-nPA/WSN). Actin beta and viral PA proteins were separated on 10% polyacrylamide gel and immunoblotted with monoclonal mouse anti-HA (for HA-ACTB), monoclonal anti actin (for ACTB and HA-ACTB) and rabbit polyclonal anti-PA antibodies.
    Figure Legend Snippet: Western blot analysis of native actin beta and HA-actin proteins in transiently transfected HEK293 cells. The cells were cotransfected with pCHA-ACTB plasmid and a plasmid expressing deleted PA protein (pCAGGS-cPA/DkPen, pCAGGS-cPA/WSN, pCAGGS-nPA/DkPen or pCAGGS-nPA/WSN). Actin beta and viral PA proteins were separated on 10% polyacrylamide gel and immunoblotted with monoclonal mouse anti-HA (for HA-ACTB), monoclonal anti actin (for ACTB and HA-ACTB) and rabbit polyclonal anti-PA antibodies.

    Techniques Used: Western Blot, Transfection, Plasmid Preparation, Expressing

    The expression profiles of interferon response genes in HEK293 cells infected with influenza A viruses (WSN or DkPen) and noninfected cells (HEK293).
    Figure Legend Snippet: The expression profiles of interferon response genes in HEK293 cells infected with influenza A viruses (WSN or DkPen) and noninfected cells (HEK293).

    Techniques Used: Expressing, Infection

    20) Product Images from "Membrane Trafficking of Large Conductance Calcium-activated Potassium Channels Is Regulated by Alternative Splicing of a Transplantable, Acidic Trafficking Motif in the RCK1-RCK2 Linker *"

    Article Title: Membrane Trafficking of Large Conductance Calcium-activated Potassium Channels Is Regulated by Alternative Splicing of a Transplantable, Acidic Trafficking Motif in the RCK1-RCK2 Linker *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.139758

    Exon 18 and exon 19, but not exon 23, are essential for BK channel cell surface expression. a , in-frame deletion mutants of exons 18, 19, and 23 that are excluded in the hSloΔ 579–664 trafficking-deficient variant were expressed in HEK293 cells. Constructs contained an N-terminal extracellular FLAG tag and a C-terminal HA tag. Cell surface expression was assayed in imaging experiments under nonpermeabilized conditions by probing for the FLAG tag as in Fig. 3 . The data are expressed as percentages of control FLAG-zero-HA cell surface expression. b , inset , representative Western blots of HA immunoreactivity from cell surface biotinylation assays of HEK293 cells expressing the zero or e18 + e19 constructs and corresponding whole cell lysates. All of the data are the means ± S.E. from a minimum of three independent experiments with > 650 cells analyzed/group. **, p
    Figure Legend Snippet: Exon 18 and exon 19, but not exon 23, are essential for BK channel cell surface expression. a , in-frame deletion mutants of exons 18, 19, and 23 that are excluded in the hSloΔ 579–664 trafficking-deficient variant were expressed in HEK293 cells. Constructs contained an N-terminal extracellular FLAG tag and a C-terminal HA tag. Cell surface expression was assayed in imaging experiments under nonpermeabilized conditions by probing for the FLAG tag as in Fig. 3 . The data are expressed as percentages of control FLAG-zero-HA cell surface expression. b , inset , representative Western blots of HA immunoreactivity from cell surface biotinylation assays of HEK293 cells expressing the zero or e18 + e19 constructs and corresponding whole cell lysates. All of the data are the means ± S.E. from a minimum of three independent experiments with > 650 cells analyzed/group. **, p

    Techniques Used: Expressing, Variant Assay, Construct, FLAG-tag, Imaging, Western Blot

    An acidic cluster-like motif in exon 19 is essential for cell surface expression. a , ClustalW sequence alignment of exons 18 and exon 19 from human ( Homo sapiens , accession number AAD31173 ), mouse ( Mus musculus , accession number AAL69971 ), chicken ( Gallus gallus , accession number NP_989555 ), turtle ( Trachemys scripta , accession number AAC41281 ), worm ( Caenorhabditis elegans , accession number NP_001024259 ), and fly ( Drosophila melanogaster , accession number NP_524486 ). The exons form the extreme C terminus of the computationally predicted RCK1 domain and the start of the unstructured RCK1-RCK2 linker. Three putative trafficking/sorting motifs predicted in this region are shown with only the acidic DD XX D XXX I motif fully conserved across phyla. Amino acid numbering is based on the amino acid sequence of the human sequence AAD31173 that starts with MDALI. b , representative confocal sections from HEK293 cells transfected with the DD XX D XXX I mutants (D617A/D618A and I625A) and zero channels with the N-terminal epitope labeled under nonpermeabilized conditions and the intracellular C-terminal HA tag under permeabilized conditions. The scale bars are 2 μm. c , summary bar chart of cell surface FLAG expression of trafficking/sorting motif mutants in which amino acids within the proposed motifs are mutated to alanine. Cell surface expression is expressed as a percentage of the FLAG-zero-HA construct using FLAG surface expression in nonpermeabilized assays as in Fig. 2 . d , representative Western blots of HA immunoreactivity from cell surface biotinylation assays of HEK293 cells expressing the corresponding constructs and whole cell lysates. e , summary bar chart of cell surface biotinylation data as in d . All of the data are the means ± S.E. from a minimum of three independent experiments with > 720 cells analyzed/group in c . **, p
    Figure Legend Snippet: An acidic cluster-like motif in exon 19 is essential for cell surface expression. a , ClustalW sequence alignment of exons 18 and exon 19 from human ( Homo sapiens , accession number AAD31173 ), mouse ( Mus musculus , accession number AAL69971 ), chicken ( Gallus gallus , accession number NP_989555 ), turtle ( Trachemys scripta , accession number AAC41281 ), worm ( Caenorhabditis elegans , accession number NP_001024259 ), and fly ( Drosophila melanogaster , accession number NP_524486 ). The exons form the extreme C terminus of the computationally predicted RCK1 domain and the start of the unstructured RCK1-RCK2 linker. Three putative trafficking/sorting motifs predicted in this region are shown with only the acidic DD XX D XXX I motif fully conserved across phyla. Amino acid numbering is based on the amino acid sequence of the human sequence AAD31173 that starts with MDALI. b , representative confocal sections from HEK293 cells transfected with the DD XX D XXX I mutants (D617A/D618A and I625A) and zero channels with the N-terminal epitope labeled under nonpermeabilized conditions and the intracellular C-terminal HA tag under permeabilized conditions. The scale bars are 2 μm. c , summary bar chart of cell surface FLAG expression of trafficking/sorting motif mutants in which amino acids within the proposed motifs are mutated to alanine. Cell surface expression is expressed as a percentage of the FLAG-zero-HA construct using FLAG surface expression in nonpermeabilized assays as in Fig. 2 . d , representative Western blots of HA immunoreactivity from cell surface biotinylation assays of HEK293 cells expressing the corresponding constructs and whole cell lysates. e , summary bar chart of cell surface biotinylation data as in d . All of the data are the means ± S.E. from a minimum of three independent experiments with > 720 cells analyzed/group in c . **, p

    Techniques Used: Expressing, Sequencing, Transfection, Labeling, Construct, Western Blot

    Lack of cell surface expression of the hSloΔ 579–664 variant. a , representative confocal images of HEK293 cells expressing FLAG-hSloΔ 579–664 -HA ( top panels ), FLAG-e22-HA ( middle panels ), or FLAG-zero-HA ( bottom panels ). The extracellular FLAG epitope was labeled ( red ) under nonpermeabilized conditions (cell surface) with the C-terminal HA epitope tag ( green ) labeled following cell permeabilization. FLAG and HA labeling from the same cell are then overlaid ( Merge ). The FLAG-e22-HA construct is a splice variant with an alternatively spliced exon (exon 22) included between exons 19 and 23. The scale bars are 2 μm. b , quantification of surface expression expressed as a percentage of the total number of transfected cells with detectable cell surface (FLAG) expression for experiments as performed in a . The data are the means ± S.E. from a minimum of three independent experiments with > 600 cells analyzed/group. **, p
    Figure Legend Snippet: Lack of cell surface expression of the hSloΔ 579–664 variant. a , representative confocal images of HEK293 cells expressing FLAG-hSloΔ 579–664 -HA ( top panels ), FLAG-e22-HA ( middle panels ), or FLAG-zero-HA ( bottom panels ). The extracellular FLAG epitope was labeled ( red ) under nonpermeabilized conditions (cell surface) with the C-terminal HA epitope tag ( green ) labeled following cell permeabilization. FLAG and HA labeling from the same cell are then overlaid ( Merge ). The FLAG-e22-HA construct is a splice variant with an alternatively spliced exon (exon 22) included between exons 19 and 23. The scale bars are 2 μm. b , quantification of surface expression expressed as a percentage of the total number of transfected cells with detectable cell surface (FLAG) expression for experiments as performed in a . The data are the means ± S.E. from a minimum of three independent experiments with > 600 cells analyzed/group. **, p

    Techniques Used: Expressing, Variant Assay, FLAG-tag, Labeling, Construct, Transfection

    Acidic cluster sequence (DD XX D XXX I) is a transplantable ER export motif. a , representative single confocal sections from HEK293 cells expressing the GABA B R1a receptor with ( GABA B R1a(ASRR)+DDXXDXXXI ) and without ( GABA B R1a(ASRR) ) the DD XX D XXX I motif engineered onto the C terminus. The N-terminal (extracellular) HA epitope tag was labeled under nonpermeabilized ( left panels , red ) and permeabilized ( middle panels , green ) conditions in the same cell with the merged images shown in the right-hand panels . The GABA B R1a receptor contained an arginine to alanine mutation (ASRR) compared with the wild type GABA B R1a receptor. The scale bars are 2 μm. b , summary bar graph of quantitative surface/intracellular HA expression normalized to the ratio for the GABA B R1a(ASRR) expressing cells (100%). **, p
    Figure Legend Snippet: Acidic cluster sequence (DD XX D XXX I) is a transplantable ER export motif. a , representative single confocal sections from HEK293 cells expressing the GABA B R1a receptor with ( GABA B R1a(ASRR)+DDXXDXXXI ) and without ( GABA B R1a(ASRR) ) the DD XX D XXX I motif engineered onto the C terminus. The N-terminal (extracellular) HA epitope tag was labeled under nonpermeabilized ( left panels , red ) and permeabilized ( middle panels , green ) conditions in the same cell with the merged images shown in the right-hand panels . The GABA B R1a receptor contained an arginine to alanine mutation (ASRR) compared with the wild type GABA B R1a receptor. The scale bars are 2 μm. b , summary bar graph of quantitative surface/intracellular HA expression normalized to the ratio for the GABA B R1a(ASRR) expressing cells (100%). **, p

    Techniques Used: Sequencing, Expressing, Labeling, Mutagenesis

    Acidic cluster sequence (DD XX D XXX I) is required for efficient functional channel expression. a , time course plots of change in relative fluorescence units ( r.f.u ) of the FLIPR blue membrane potential dye in HEK293 cells expressing zero (○), hSloΔ 579–664 (▿), D617A/D618A (▵), I625A (■), or mock transfected HEK293 cells (◇) in response to calcium influx induced by 1 μ m ionomycin. A decrease in fluorescence relative to the HEK293 cell response indicates a net hyperpolarization of the membrane potential resulting from activation of BK channels. b , summary bar chart of the membrane potential change for each construct in a expressed as a percentage of the maximal hyperpolarization elicited in HEK293 cells expressing the zero variant (100%). The data were determined at t = 70 s in the time course plots in a . All of the data are the means ± S.E. ( n = 9–12). **, p
    Figure Legend Snippet: Acidic cluster sequence (DD XX D XXX I) is required for efficient functional channel expression. a , time course plots of change in relative fluorescence units ( r.f.u ) of the FLIPR blue membrane potential dye in HEK293 cells expressing zero (○), hSloΔ 579–664 (▿), D617A/D618A (▵), I625A (■), or mock transfected HEK293 cells (◇) in response to calcium influx induced by 1 μ m ionomycin. A decrease in fluorescence relative to the HEK293 cell response indicates a net hyperpolarization of the membrane potential resulting from activation of BK channels. b , summary bar chart of the membrane potential change for each construct in a expressed as a percentage of the maximal hyperpolarization elicited in HEK293 cells expressing the zero variant (100%). The data were determined at t = 70 s in the time course plots in a . All of the data are the means ± S.E. ( n = 9–12). **, p

    Techniques Used: Sequencing, Functional Assay, Expressing, Fluorescence, Transfection, Activation Assay, Construct, Variant Assay

    The hSloΔ 579–664 variant is a dominant negative of cell surface expression. a , Western blot of total cell lysates from the FLAG-hSloΔ 579–664 -HA and FLAG-zero-HA variants expressed in HEK293 cells. b , representative blots from co-immunoprecipitation ( IP ) experiments from HEK293 cells co-expressing the zero-HA variant with either the FLAG-hSloΔ 579–664 or FLAG-e22 variants. Left panel , channels were immunoprecipitated with mouse anti-FLAG M2 antibody, and blots were probed with rabbit anti-HA antibody. Right panel , channels were immunoprecipitated with rabbit anti-HA antibody and probed with mouse anti-FLAG M2 antibody. c , zero-HA subunits were co-expressed with either FLAG-hSloΔ 579–664 -eYFP, FLAG-e22-eYFP, or FLAG-zero-eYFP channels in HEK293 cells, and cell surface (FLAG) expression was quantified. d , FLAG-zero-eYFP subunits were co-expressed with either hSloΔ 579–664 -HA, e22-HA or zero-HA channels in HEK293 cells. In c and d , the data are the cell surface FLAG staining values expressed as percentages of control (surface FLAG-zero-eYFP levels when co-expressed with the zero-HA construct) under nonpermeabilized conditions. e , representative Western blots of HA or green fluorescent protein immunoreactivity from cell surface biotinylation assays of HEK293 cells expressing the indicated constructs and corresponding whole cell lysates. All data are the means ± S.E. from a minimum of six independent experiments with > 700 cells analyzed/group in c and d and three independent experiments in a , b , and e . **, p
    Figure Legend Snippet: The hSloΔ 579–664 variant is a dominant negative of cell surface expression. a , Western blot of total cell lysates from the FLAG-hSloΔ 579–664 -HA and FLAG-zero-HA variants expressed in HEK293 cells. b , representative blots from co-immunoprecipitation ( IP ) experiments from HEK293 cells co-expressing the zero-HA variant with either the FLAG-hSloΔ 579–664 or FLAG-e22 variants. Left panel , channels were immunoprecipitated with mouse anti-FLAG M2 antibody, and blots were probed with rabbit anti-HA antibody. Right panel , channels were immunoprecipitated with rabbit anti-HA antibody and probed with mouse anti-FLAG M2 antibody. c , zero-HA subunits were co-expressed with either FLAG-hSloΔ 579–664 -eYFP, FLAG-e22-eYFP, or FLAG-zero-eYFP channels in HEK293 cells, and cell surface (FLAG) expression was quantified. d , FLAG-zero-eYFP subunits were co-expressed with either hSloΔ 579–664 -HA, e22-HA or zero-HA channels in HEK293 cells. In c and d , the data are the cell surface FLAG staining values expressed as percentages of control (surface FLAG-zero-eYFP levels when co-expressed with the zero-HA construct) under nonpermeabilized conditions. e , representative Western blots of HA or green fluorescent protein immunoreactivity from cell surface biotinylation assays of HEK293 cells expressing the indicated constructs and corresponding whole cell lysates. All data are the means ± S.E. from a minimum of six independent experiments with > 700 cells analyzed/group in c and d and three independent experiments in a , b , and e . **, p

    Techniques Used: Variant Assay, Dominant Negative Mutation, Expressing, Western Blot, Immunoprecipitation, Staining, Construct

    21) Product Images from "Cystatin A, a Potential Common Link for Mutant Myocilin Causative Glaucoma"

    Article Title: Cystatin A, a Potential Common Link for Mutant Myocilin Causative Glaucoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0036301

    Cystatin A inhibits the processing of MYOC wild-type in cultured cells. Recombinant expression plasmids containing tag-fused full coding wild-type MYOC (pMG29), CSTA, and controls plasmids, inactive mutated CSTA (CSTAm) and pEmpty, were generated as indicated in Methods . pMG29 was co-transfected with either pCSTA, pCSTAm or pEmpty (1∶2) and harvested at 48 h post-transfection. Equivalent volumes of cell extracts and of their supernatants were loaded onto 4–15% SDS-PAGE gels, transferred to PVDF membranes and analyzed by immunoblotting. Different MYOC protein forms (full length and processed) were detected with an anti-V5 mouse monoclonal followed by an anti-mouse horseradish peroxidase antibodies. Blots were re-probed with β-actin and DDK antibodies for loading and identification controls. Percent of the MYOC processed band was calculated by densitometry. A) schematic representation of the expression cassettes of the recombinant plasmids. B, C and D: Representative western blots with extracts from transfected cells. B) extracts from HEK293 co-transfected by calcium phosphate. C and D) extracts from primary HTM-137 cells co-transfected by nucleofector electroporation.
    Figure Legend Snippet: Cystatin A inhibits the processing of MYOC wild-type in cultured cells. Recombinant expression plasmids containing tag-fused full coding wild-type MYOC (pMG29), CSTA, and controls plasmids, inactive mutated CSTA (CSTAm) and pEmpty, were generated as indicated in Methods . pMG29 was co-transfected with either pCSTA, pCSTAm or pEmpty (1∶2) and harvested at 48 h post-transfection. Equivalent volumes of cell extracts and of their supernatants were loaded onto 4–15% SDS-PAGE gels, transferred to PVDF membranes and analyzed by immunoblotting. Different MYOC protein forms (full length and processed) were detected with an anti-V5 mouse monoclonal followed by an anti-mouse horseradish peroxidase antibodies. Blots were re-probed with β-actin and DDK antibodies for loading and identification controls. Percent of the MYOC processed band was calculated by densitometry. A) schematic representation of the expression cassettes of the recombinant plasmids. B, C and D: Representative western blots with extracts from transfected cells. B) extracts from HEK293 co-transfected by calcium phosphate. C and D) extracts from primary HTM-137 cells co-transfected by nucleofector electroporation.

    Techniques Used: Cell Culture, Recombinant, Expressing, Generated, Transfection, SDS Page, Western Blot, Electroporation

    22) Product Images from "Control of NOD2 and Rip2 dependent innate immune activation by GEF-H1"

    Article Title: Control of NOD2 and Rip2 dependent innate immune activation by GEF-H1

    Journal: Inflammatory bowel diseases

    doi: 10.1002/ibd.21851

    GEF-H1 interacts with Rip kinases and specifically phosphorylates Rip2 (A) Analysis of immune complexes containing GEF-H1, which form in the presence of Rip kinases. GEF-H1 was immunoprecipitated from HEK293 cells after transfection with GEF-H1, Rip1, Rip2 and Rip3 expression constructs and analyzed by Western blotting with indicated antibodies. (B) Analysis of Rip2-containing immunocomplexes in the presence of NOD2 or the 3020insC NOD2 variant in HEK293 cells. (C) Alignment of Rip kinase protein sequences containing the new tyrosine phosphorylation sites in Rip2 responsible for GEF-H1-mediated NF-κB activation. (D) Analysis of immune complexes forming in the presence of GEF-H1, Rip2 or the Rip2 Y381A mutant in HEK293 cells.
    Figure Legend Snippet: GEF-H1 interacts with Rip kinases and specifically phosphorylates Rip2 (A) Analysis of immune complexes containing GEF-H1, which form in the presence of Rip kinases. GEF-H1 was immunoprecipitated from HEK293 cells after transfection with GEF-H1, Rip1, Rip2 and Rip3 expression constructs and analyzed by Western blotting with indicated antibodies. (B) Analysis of Rip2-containing immunocomplexes in the presence of NOD2 or the 3020insC NOD2 variant in HEK293 cells. (C) Alignment of Rip kinase protein sequences containing the new tyrosine phosphorylation sites in Rip2 responsible for GEF-H1-mediated NF-κB activation. (D) Analysis of immune complexes forming in the presence of GEF-H1, Rip2 or the Rip2 Y381A mutant in HEK293 cells.

    Techniques Used: Immunoprecipitation, Transfection, Expressing, Construct, Western Blot, Variant Assay, Activation Assay, Mutagenesis

    GEF-H1-mediated phosphorylation of Rip2 is dependent on Src kinases (A) Analysis of Rip2-containing immune complexes in the presence of GEF-H1 and various amounts the SFK inhibitor PP2. (B) NF-κB activation in HEK293 cells by GEF-H1 in the absence or presence of Rip2 and CSK (* indicates p
    Figure Legend Snippet: GEF-H1-mediated phosphorylation of Rip2 is dependent on Src kinases (A) Analysis of Rip2-containing immune complexes in the presence of GEF-H1 and various amounts the SFK inhibitor PP2. (B) NF-κB activation in HEK293 cells by GEF-H1 in the absence or presence of Rip2 and CSK (* indicates p

    Techniques Used: Activation Assay

    23) Product Images from "Osteogenic Potential of Mouse Adipose-Derived Stem Cells Sorted for CD90 and CD105 In Vitro"

    Article Title: Osteogenic Potential of Mouse Adipose-Derived Stem Cells Sorted for CD90 and CD105 In Vitro

    Journal: Stem Cells International

    doi: 10.1155/2014/576358

    Definition of viral titers. (a) pAdenoX encoding human bmp2 was identified using 2% agarose gel electrophoresis after PCR amplification with two primer sets. Lane 1: λ - Hind III digest; lane 2: φ X 174- Hae III digest; lane 3: full sequence human bmp2 (1191 bp); lane 4: partial sequence human bmp2 (225 bp). (b) HEK293 cells were infected with adenovirus encoding bmp2 , which demonstrated a titer of 5.85 × 10 6 ifu/mL.
    Figure Legend Snippet: Definition of viral titers. (a) pAdenoX encoding human bmp2 was identified using 2% agarose gel electrophoresis after PCR amplification with two primer sets. Lane 1: λ - Hind III digest; lane 2: φ X 174- Hae III digest; lane 3: full sequence human bmp2 (1191 bp); lane 4: partial sequence human bmp2 (225 bp). (b) HEK293 cells were infected with adenovirus encoding bmp2 , which demonstrated a titer of 5.85 × 10 6 ifu/mL.

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Sequencing, Infection

    24) Product Images from "Palmitoylation and Membrane Association of the Stress Axis Regulated Insert (STREX) Controls BK Channel Regulation by Protein Kinase C *"

    Article Title: Palmitoylation and Membrane Association of the Stress Axis Regulated Insert (STREX) Controls BK Channel Regulation by Protein Kinase C *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.386359

    The STREX insert prevents the inhibitory effect of PKC on BK channel activity. A–D , conductance-voltage relationships obtained from inside-out membrane patches of HEK293 cells expressing STREX or ZERO BK channels. A and B , curves before ( Ctr )
    Figure Legend Snippet: The STREX insert prevents the inhibitory effect of PKC on BK channel activity. A–D , conductance-voltage relationships obtained from inside-out membrane patches of HEK293 cells expressing STREX or ZERO BK channels. A and B , curves before ( Ctr )

    Techniques Used: Activity Assay, Expressing

    Removal of STREX membrane localization by PKA re-established PKC-dependent channel inhibition. A and B , conductance-voltage relationships obtained from inside-out membrane patches of HEK293 cells expressing STREX BK channels. Control patches ( Ctr ) were
    Figure Legend Snippet: Removal of STREX membrane localization by PKA re-established PKC-dependent channel inhibition. A and B , conductance-voltage relationships obtained from inside-out membrane patches of HEK293 cells expressing STREX BK channels. Control patches ( Ctr ) were

    Techniques Used: Inhibition, Expressing

    Phosphorylation of serine 1156 determines the sensitivity of STREX channels to protein kinases. A–D , bars represent conductances at +20 mV from inside-out membrane patches obtained from HEK293 cells expressing either the phosphomimetic S1156D
    Figure Legend Snippet: Phosphorylation of serine 1156 determines the sensitivity of STREX channels to protein kinases. A–D , bars represent conductances at +20 mV from inside-out membrane patches obtained from HEK293 cells expressing either the phosphomimetic S1156D

    Techniques Used: Expressing

    Prevention of STREX palmitoylation established PKC-dependent channel inhibition. Bars represent conductances at +40 mV from inside-out membrane patches obtained from HEK293 cells expressing STREX ( A ), the S700A- ( B ) or the C645A/C646A ( C ) mutant channel.
    Figure Legend Snippet: Prevention of STREX palmitoylation established PKC-dependent channel inhibition. Bars represent conductances at +40 mV from inside-out membrane patches obtained from HEK293 cells expressing STREX ( A ), the S700A- ( B ) or the C645A/C646A ( C ) mutant channel.

    Techniques Used: Inhibition, Expressing, Mutagenesis

    25) Product Images from "Identification of Target Binding Site in Photoreceptor Guanylyl Cyclase-activating Protein 1 (GCAP1) *"

    Article Title: Identification of Target Binding Site in Photoreceptor Guanylyl Cyclase-activating Protein 1 (GCAP1) *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.540716

    Effect of mutations in EF-hand 1 on direct binding of GCAP1 to RetGC1 in HEK293 cells. A ): left panel , GCAP1-GFP fluorescence, middle panel , fluorescence superimposed on differential interference contrast ( DIC ) image; right panel , distribution of GCAP1 fluorescence (arbitrary scale) across the cell scanned along the dashed line shown in the middle panel. B–I , expression vectors coding for GCAP1-GFP ( green ) and mOrange-RetGC1 ( red ). Each row of panels presents ( left to right ), respectively, the fluorescence image of GCAP1, the fluorescence image of RetGC1, a merged image of the two, and the distribution of the corresponding fluorochrome brightness (arbitrary scale) scanned across the cell along the dashed line shown in the merged image. B , wild type; C , Y22D; D , K24D; E , M26R; F , T27K; G , S31Y; H , G32N; and I , E38R. A minor γ adjustment for better clarity of perception in some panels was applied to the whole view field. The fluorescence intensity distribution in all cases was recorded from the original image within proportional range of the photomultiplier without any adjustments to the image itself. Objective, ×60; the green fluorescence was excited by 488 nm and the red fluorescence by 543 nm laser, respectively.
    Figure Legend Snippet: Effect of mutations in EF-hand 1 on direct binding of GCAP1 to RetGC1 in HEK293 cells. A ): left panel , GCAP1-GFP fluorescence, middle panel , fluorescence superimposed on differential interference contrast ( DIC ) image; right panel , distribution of GCAP1 fluorescence (arbitrary scale) across the cell scanned along the dashed line shown in the middle panel. B–I , expression vectors coding for GCAP1-GFP ( green ) and mOrange-RetGC1 ( red ). Each row of panels presents ( left to right ), respectively, the fluorescence image of GCAP1, the fluorescence image of RetGC1, a merged image of the two, and the distribution of the corresponding fluorochrome brightness (arbitrary scale) scanned across the cell along the dashed line shown in the merged image. B , wild type; C , Y22D; D , K24D; E , M26R; F , T27K; G , S31Y; H , G32N; and I , E38R. A minor γ adjustment for better clarity of perception in some panels was applied to the whole view field. The fluorescence intensity distribution in all cases was recorded from the original image within proportional range of the photomultiplier without any adjustments to the image itself. Objective, ×60; the green fluorescence was excited by 488 nm and the red fluorescence by 543 nm laser, respectively.

    Techniques Used: Binding Assay, Fluorescence, Expressing

    26) Product Images from "Targeted deletion of the aquaglyceroporin AQP9 is protective in a mouse model of Parkinson’s disease"

    Article Title: Targeted deletion of the aquaglyceroporin AQP9 is protective in a mouse model of Parkinson’s disease

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0194896

    HEK293 cells expressing h AQP9 or h DAT reveal higher sensitivity to MPP + . A-B) Immunofluorescence images of HEK293 cells expressing EGFP- h AQP9 (A) or YFP- h DAT (B) grown on coverslips confirm plasma membrane localization of the respective constructs (identified by antibodies to AQP9 or DAT). The cells were counterstained with Hoechst to visualize nuclei. C-G) Native HEK293 cells and HEK293 cells expressing EGFP- h AQP9 or YFP- h DAT were grown in 96-well plates and exposed to different concentrations of MPP + (four wells for each concentration). Cell viability was assessed after 24 hours using the MTT assay. Data were collected from independent plates (n = 3 for each construct) and normalized to respective untreated cells. Native HEK293 cells show sensitivity to MPP + only at very high concentrations (~100 μM). Cells expressing h DAT become sensitive at 1 μM MPP + , compared with 0.1 μM for cells expressing h AQP9. Overlay of the dose/response curve for the three groups (D) and the individual curves for native HEK293 cells (E), YFP- h DAT expressing (F) and EGFP- h AQP9 expressing HEK293 cells (G) are shown. Bars are mean ± SEM. Asterisks: significantly different from untreated controls; *p
    Figure Legend Snippet: HEK293 cells expressing h AQP9 or h DAT reveal higher sensitivity to MPP + . A-B) Immunofluorescence images of HEK293 cells expressing EGFP- h AQP9 (A) or YFP- h DAT (B) grown on coverslips confirm plasma membrane localization of the respective constructs (identified by antibodies to AQP9 or DAT). The cells were counterstained with Hoechst to visualize nuclei. C-G) Native HEK293 cells and HEK293 cells expressing EGFP- h AQP9 or YFP- h DAT were grown in 96-well plates and exposed to different concentrations of MPP + (four wells for each concentration). Cell viability was assessed after 24 hours using the MTT assay. Data were collected from independent plates (n = 3 for each construct) and normalized to respective untreated cells. Native HEK293 cells show sensitivity to MPP + only at very high concentrations (~100 μM). Cells expressing h DAT become sensitive at 1 μM MPP + , compared with 0.1 μM for cells expressing h AQP9. Overlay of the dose/response curve for the three groups (D) and the individual curves for native HEK293 cells (E), YFP- h DAT expressing (F) and EGFP- h AQP9 expressing HEK293 cells (G) are shown. Bars are mean ± SEM. Asterisks: significantly different from untreated controls; *p

    Techniques Used: Expressing, Immunofluorescence, Construct, Concentration Assay, MTT Assay

    HEK293 cells expressing EGFP- h AQP9 are more sensitive to arsenite than HEK293 cells expressing YFP- h DAT. A-C) Native HEK293 cells and HEK293 cells expressing EGFP- h AQP9 or YFP- h DAT were grown in 96-well plates and exposed to different concentrations of arsenite (eight wells for each concentration). Cell viability was assessed after 24 hours using the MTT assay. Data were collected from independent plates (n = 3 for each construct) and normalized to respective untreated cells. Both EGFP- h AQP9 and YFP- h DAT expressing cells showed higher sensitivity to arsenite, than native HEK293 cells, with EGFP- h AQP9 cells being the most sensitive. At the arsenite concentration of 10 μM, stably transfected EGFP- h AQP9 were the only cells showing toxin sensitivity (A). The curve showing IC50 values for arsenite calculated by nonlinear regression, log(inhibitor) vs response (three parameters) is shown (B). For log transformed data, the concentration 0 was set to 1 nM. Comparison of the IC50 values shows a significantly lower IC50 value for the HEK293 cells expressing EGFP- h AQP9 compared to the native HEK293 cells or HEK293 cells expressing YFP- h DAT (C). Bars are mean ± SEM. Asterisks: significantly different from untreated controls; *p
    Figure Legend Snippet: HEK293 cells expressing EGFP- h AQP9 are more sensitive to arsenite than HEK293 cells expressing YFP- h DAT. A-C) Native HEK293 cells and HEK293 cells expressing EGFP- h AQP9 or YFP- h DAT were grown in 96-well plates and exposed to different concentrations of arsenite (eight wells for each concentration). Cell viability was assessed after 24 hours using the MTT assay. Data were collected from independent plates (n = 3 for each construct) and normalized to respective untreated cells. Both EGFP- h AQP9 and YFP- h DAT expressing cells showed higher sensitivity to arsenite, than native HEK293 cells, with EGFP- h AQP9 cells being the most sensitive. At the arsenite concentration of 10 μM, stably transfected EGFP- h AQP9 were the only cells showing toxin sensitivity (A). The curve showing IC50 values for arsenite calculated by nonlinear regression, log(inhibitor) vs response (three parameters) is shown (B). For log transformed data, the concentration 0 was set to 1 nM. Comparison of the IC50 values shows a significantly lower IC50 value for the HEK293 cells expressing EGFP- h AQP9 compared to the native HEK293 cells or HEK293 cells expressing YFP- h DAT (C). Bars are mean ± SEM. Asterisks: significantly different from untreated controls; *p

    Techniques Used: Expressing, Concentration Assay, MTT Assay, Construct, Stable Transfection, Transfection, Transformation Assay

    27) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.215426

    Mapping of the regions involved in the association of TRAF7 with NEMO. A , schematic representation of the TRAF7 constructs used in this study. B , HEK293 cells were transfected with a vector expressing the indicated FLAG-tagged TRAF7 polypeptides. 24 h later, cell lysates were immunoprecipitated ( IP ) with anti-NEMO antibody and analyzed by immunoblot probed with anti-FLAG mAb. WB , Western blot. C , HEK293 cells were transfected with a vector expressing the indicated FLAG- and HA-tagged polypeptides. 24 h later, cell lysates were immunoprecipitated with anti-HA and analyzed by immunoblot probed with anti-FLAG mAb.
    Figure Legend Snippet: Mapping of the regions involved in the association of TRAF7 with NEMO. A , schematic representation of the TRAF7 constructs used in this study. B , HEK293 cells were transfected with a vector expressing the indicated FLAG-tagged TRAF7 polypeptides. 24 h later, cell lysates were immunoprecipitated ( IP ) with anti-NEMO antibody and analyzed by immunoblot probed with anti-FLAG mAb. WB , Western blot. C , HEK293 cells were transfected with a vector expressing the indicated FLAG- and HA-tagged polypeptides. 24 h later, cell lysates were immunoprecipitated with anti-HA and analyzed by immunoblot probed with anti-FLAG mAb.

    Techniques Used: Construct, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Western Blot

    TRAF7 promotes cell death. A , HEK293 cells were transfected with the indicated expression plasmids. Twenty four hours after transfection, cell viability was determined with an MTT assay. B , phase contrast micrographs of HEK293 cells transfected with a vector empty or encoding for TRAF7 or with a lentiviral vector expressing a TRAF7-silencing shRNA, left untreated, or exposed for 24 h to TNFα (10 ng/ml).
    Figure Legend Snippet: TRAF7 promotes cell death. A , HEK293 cells were transfected with the indicated expression plasmids. Twenty four hours after transfection, cell viability was determined with an MTT assay. B , phase contrast micrographs of HEK293 cells transfected with a vector empty or encoding for TRAF7 or with a lentiviral vector expressing a TRAF7-silencing shRNA, left untreated, or exposed for 24 h to TNFα (10 ng/ml).

    Techniques Used: Transfection, Expressing, MTT Assay, Plasmid Preparation, shRNA

    Fluorescence micrographs of p65. A , HEK293 cells expressing p65 alone or in the presence of an excess of TRAF7 were stained with anti-p65Ab followed by FITC-conjugated anti-mouse IgG. B , HeLa cells expressing an shRNA targeting TRAF7 or a control shRNA were stained with anti-p65Ab followed by FITC-conjugated anti-goat IgG.
    Figure Legend Snippet: Fluorescence micrographs of p65. A , HEK293 cells expressing p65 alone or in the presence of an excess of TRAF7 were stained with anti-p65Ab followed by FITC-conjugated anti-mouse IgG. B , HeLa cells expressing an shRNA targeting TRAF7 or a control shRNA were stained with anti-p65Ab followed by FITC-conjugated anti-goat IgG.

    Techniques Used: Fluorescence, Expressing, Staining, shRNA

    TRAF7 promotes ubiquitination of NEMO. A , HEK293 cells were transfected with the indicated amount of a vector expressing TRAF7. 24 h later, cell lysates were immunoprecipitated ( IP ) with anti-NEMO antibody, separated by SDS-PAGE, and transferred onto membranes subsequently probed with anti-ubiquitin. WB , Western blot. B , HEK293 cells were cotransfected with the indicated constructs; cell lysates were immunoprecipitated with anti-NEMO antibody and analyzed by immunoblot probed with anti-HA. C , HEK293 cells were cotransfected with FLAG-tagged TRAF7 and HA-tagged ubiquitin ( Ub ) mutants; the number ( n ) indicates the only lysine residue remaining in the ubiquitin molecule. Immunoprecipitates with anti-NEMO antibody were resolved by SDS-PAGE and blotted onto a membrane subsequently probed with anti-HA. A fraction of cell lysates was probed with anti-HA antibody to monitor expression and usage of all ubiquitin mutants. D , HEK293 cells were cotransfected with TRAF6 and HA-tagged ubiquitin mutants as in C , and anti-NEMO immunoprecipitates were analyzed by immunoblot probed with anti-HA.
    Figure Legend Snippet: TRAF7 promotes ubiquitination of NEMO. A , HEK293 cells were transfected with the indicated amount of a vector expressing TRAF7. 24 h later, cell lysates were immunoprecipitated ( IP ) with anti-NEMO antibody, separated by SDS-PAGE, and transferred onto membranes subsequently probed with anti-ubiquitin. WB , Western blot. B , HEK293 cells were cotransfected with the indicated constructs; cell lysates were immunoprecipitated with anti-NEMO antibody and analyzed by immunoblot probed with anti-HA. C , HEK293 cells were cotransfected with FLAG-tagged TRAF7 and HA-tagged ubiquitin ( Ub ) mutants; the number ( n ) indicates the only lysine residue remaining in the ubiquitin molecule. Immunoprecipitates with anti-NEMO antibody were resolved by SDS-PAGE and blotted onto a membrane subsequently probed with anti-HA. A fraction of cell lysates was probed with anti-HA antibody to monitor expression and usage of all ubiquitin mutants. D , HEK293 cells were cotransfected with TRAF6 and HA-tagged ubiquitin mutants as in C , and anti-NEMO immunoprecipitates were analyzed by immunoblot probed with anti-HA.

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, SDS Page, Western Blot, Construct

    28) Product Images from "Human sorting nexin 2 protein interacts with Influenza A virus PA protein and has a negative regulatory effect on the virus replication"

    Article Title: Human sorting nexin 2 protein interacts with Influenza A virus PA protein and has a negative regulatory effect on the virus replication

    Journal: Molecular Biology Reports

    doi: 10.1007/s11033-021-06906-9

    Rescue effect of SNX2 proteins on influenza A/DkPen PA inhibitory activity to host gene expression. The HEK293 cells were grown in a 24-well plate (5 × 10 4 cells/well) for 24 h in standard culture conditions and transfected with constant amounts of pCAGGS-PA(D) (1 ng/well) and pSEAP (20 ng/well) plasmids and indicated amounts of the SNX2 plasmids in the figure. After 24 h of transfection, the reporter SEAP activities in culture media was detected with a commercial kit
    Figure Legend Snippet: Rescue effect of SNX2 proteins on influenza A/DkPen PA inhibitory activity to host gene expression. The HEK293 cells were grown in a 24-well plate (5 × 10 4 cells/well) for 24 h in standard culture conditions and transfected with constant amounts of pCAGGS-PA(D) (1 ng/well) and pSEAP (20 ng/well) plasmids and indicated amounts of the SNX2 plasmids in the figure. After 24 h of transfection, the reporter SEAP activities in culture media was detected with a commercial kit

    Techniques Used: Activity Assay, Expressing, Transfection

    29) Product Images from "Human DDX56 protein interacts with influenza A virus NS1 protein and stimulates the virus replication"

    Article Title: Human DDX56 protein interacts with influenza A virus NS1 protein and stimulates the virus replication

    Journal: Genetics and Molecular Biology

    doi: 10.1590/1678-4685-GMB-2020-0158

    The quantitation of human DDX56 transcript and viral M1 protein in the cells transfected with siRNA or H-DDX56 encoding plasmid DNA (pCHA-DDX56) and infected with influenza A viruses. A . Quantity of human DDX56 transcript in the non-transfected HeLa cells (N.T. cells), the HeLa cells transfected with DDX56 specific siRNA (siRNA/DDX56), and negative control siRNA (cont.siRNA). B . Viral M1 protein quantity in HeLa cells transfected with siRNAs and then infected with viruses. C. The quantity (plaque forming unit; pfu/ml) of influenza A virus in medium of knock down/infected cell culture . D . The M1 protein quantity in non-transfected HEK293 cells (N.T. cells) and in HEK293 cells transfected with pCHA-DDX56 or pCHA (Vector) plasmid and then infected with human influenza A (WSN) viruses. After 8 h p.i. the cells harvested and the proteins in the cell lysates were separated on 10% PAGE. As primary antibodies, mouse monoclonal anti-HA, rabbit polyclonal anti-M1 serum, and mouse monoclonal anti-actin beta antibodies were used.
    Figure Legend Snippet: The quantitation of human DDX56 transcript and viral M1 protein in the cells transfected with siRNA or H-DDX56 encoding plasmid DNA (pCHA-DDX56) and infected with influenza A viruses. A . Quantity of human DDX56 transcript in the non-transfected HeLa cells (N.T. cells), the HeLa cells transfected with DDX56 specific siRNA (siRNA/DDX56), and negative control siRNA (cont.siRNA). B . Viral M1 protein quantity in HeLa cells transfected with siRNAs and then infected with viruses. C. The quantity (plaque forming unit; pfu/ml) of influenza A virus in medium of knock down/infected cell culture . D . The M1 protein quantity in non-transfected HEK293 cells (N.T. cells) and in HEK293 cells transfected with pCHA-DDX56 or pCHA (Vector) plasmid and then infected with human influenza A (WSN) viruses. After 8 h p.i. the cells harvested and the proteins in the cell lysates were separated on 10% PAGE. As primary antibodies, mouse monoclonal anti-HA, rabbit polyclonal anti-M1 serum, and mouse monoclonal anti-actin beta antibodies were used.

    Techniques Used: Quantitation Assay, Transfection, Plasmid Preparation, Infection, Negative Control, Cell Culture, Polyacrylamide Gel Electrophoresis

    30) Product Images from "Expression profiles of interferon-related genes in cells infected with influenza A viruses or transiently transfected with plasmids encoding viral RNA polymerase"

    Article Title: Expression profiles of interferon-related genes in cells infected with influenza A viruses or transiently transfected with plasmids encoding viral RNA polymerase

    Journal: Turkish Journal of Biology

    doi: 10.3906/biy-2005-73

    The effects of influenza A virus PA proteins on the expression of SEAP reporter in transiently transfected HEK293 cells. A. The cells expressing native PA proteins. B. The cells expressing chimeric PA proteins. C. The cells expressing influenza PA proteins with WSN PB2 and PB1. D. The cells expressing influenza PA proteins with DkPen, PB2 and PB1. Total plasmid DNA adjusted to 250 ng/well with pCAGGS plasmid DNA (Niwa et al., 1991).
    Figure Legend Snippet: The effects of influenza A virus PA proteins on the expression of SEAP reporter in transiently transfected HEK293 cells. A. The cells expressing native PA proteins. B. The cells expressing chimeric PA proteins. C. The cells expressing influenza PA proteins with WSN PB2 and PB1. D. The cells expressing influenza PA proteins with DkPen, PB2 and PB1. Total plasmid DNA adjusted to 250 ng/well with pCAGGS plasmid DNA (Niwa et al., 1991).

    Techniques Used: Expressing, Transfection, Plasmid Preparation

    The heatmaps and Venn diagram of the genes related to the interferon response in HEK293 cells infected with influenza A viruses (WSN or DkPen) and noninfected cells (HEK293).
    Figure Legend Snippet: The heatmaps and Venn diagram of the genes related to the interferon response in HEK293 cells infected with influenza A viruses (WSN or DkPen) and noninfected cells (HEK293).

    Techniques Used: Infection

    The heatmaps and Venn diagram of the interferon response genes in HEK293 cells transiently transected with plasmid DNAs expressing influenza A virus RdRP enzyme (3P).
    Figure Legend Snippet: The heatmaps and Venn diagram of the interferon response genes in HEK293 cells transiently transected with plasmid DNAs expressing influenza A virus RdRP enzyme (3P).

    Techniques Used: Plasmid Preparation, Expressing

    The heatmaps and Venn diagram of the interferon response genes in HEK293 cells transiently transected with plasmid DNAs expressing the PA subunit of influenza A virus RdRP.
    Figure Legend Snippet: The heatmaps and Venn diagram of the interferon response genes in HEK293 cells transiently transected with plasmid DNAs expressing the PA subunit of influenza A virus RdRP.

    Techniques Used: Plasmid Preparation, Expressing

    Western blot analysis of native actin beta and HA-actin proteins in transiently transfected HEK293 cells. The cells were cotransfected with pCHA-ACTB plasmid and a plasmid expressing deleted PA protein (pCAGGS-cPA/DkPen, pCAGGS-cPA/WSN, pCAGGS-nPA/DkPen or pCAGGS-nPA/WSN). Actin beta and viral PA proteins were separated on 10% polyacrylamide gel and immunoblotted with monoclonal mouse anti-HA (for HA-ACTB), monoclonal anti actin (for ACTB and HA-ACTB) and rabbit polyclonal anti-PA antibodies.
    Figure Legend Snippet: Western blot analysis of native actin beta and HA-actin proteins in transiently transfected HEK293 cells. The cells were cotransfected with pCHA-ACTB plasmid and a plasmid expressing deleted PA protein (pCAGGS-cPA/DkPen, pCAGGS-cPA/WSN, pCAGGS-nPA/DkPen or pCAGGS-nPA/WSN). Actin beta and viral PA proteins were separated on 10% polyacrylamide gel and immunoblotted with monoclonal mouse anti-HA (for HA-ACTB), monoclonal anti actin (for ACTB and HA-ACTB) and rabbit polyclonal anti-PA antibodies.

    Techniques Used: Western Blot, Transfection, Plasmid Preparation, Expressing

    The expression profiles of interferon response genes in HEK293 cells infected with influenza A viruses (WSN or DkPen) and noninfected cells (HEK293).
    Figure Legend Snippet: The expression profiles of interferon response genes in HEK293 cells infected with influenza A viruses (WSN or DkPen) and noninfected cells (HEK293).

    Techniques Used: Expressing, Infection

    31) Product Images from "Expression profiles of interferon-related genes in cells infected with influenza A viruses or transiently transfected with plasmids encoding viral RNA polymerase"

    Article Title: Expression profiles of interferon-related genes in cells infected with influenza A viruses or transiently transfected with plasmids encoding viral RNA polymerase

    Journal: Turkish Journal of Biology

    doi: 10.3906/biy-2005-73

    The effects of influenza A virus PA proteins on the expression of SEAP reporter in transiently transfected HEK293 cells. A. The cells expressing native PA proteins. B. The cells expressing chimeric PA proteins. C. The cells expressing influenza PA proteins with WSN PB2 and PB1. D. The cells expressing influenza PA proteins with DkPen, PB2 and PB1. Total plasmid DNA adjusted to 250 ng/well with pCAGGS plasmid DNA (Niwa et al., 1991).
    Figure Legend Snippet: The effects of influenza A virus PA proteins on the expression of SEAP reporter in transiently transfected HEK293 cells. A. The cells expressing native PA proteins. B. The cells expressing chimeric PA proteins. C. The cells expressing influenza PA proteins with WSN PB2 and PB1. D. The cells expressing influenza PA proteins with DkPen, PB2 and PB1. Total plasmid DNA adjusted to 250 ng/well with pCAGGS plasmid DNA (Niwa et al., 1991).

    Techniques Used: Expressing, Transfection, Plasmid Preparation

    The heatmaps and Venn diagram of the genes related to the interferon response in HEK293 cells infected with influenza A viruses (WSN or DkPen) and noninfected cells (HEK293).
    Figure Legend Snippet: The heatmaps and Venn diagram of the genes related to the interferon response in HEK293 cells infected with influenza A viruses (WSN or DkPen) and noninfected cells (HEK293).

    Techniques Used: Infection

    The heatmaps and Venn diagram of the interferon response genes in HEK293 cells transiently transected with plasmid DNAs expressing influenza A virus RdRP enzyme (3P).
    Figure Legend Snippet: The heatmaps and Venn diagram of the interferon response genes in HEK293 cells transiently transected with plasmid DNAs expressing influenza A virus RdRP enzyme (3P).

    Techniques Used: Plasmid Preparation, Expressing

    The heatmaps and Venn diagram of the interferon response genes in HEK293 cells transiently transected with plasmid DNAs expressing the PA subunit of influenza A virus RdRP.
    Figure Legend Snippet: The heatmaps and Venn diagram of the interferon response genes in HEK293 cells transiently transected with plasmid DNAs expressing the PA subunit of influenza A virus RdRP.

    Techniques Used: Plasmid Preparation, Expressing

    Western blot analysis of native actin beta and HA-actin proteins in transiently transfected HEK293 cells. The cells were cotransfected with pCHA-ACTB plasmid and a plasmid expressing deleted PA protein (pCAGGS-cPA/DkPen, pCAGGS-cPA/WSN, pCAGGS-nPA/DkPen or pCAGGS-nPA/WSN). Actin beta and viral PA proteins were separated on 10% polyacrylamide gel and immunoblotted with monoclonal mouse anti-HA (for HA-ACTB), monoclonal anti actin (for ACTB and HA-ACTB) and rabbit polyclonal anti-PA antibodies.
    Figure Legend Snippet: Western blot analysis of native actin beta and HA-actin proteins in transiently transfected HEK293 cells. The cells were cotransfected with pCHA-ACTB plasmid and a plasmid expressing deleted PA protein (pCAGGS-cPA/DkPen, pCAGGS-cPA/WSN, pCAGGS-nPA/DkPen or pCAGGS-nPA/WSN). Actin beta and viral PA proteins were separated on 10% polyacrylamide gel and immunoblotted with monoclonal mouse anti-HA (for HA-ACTB), monoclonal anti actin (for ACTB and HA-ACTB) and rabbit polyclonal anti-PA antibodies.

    Techniques Used: Western Blot, Transfection, Plasmid Preparation, Expressing

    The expression profiles of interferon response genes in HEK293 cells infected with influenza A viruses (WSN or DkPen) and noninfected cells (HEK293).
    Figure Legend Snippet: The expression profiles of interferon response genes in HEK293 cells infected with influenza A viruses (WSN or DkPen) and noninfected cells (HEK293).

    Techniques Used: Expressing, Infection

    32) Product Images from "Regulation of mitochondrial iron homeostasis by sideroflexin 2"

    Article Title: Regulation of mitochondrial iron homeostasis by sideroflexin 2

    Journal: The Journal of Physiological Sciences

    doi: 10.1007/s12576-018-0652-2

    Mitochondrial localization of SFXN2. mCherry-SFXN2 ( a ) and SFXN2-mCherry ( b ) colocalized with MitoTracker. Bar = 5 μm. mCherry-SFXN2 ( c ) and SFXN2-mCherry ( d ) colocalized with MitoTracker and endogenous Tomm20. Arrows indicate mCherry-SFXN2 or SFXN2-mCherry, while arrowheads indicate Tomm20. The fluorescence intensities along the dashed lines are shown as line profile graphs. Bars = 1 μm. e Mitochondria were isolated from HEK293 cells transfected with SFXN2-mCherry and then digested with trypsin. SFXN2, Mitofusin1, and Timm50 were detected by Western blotting. The arrow indicates bands corresponding to SFXN2-mCherry. f Quantification of SFXN2-mCherry, Mitofusin1, and Timm50. n = 3–4 each. * p
    Figure Legend Snippet: Mitochondrial localization of SFXN2. mCherry-SFXN2 ( a ) and SFXN2-mCherry ( b ) colocalized with MitoTracker. Bar = 5 μm. mCherry-SFXN2 ( c ) and SFXN2-mCherry ( d ) colocalized with MitoTracker and endogenous Tomm20. Arrows indicate mCherry-SFXN2 or SFXN2-mCherry, while arrowheads indicate Tomm20. The fluorescence intensities along the dashed lines are shown as line profile graphs. Bars = 1 μm. e Mitochondria were isolated from HEK293 cells transfected with SFXN2-mCherry and then digested with trypsin. SFXN2, Mitofusin1, and Timm50 were detected by Western blotting. The arrow indicates bands corresponding to SFXN2-mCherry. f Quantification of SFXN2-mCherry, Mitofusin1, and Timm50. n = 3–4 each. * p

    Techniques Used: Fluorescence, Isolation, Transfection, Western Blot

    33) Product Images from "Different Contribution of Redox-Sensitive Transient Receptor Potential Channels to Acetaminophen-Induced Death of Human Hepatoma Cell Line"

    Article Title: Different Contribution of Redox-Sensitive Transient Receptor Potential Channels to Acetaminophen-Induced Death of Human Hepatoma Cell Line

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2016.00019

    Expression of redox-sensitive TRP channels in HepG2 cells and their responses when expressed in HEK293 cells . (A) Expression of redox-sensitive TRP channel mRNAs [TRPV1-4, TRPC1, TRPC4, TRPC5, TRPM2, TRPM7, TRPA1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] detected by RT-PCR in total RNA isolated from HepG2 cells. Specific PCR primers used are listed in Table 1 . (B,C) [Ca 2+ ] i responsesevoked by 20 mM APAP (B) or 1 mM H 2 O 2 (C) in HEK293 cells expressing human TRPV1, human TRPV2, human TRPV3, human TRPV4, mouse TRPC1, mouse TRPC4β, mouse TRPC5, human TRPM2, human TRPM7, human TRPA1, or vector. Average time courses (left) and Δ[Ca 2+ ] i (right) ( n = 16–64). Data points are mean ± SEM. P ≥0.05, * P
    Figure Legend Snippet: Expression of redox-sensitive TRP channels in HepG2 cells and their responses when expressed in HEK293 cells . (A) Expression of redox-sensitive TRP channel mRNAs [TRPV1-4, TRPC1, TRPC4, TRPC5, TRPM2, TRPM7, TRPA1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] detected by RT-PCR in total RNA isolated from HepG2 cells. Specific PCR primers used are listed in Table 1 . (B,C) [Ca 2+ ] i responsesevoked by 20 mM APAP (B) or 1 mM H 2 O 2 (C) in HEK293 cells expressing human TRPV1, human TRPV2, human TRPV3, human TRPV4, mouse TRPC1, mouse TRPC4β, mouse TRPC5, human TRPM2, human TRPM7, human TRPA1, or vector. Average time courses (left) and Δ[Ca 2+ ] i (right) ( n = 16–64). Data points are mean ± SEM. P ≥0.05, * P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Polymerase Chain Reaction, Plasmid Preparation

    34) Product Images from "The Human Cytomegalovirus IE1 Protein Antagonizes PML Nuclear Body-Mediated Intrinsic Immunity via the Inhibition of PML De Novo SUMOylation"

    Article Title: The Human Cytomegalovirus IE1 Protein Antagonizes PML Nuclear Body-Mediated Intrinsic Immunity via the Inhibition of PML De Novo SUMOylation

    Journal: Journal of Virology

    doi: 10.1128/JVI.02049-16

    IE1 prevents PML de novo SUMOylation in vivo . (A and B) HEK293-IE1 cells were not induced or induced with doxycycline (0.5 μg/ml). (A) At the indicated time points, the cells were harvested and analyzed by Western blotting for IE1 and β-actin.
    Figure Legend Snippet: IE1 prevents PML de novo SUMOylation in vivo . (A and B) HEK293-IE1 cells were not induced or induced with doxycycline (0.5 μg/ml). (A) At the indicated time points, the cells were harvested and analyzed by Western blotting for IE1 and β-actin.

    Techniques Used: In Vivo, Western Blot

    35) Product Images from "Intronic regulation of Aire expression by Jmjd6 for self-tolerance induction in the thymus"

    Article Title: Intronic regulation of Aire expression by Jmjd6 for self-tolerance induction in the thymus

    Journal: Nature Communications

    doi: 10.1038/ncomms9820

    The effect of immature Aire protein on localization and stability of mature Aire protein. ( a ). Subcellular localization of GFP-tagged mature Aire protein and mCherry-tagged immature Aire protein generated by intron 2 retention. Data are representative of two independent experiments. ( b ) Schematic representation of pCI-GFP-mature Aire and pTRE2hyg-HA-immature Aire. ( c , d ) After treatment with doxycycline (Dox) for the indicated times in the presence or absence of MG132, expressions of mature and immature Aire proteins were analysed for HEK293 cells stably expressing both pCI-GFP-mature Aire and pTRE2hyg-HA-immature Aire or pTRE2hyg. Data (mean±s.d., n =3) are expressed as the level of mature Aire protein after normalization of the 0 h value to an arbitrary value of 1. ** P
    Figure Legend Snippet: The effect of immature Aire protein on localization and stability of mature Aire protein. ( a ). Subcellular localization of GFP-tagged mature Aire protein and mCherry-tagged immature Aire protein generated by intron 2 retention. Data are representative of two independent experiments. ( b ) Schematic representation of pCI-GFP-mature Aire and pTRE2hyg-HA-immature Aire. ( c , d ) After treatment with doxycycline (Dox) for the indicated times in the presence or absence of MG132, expressions of mature and immature Aire proteins were analysed for HEK293 cells stably expressing both pCI-GFP-mature Aire and pTRE2hyg-HA-immature Aire or pTRE2hyg. Data (mean±s.d., n =3) are expressed as the level of mature Aire protein after normalization of the 0 h value to an arbitrary value of 1. ** P

    Techniques Used: Generated, Stable Transfection, Expressing

    36) Product Images from "Ubiquitin ligase RIPLET mediates polyubiquitination of RIG-I and LGP2 and regulates the innate immune responses to SARS-CoV-2 infection"

    Article Title: Ubiquitin ligase RIPLET mediates polyubiquitination of RIG-I and LGP2 and regulates the innate immune responses to SARS-CoV-2 infection

    Journal: bioRxiv

    doi: 10.1101/2021.01.25.428042

    Riplet plays a crucial role in antiviral response against SARS-CoV-2 (a) Total RNAs were extracted from VeroE6/TMPRSS2 cells infected with or without SARS-CoV-2 for 24 hr. 1 µg of extracted RNA was transfected into HEK293 cells, and the cytokine expression at indicated time points were determined by RT-qPCR (n = 3). (b) 1 µg of Total RNA from VeroE6/TMPR22S infected with SARS-CoV-2 were transfected into WT, RIG-I KO, and MDA5 KO cells. The cytokine expression at indicated time points were determined by RT-qPCR (n = 3, *p
    Figure Legend Snippet: Riplet plays a crucial role in antiviral response against SARS-CoV-2 (a) Total RNAs were extracted from VeroE6/TMPRSS2 cells infected with or without SARS-CoV-2 for 24 hr. 1 µg of extracted RNA was transfected into HEK293 cells, and the cytokine expression at indicated time points were determined by RT-qPCR (n = 3). (b) 1 µg of Total RNA from VeroE6/TMPR22S infected with SARS-CoV-2 were transfected into WT, RIG-I KO, and MDA5 KO cells. The cytokine expression at indicated time points were determined by RT-qPCR (n = 3, *p

    Techniques Used: Infection, Transfection, Expressing, Quantitative RT-PCR

    Ubiquitination sites of LGP2 (a) Liquid chromatography-tandem mass spectrometry (LC/LC-MS/MS) of LGP2 in Riplet-expressing cells. Schematic representation of ubiquitination sites of RIG-I and LGP2 were shown in upper panel. (b) FLAG-tagged LGP2 fragments and HA-tagged Riplet expression vectors were transfected into HEK293FT cells. WCEs were prepared 24 h after transfection, and immunoprecipitation was performed with anti-FLAG Ab. The proteins were subjected to SDS-PAGE and detected by western blotting, as indicated Abs. (c) HA-tagged ubiquitin, FLAG-tagged LGP2 CTD, and FLAG-tagged LGP2 CTD 3KR expression vectors were transfected into HEK293FT cells. WCE were prepared 24 h after transfection, and immunoprecipitation was performed with anti-FLAG Ab. The proteins were subjected to SDS-PAGE and detected by western blotting, as indicated Abs. (e) HA-tagged ubiquitin and FLAG-tagged wild-type and 4KR LPG2 expression vectors were transfected into HEK293FT cells. WCEs were prepared, and a pull-down assay with mock or biotin-conjugated poly I:C was performed. The proteins were subjected to SDS-PAGE and detected by western blotting, as indicated Abs. (f) RIG-I and/or wild-type and KR mutants of LGP2 were transfected into HEK293 cells together with p125luc plasmid and Renilla luciferase vector. Luciferase activities were determined 24 h after transfection. The data represent the mean ± SD (n = 3). (g) HE293 cells stably expressing GFP (control), LGP2, LGP2-4KR were generated using lentivirus vectors. The cells were transfected with p125luc plasmid and Renilla luciferase vectors and then stimulated with short poly I:C. WCEs were prepared 24 h after transfection and stimulation, and luciferase activities were determined. The proteins were subjected to SDS-PAGE and detected by western blotting with the indicated Abs. The data represent the mean ± SD (n = 3, *p
    Figure Legend Snippet: Ubiquitination sites of LGP2 (a) Liquid chromatography-tandem mass spectrometry (LC/LC-MS/MS) of LGP2 in Riplet-expressing cells. Schematic representation of ubiquitination sites of RIG-I and LGP2 were shown in upper panel. (b) FLAG-tagged LGP2 fragments and HA-tagged Riplet expression vectors were transfected into HEK293FT cells. WCEs were prepared 24 h after transfection, and immunoprecipitation was performed with anti-FLAG Ab. The proteins were subjected to SDS-PAGE and detected by western blotting, as indicated Abs. (c) HA-tagged ubiquitin, FLAG-tagged LGP2 CTD, and FLAG-tagged LGP2 CTD 3KR expression vectors were transfected into HEK293FT cells. WCE were prepared 24 h after transfection, and immunoprecipitation was performed with anti-FLAG Ab. The proteins were subjected to SDS-PAGE and detected by western blotting, as indicated Abs. (e) HA-tagged ubiquitin and FLAG-tagged wild-type and 4KR LPG2 expression vectors were transfected into HEK293FT cells. WCEs were prepared, and a pull-down assay with mock or biotin-conjugated poly I:C was performed. The proteins were subjected to SDS-PAGE and detected by western blotting, as indicated Abs. (f) RIG-I and/or wild-type and KR mutants of LGP2 were transfected into HEK293 cells together with p125luc plasmid and Renilla luciferase vector. Luciferase activities were determined 24 h after transfection. The data represent the mean ± SD (n = 3). (g) HE293 cells stably expressing GFP (control), LGP2, LGP2-4KR were generated using lentivirus vectors. The cells were transfected with p125luc plasmid and Renilla luciferase vectors and then stimulated with short poly I:C. WCEs were prepared 24 h after transfection and stimulation, and luciferase activities were determined. The proteins were subjected to SDS-PAGE and detected by western blotting with the indicated Abs. The data represent the mean ± SD (n = 3, *p

    Techniques Used: Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Expressing, Transfection, Immunoprecipitation, SDS Page, Western Blot, Pull Down Assay, Plasmid Preparation, Luciferase, Stable Transfection, Generated

    Accessory proteins modulate the Riplet- and TRIM25-medaited RIG-I activation (a–c) Wild-type, TRIM25 KO, and/or Riplet KO HEK293 cells were transfected with RIG-I, NLRP12, and/or NDR2 expression vectors together with p125luc plasmid and Renilla luciferase vector. 24 h after transfection, luciferase activities were determined. The data represent the mean ± SD (n = 3, *p
    Figure Legend Snippet: Accessory proteins modulate the Riplet- and TRIM25-medaited RIG-I activation (a–c) Wild-type, TRIM25 KO, and/or Riplet KO HEK293 cells were transfected with RIG-I, NLRP12, and/or NDR2 expression vectors together with p125luc plasmid and Renilla luciferase vector. 24 h after transfection, luciferase activities were determined. The data represent the mean ± SD (n = 3, *p

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

    K63-linked polyubiquitination of LGP2 (a–d) Wild-type and LGP2-KO HEK293 cells were transfected with 200 ng/ml of short poly I:C or infected with Sendai virus (SeV). Total RNAs were extracted as the indicated time points, and IFN-β and IP-10 mRNA expression was determined by RT-qPCR. The data represent the mean ± SD (n = 3, *p
    Figure Legend Snippet: K63-linked polyubiquitination of LGP2 (a–d) Wild-type and LGP2-KO HEK293 cells were transfected with 200 ng/ml of short poly I:C or infected with Sendai virus (SeV). Total RNAs were extracted as the indicated time points, and IFN-β and IP-10 mRNA expression was determined by RT-qPCR. The data represent the mean ± SD (n = 3, *p

    Techniques Used: Transfection, Infection, Expressing, Quantitative RT-PCR

    Riplet KO exhibits severe defect in RIG-I-dependent cytokine expression (a–e) p125luc (IFN-β promoter reporter) plasmid and Renilla luciferase vector (internal control) together with indicated expression vectors were transfected into WT and mutant HEK293 cells. The amounts of total DNA in each sample were normalized using an empty vector. After 24 h of transfection, the cells were lysed, and luciferase activities were determined. Western blotting was performed with the indicated antibodies (Abs). The reporter gene activities were measured in three independently isolated TRIM25-KO HEK293 cells (b). The data represent the mean ± SD (n = 3, *p
    Figure Legend Snippet: Riplet KO exhibits severe defect in RIG-I-dependent cytokine expression (a–e) p125luc (IFN-β promoter reporter) plasmid and Renilla luciferase vector (internal control) together with indicated expression vectors were transfected into WT and mutant HEK293 cells. The amounts of total DNA in each sample were normalized using an empty vector. After 24 h of transfection, the cells were lysed, and luciferase activities were determined. Western blotting was performed with the indicated antibodies (Abs). The reporter gene activities were measured in three independently isolated TRIM25-KO HEK293 cells (b). The data represent the mean ± SD (n = 3, *p

    Techniques Used: Expressing, Plasmid Preparation, Luciferase, Transfection, Mutagenesis, Western Blot, Isolation

    Ubiquitination of LGP2 attenuates the cytokine expression at a late phase (a–f) HEK293 cells stably expressing GFP, LGP2, and LGP2-4KR were transfected with 200 ng/ml of short poly I:C or infected with Sendai virus (SeV) at MOI = 5. Total RNAs were extracted at the indicated time points. The expression of IFN-β mRNA was determined by RT-qPCR. The data represent the mean ± SD (n = 3, *p
    Figure Legend Snippet: Ubiquitination of LGP2 attenuates the cytokine expression at a late phase (a–f) HEK293 cells stably expressing GFP, LGP2, and LGP2-4KR were transfected with 200 ng/ml of short poly I:C or infected with Sendai virus (SeV) at MOI = 5. Total RNAs were extracted at the indicated time points. The expression of IFN-β mRNA was determined by RT-qPCR. The data represent the mean ± SD (n = 3, *p

    Techniques Used: Expressing, Stable Transfection, Transfection, Infection, Quantitative RT-PCR

    37) Product Images from "A Novel Degradation Signal Derived from Distal C-terminal Frameshift Mutations of KCNQ2 Protein Which Cause Neonatal Epilepsy *"

    Article Title: A Novel Degradation Signal Derived from Distal C-terminal Frameshift Mutations of KCNQ2 Protein Which Cause Neonatal Epilepsy *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.287268

    The degradation function of ExtraC is transferable to CD4 protein. A , HEK293 cells transfected with HA-tagged plasmids of CD4 ( left panels , as a blank control), CD4-GFP60aa ( middle left panels , as a negative control), or CD4-ExtraC ( middle right panels
    Figure Legend Snippet: The degradation function of ExtraC is transferable to CD4 protein. A , HEK293 cells transfected with HA-tagged plasmids of CD4 ( left panels , as a blank control), CD4-GFP60aa ( middle left panels , as a negative control), or CD4-ExtraC ( middle right panels

    Techniques Used: Transfection, Negative Control

    Current reduction in KCNQ channel mutations containing the ExtraC peptide. A , current traces were recorded by whole-cell patch clamp in HEK293 cells expressing KCNQ2 ( left panel ) or fsKCNQ2 ( middle panel ). Cells were held at −80 mV in response
    Figure Legend Snippet: Current reduction in KCNQ channel mutations containing the ExtraC peptide. A , current traces were recorded by whole-cell patch clamp in HEK293 cells expressing KCNQ2 ( left panel ) or fsKCNQ2 ( middle panel ). Cells were held at −80 mV in response

    Techniques Used: Patch Clamp, Expressing

    Functional identification of the RCLRG motif critical for current reduction and accelerated degradation. A , left panels , shown are current densities of HEK293 cells expressing KCNQ2, fsKCNQ2, or fsKCNQ2 truncation mutants: fsQ2–845X, -853X, -860X,
    Figure Legend Snippet: Functional identification of the RCLRG motif critical for current reduction and accelerated degradation. A , left panels , shown are current densities of HEK293 cells expressing KCNQ2, fsKCNQ2, or fsKCNQ2 truncation mutants: fsQ2–845X, -853X, -860X,

    Techniques Used: Functional Assay, Expressing

    Reduced surface expression of fsKCNQ2 is a result of total protein reduction and is not due to ER retention. A , left panels , confocal imaging is shown of HEK293 cells co-transfected with plasmids of either EGFP-KCNQ2 or EGFP-fsKCNQ2 ( green ) and pDsRed2-ER
    Figure Legend Snippet: Reduced surface expression of fsKCNQ2 is a result of total protein reduction and is not due to ER retention. A , left panels , confocal imaging is shown of HEK293 cells co-transfected with plasmids of either EGFP-KCNQ2 or EGFP-fsKCNQ2 ( green ) and pDsRed2-ER

    Techniques Used: Expressing, Imaging, Transfection

    38) Product Images from "Expression of Adenosine Receptors in Rodent Pancreas"

    Article Title: Expression of Adenosine Receptors in Rodent Pancreas

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20215329

    Immunoblot of A 2A ( A ) and A 2B ( B ) adenosine receptors from the rat pancreatic duct. Protein samples were resolved by SDS-PAGE. Arrowheads indicate adenosine receptor proteins detected by immunoblotting using anti-ADORA2A ( A , 1:200, sc-13937) or anti-ADORA2B ( B , 1:1000, AAR-003) antibody. Representative membranes from two independent experiments are shown. M, marker; D, duct; C, Capan-1; H, HEK293.
    Figure Legend Snippet: Immunoblot of A 2A ( A ) and A 2B ( B ) adenosine receptors from the rat pancreatic duct. Protein samples were resolved by SDS-PAGE. Arrowheads indicate adenosine receptor proteins detected by immunoblotting using anti-ADORA2A ( A , 1:200, sc-13937) or anti-ADORA2B ( B , 1:1000, AAR-003) antibody. Representative membranes from two independent experiments are shown. M, marker; D, duct; C, Capan-1; H, HEK293.

    Techniques Used: SDS Page, Marker

    39) Product Images from "Human DDX56 protein interacts with influenza A virus NS1 protein and stimulates the virus replication"

    Article Title: Human DDX56 protein interacts with influenza A virus NS1 protein and stimulates the virus replication

    Journal: Genetics and Molecular Biology

    doi: 10.1590/1678-4685-GMB-2020-0158

    The quantitation of human DDX56 transcript and viral M1 protein in the cells transfected with siRNA or H-DDX56 encoding plasmid DNA (pCHA-DDX56) and infected with influenza A viruses. A . Quantity of human DDX56 transcript in the non-transfected HeLa cells (N.T. cells), the HeLa cells transfected with DDX56 specific siRNA (siRNA/DDX56), and negative control siRNA (cont.siRNA). B . Viral M1 protein quantity in HeLa cells transfected with siRNAs and then infected with viruses. C. The quantity (plaque forming unit; pfu/ml) of influenza A virus in medium of knock down/infected cell culture . D . The M1 protein quantity in non-transfected HEK293 cells (N.T. cells) and in HEK293 cells transfected with pCHA-DDX56 or pCHA (Vector) plasmid and then infected with human influenza A (WSN) viruses. After 8 h p.i. the cells harvested and the proteins in the cell lysates were separated on 10% PAGE. As primary antibodies, mouse monoclonal anti-HA, rabbit polyclonal anti-M1 serum, and mouse monoclonal anti-actin beta antibodies were used.
    Figure Legend Snippet: The quantitation of human DDX56 transcript and viral M1 protein in the cells transfected with siRNA or H-DDX56 encoding plasmid DNA (pCHA-DDX56) and infected with influenza A viruses. A . Quantity of human DDX56 transcript in the non-transfected HeLa cells (N.T. cells), the HeLa cells transfected with DDX56 specific siRNA (siRNA/DDX56), and negative control siRNA (cont.siRNA). B . Viral M1 protein quantity in HeLa cells transfected with siRNAs and then infected with viruses. C. The quantity (plaque forming unit; pfu/ml) of influenza A virus in medium of knock down/infected cell culture . D . The M1 protein quantity in non-transfected HEK293 cells (N.T. cells) and in HEK293 cells transfected with pCHA-DDX56 or pCHA (Vector) plasmid and then infected with human influenza A (WSN) viruses. After 8 h p.i. the cells harvested and the proteins in the cell lysates were separated on 10% PAGE. As primary antibodies, mouse monoclonal anti-HA, rabbit polyclonal anti-M1 serum, and mouse monoclonal anti-actin beta antibodies were used.

    Techniques Used: Quantitation Assay, Transfection, Plasmid Preparation, Infection, Negative Control, Cell Culture, Polyacrylamide Gel Electrophoresis

    40) Product Images from "The ?-tail domain (?TD) regulates physiologic ligand binding to integrin CD11b/CD18"

    Article Title: The ?-tail domain (?TD) regulates physiologic ligand binding to integrin CD11b/CD18

    Journal:

    doi: 10.1182/blood-2005-11-056689

    Surface expression and iC3b binding of WT and mutant CD11b/CD18 in HEK293 cells . (A) Histograms showing the relative binding of the heterodimer-specific mAb IB4 and the anti-CD11b mAb 44a to HEK293 cells expressing WT and βTD mutant CD11b/CD18
    Figure Legend Snippet: Surface expression and iC3b binding of WT and mutant CD11b/CD18 in HEK293 cells . (A) Histograms showing the relative binding of the heterodimer-specific mAb IB4 and the anti-CD11b mAb 44a to HEK293 cells expressing WT and βTD mutant CD11b/CD18

    Techniques Used: Expressing, Binding Assay, Mutagenesis

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    ADAM10 expression is repressed by the G-quadruplex motif. A , <t>HEK293</t> cells were transiently transfected with the indicated ADAM10 cDNA constructs, and lysates were analyzed by immunoblotting for V5-tagged ADAM10, endogenous APP, β-actin as loading control, and GFP as transfection control. Supernatants were analyzed for APPsα secretion using antibody 2D8. Cellular APP is present in low molecular weight immature forms ( im ) and high molecular weight mature form ( m ). ADAM10 is present as a mature ( m ) form and predominantly as an immature ( im ) form. B , quantification of ADAM10 protein ( black bars ) and mRNA levels ( white bars ) from cells transfected with ADAM10 cDNA constructs shown in A . ADAM10 protein levels were normalized to GFP and actin levels. The signal for ADAM10 with the wild-type G-quadruplex GQ-WT ADAM10 was set to 1. Results are expressed as the means ± S.D. from three experiments made in triplicate. ADAM10 mRNA was normalized to glycerolaldehyde-3-phosphate-dehydrogenase mRNA levels, and the signal for GQ-WT ADAM10 was set to 1. Results are expressed as the means ± S.D. from three experiments. C , quantification of secreted APPsα from cells transfected with the indicated ADAM10 variants were shown in A . The signal for APPsα from GQ-WT ADAM10 transfected cells was set to 100%. Results are expressed as the means ± S.D. from three experiments. Asterisks indicate statistical significance (one-way analysis of variance with Dunnett's post test) relative to GQ-WT ADAM10 transfected cells (*, p
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    A population of NSP4-EGFP fusion protein localizes in the ER or ERGIC compartment in <t>HEK</t> 293 cells. Cells were induced for 24 h, fixed, and stained with rabbit anti-calnexin and mouse monoclonal anti-ERGIC-53 antibodies, followed with Alexa 568-conjugated
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    ADAM10 expression is repressed by the G-quadruplex motif. A , HEK293 cells were transiently transfected with the indicated ADAM10 cDNA constructs, and lysates were analyzed by immunoblotting for V5-tagged ADAM10, endogenous APP, β-actin as loading control, and GFP as transfection control. Supernatants were analyzed for APPsα secretion using antibody 2D8. Cellular APP is present in low molecular weight immature forms ( im ) and high molecular weight mature form ( m ). ADAM10 is present as a mature ( m ) form and predominantly as an immature ( im ) form. B , quantification of ADAM10 protein ( black bars ) and mRNA levels ( white bars ) from cells transfected with ADAM10 cDNA constructs shown in A . ADAM10 protein levels were normalized to GFP and actin levels. The signal for ADAM10 with the wild-type G-quadruplex GQ-WT ADAM10 was set to 1. Results are expressed as the means ± S.D. from three experiments made in triplicate. ADAM10 mRNA was normalized to glycerolaldehyde-3-phosphate-dehydrogenase mRNA levels, and the signal for GQ-WT ADAM10 was set to 1. Results are expressed as the means ± S.D. from three experiments. C , quantification of secreted APPsα from cells transfected with the indicated ADAM10 variants were shown in A . The signal for APPsα from GQ-WT ADAM10 transfected cells was set to 100%. Results are expressed as the means ± S.D. from three experiments. Asterisks indicate statistical significance (one-way analysis of variance with Dunnett's post test) relative to GQ-WT ADAM10 transfected cells (*, p

    Journal: The Journal of Biological Chemistry

    Article Title: Translational Repression of the Disintegrin and Metalloprotease ADAM10 by a Stable G-quadruplex Secondary Structure in Its 5?-Untranslated Region *

    doi: 10.1074/jbc.M111.296921

    Figure Lengend Snippet: ADAM10 expression is repressed by the G-quadruplex motif. A , HEK293 cells were transiently transfected with the indicated ADAM10 cDNA constructs, and lysates were analyzed by immunoblotting for V5-tagged ADAM10, endogenous APP, β-actin as loading control, and GFP as transfection control. Supernatants were analyzed for APPsα secretion using antibody 2D8. Cellular APP is present in low molecular weight immature forms ( im ) and high molecular weight mature form ( m ). ADAM10 is present as a mature ( m ) form and predominantly as an immature ( im ) form. B , quantification of ADAM10 protein ( black bars ) and mRNA levels ( white bars ) from cells transfected with ADAM10 cDNA constructs shown in A . ADAM10 protein levels were normalized to GFP and actin levels. The signal for ADAM10 with the wild-type G-quadruplex GQ-WT ADAM10 was set to 1. Results are expressed as the means ± S.D. from three experiments made in triplicate. ADAM10 mRNA was normalized to glycerolaldehyde-3-phosphate-dehydrogenase mRNA levels, and the signal for GQ-WT ADAM10 was set to 1. Results are expressed as the means ± S.D. from three experiments. C , quantification of secreted APPsα from cells transfected with the indicated ADAM10 variants were shown in A . The signal for APPsα from GQ-WT ADAM10 transfected cells was set to 100%. Results are expressed as the means ± S.D. from three experiments. Asterisks indicate statistical significance (one-way analysis of variance with Dunnett's post test) relative to GQ-WT ADAM10 transfected cells (*, p

    Article Snippet: 1.8 × 106 HEK293 cells were plated in 6-cm dishes and transiently transfected with 8 μg of cDNA encoding ADAM10 variants and 0.1 μg of pEGFP-N1 (Clontech).

    Techniques: Expressing, Transfection, Construct, Molecular Weight

    Translational repression of a luciferase reporter by the ADAM10 G-quadruplex motif. A, Schematic representation of the plasmids used for reporter gene assays. The wild-type G-quadruplex sequence (GQ-WT) of the ADAM10 5′-UTR or mutated variants thereof were cloned directly in front of the Renilla coding region. B, 24 h after transfection of the indicated plasmids in HEK293 cells dual-luciferase assays were performed and mRNA was isolated. Renilla luciferase activity was normalized to Firefly luciferase activity and the value for GQ-WT was set to 100%. C T values for Renilla and Firefly luciferase mRNA were determined by quantitative RT-PCR and the ratio of C T Renilla/C T ). Results are expressed as means ± S.D. of at least three independent experiments made in triplicates.

    Journal: The Journal of Biological Chemistry

    Article Title: Translational Repression of the Disintegrin and Metalloprotease ADAM10 by a Stable G-quadruplex Secondary Structure in Its 5?-Untranslated Region *

    doi: 10.1074/jbc.M111.296921

    Figure Lengend Snippet: Translational repression of a luciferase reporter by the ADAM10 G-quadruplex motif. A, Schematic representation of the plasmids used for reporter gene assays. The wild-type G-quadruplex sequence (GQ-WT) of the ADAM10 5′-UTR or mutated variants thereof were cloned directly in front of the Renilla coding region. B, 24 h after transfection of the indicated plasmids in HEK293 cells dual-luciferase assays were performed and mRNA was isolated. Renilla luciferase activity was normalized to Firefly luciferase activity and the value for GQ-WT was set to 100%. C T values for Renilla and Firefly luciferase mRNA were determined by quantitative RT-PCR and the ratio of C T Renilla/C T ). Results are expressed as means ± S.D. of at least three independent experiments made in triplicates.

    Article Snippet: 1.8 × 106 HEK293 cells were plated in 6-cm dishes and transiently transfected with 8 μg of cDNA encoding ADAM10 variants and 0.1 μg of pEGFP-N1 (Clontech).

    Techniques: Luciferase, Sequencing, Clone Assay, Transfection, Isolation, Activity Assay, Quantitative RT-PCR

    HEK293 cells expressing h AQP9 or h DAT reveal higher sensitivity to MPP + . A-B) Immunofluorescence images of HEK293 cells expressing EGFP- h AQP9 (A) or YFP- h DAT (B) grown on coverslips confirm plasma membrane localization of the respective constructs (identified by antibodies to AQP9 or DAT). The cells were counterstained with Hoechst to visualize nuclei. C-G) Native HEK293 cells and HEK293 cells expressing EGFP- h AQP9 or YFP- h DAT were grown in 96-well plates and exposed to different concentrations of MPP + (four wells for each concentration). Cell viability was assessed after 24 hours using the MTT assay. Data were collected from independent plates (n = 3 for each construct) and normalized to respective untreated cells. Native HEK293 cells show sensitivity to MPP + only at very high concentrations (~100 μM). Cells expressing h DAT become sensitive at 1 μM MPP + , compared with 0.1 μM for cells expressing h AQP9. Overlay of the dose/response curve for the three groups (D) and the individual curves for native HEK293 cells (E), YFP- h DAT expressing (F) and EGFP- h AQP9 expressing HEK293 cells (G) are shown. Bars are mean ± SEM. Asterisks: significantly different from untreated controls; *p

    Journal: PLoS ONE

    Article Title: Targeted deletion of the aquaglyceroporin AQP9 is protective in a mouse model of Parkinson’s disease

    doi: 10.1371/journal.pone.0194896

    Figure Lengend Snippet: HEK293 cells expressing h AQP9 or h DAT reveal higher sensitivity to MPP + . A-B) Immunofluorescence images of HEK293 cells expressing EGFP- h AQP9 (A) or YFP- h DAT (B) grown on coverslips confirm plasma membrane localization of the respective constructs (identified by antibodies to AQP9 or DAT). The cells were counterstained with Hoechst to visualize nuclei. C-G) Native HEK293 cells and HEK293 cells expressing EGFP- h AQP9 or YFP- h DAT were grown in 96-well plates and exposed to different concentrations of MPP + (four wells for each concentration). Cell viability was assessed after 24 hours using the MTT assay. Data were collected from independent plates (n = 3 for each construct) and normalized to respective untreated cells. Native HEK293 cells show sensitivity to MPP + only at very high concentrations (~100 μM). Cells expressing h DAT become sensitive at 1 μM MPP + , compared with 0.1 μM for cells expressing h AQP9. Overlay of the dose/response curve for the three groups (D) and the individual curves for native HEK293 cells (E), YFP- h DAT expressing (F) and EGFP- h AQP9 expressing HEK293 cells (G) are shown. Bars are mean ± SEM. Asterisks: significantly different from untreated controls; *p

    Article Snippet: HEK293 cells stably transfected with EGFP-h AQP9 were seeded in media containing 1μg/mL doxycycline (Clontech Labs, Mountain View, CA, USA) for induction of AQP9 expression at a density of 50 000 cells per well due to impaired growth.

    Techniques: Expressing, Immunofluorescence, Construct, Concentration Assay, MTT Assay

    HEK293 cells expressing EGFP- h AQP9 are more sensitive to arsenite than HEK293 cells expressing YFP- h DAT. A-C) Native HEK293 cells and HEK293 cells expressing EGFP- h AQP9 or YFP- h DAT were grown in 96-well plates and exposed to different concentrations of arsenite (eight wells for each concentration). Cell viability was assessed after 24 hours using the MTT assay. Data were collected from independent plates (n = 3 for each construct) and normalized to respective untreated cells. Both EGFP- h AQP9 and YFP- h DAT expressing cells showed higher sensitivity to arsenite, than native HEK293 cells, with EGFP- h AQP9 cells being the most sensitive. At the arsenite concentration of 10 μM, stably transfected EGFP- h AQP9 were the only cells showing toxin sensitivity (A). The curve showing IC50 values for arsenite calculated by nonlinear regression, log(inhibitor) vs response (three parameters) is shown (B). For log transformed data, the concentration 0 was set to 1 nM. Comparison of the IC50 values shows a significantly lower IC50 value for the HEK293 cells expressing EGFP- h AQP9 compared to the native HEK293 cells or HEK293 cells expressing YFP- h DAT (C). Bars are mean ± SEM. Asterisks: significantly different from untreated controls; *p

    Journal: PLoS ONE

    Article Title: Targeted deletion of the aquaglyceroporin AQP9 is protective in a mouse model of Parkinson’s disease

    doi: 10.1371/journal.pone.0194896

    Figure Lengend Snippet: HEK293 cells expressing EGFP- h AQP9 are more sensitive to arsenite than HEK293 cells expressing YFP- h DAT. A-C) Native HEK293 cells and HEK293 cells expressing EGFP- h AQP9 or YFP- h DAT were grown in 96-well plates and exposed to different concentrations of arsenite (eight wells for each concentration). Cell viability was assessed after 24 hours using the MTT assay. Data were collected from independent plates (n = 3 for each construct) and normalized to respective untreated cells. Both EGFP- h AQP9 and YFP- h DAT expressing cells showed higher sensitivity to arsenite, than native HEK293 cells, with EGFP- h AQP9 cells being the most sensitive. At the arsenite concentration of 10 μM, stably transfected EGFP- h AQP9 were the only cells showing toxin sensitivity (A). The curve showing IC50 values for arsenite calculated by nonlinear regression, log(inhibitor) vs response (three parameters) is shown (B). For log transformed data, the concentration 0 was set to 1 nM. Comparison of the IC50 values shows a significantly lower IC50 value for the HEK293 cells expressing EGFP- h AQP9 compared to the native HEK293 cells or HEK293 cells expressing YFP- h DAT (C). Bars are mean ± SEM. Asterisks: significantly different from untreated controls; *p

    Article Snippet: HEK293 cells stably transfected with EGFP-h AQP9 were seeded in media containing 1μg/mL doxycycline (Clontech Labs, Mountain View, CA, USA) for induction of AQP9 expression at a density of 50 000 cells per well due to impaired growth.

    Techniques: Expressing, Concentration Assay, MTT Assay, Construct, Stable Transfection, Transfection, Transformation Assay

    The CRISPR–Cas LMNA HDR system accumulates R-loops in a DDX5-dependent manner. ( A ) Illustration of the Cas9-directed knock-in of the Clover in the LMNA coding sequence. The red and the blue arrows represent both arms used for HDR. Cells were transfected with plasmids for CRISPR–Cas LMNA HDR analysis. B, E and H denote the location of the Bsr GI, Eco RI and Hind III restriction sites. qPCR amplification region is shown at the top of the red homology arm of the LMNA used for the DRIP-qPCR. ( B ) HEK293 cells were transfected with pcDNA or RNAse H1 (RNH1) expressing vector along with the siRNAs. Cells were then transfected with the CRISPR–Cas LMNA HDR system and iRFP plasmids and subjected to DRIP-qPCR analysis at both LMNA homology arms and the EGR1 control locus. ( C ) HEK293 cells were transfected with Flag-DDX5 and subjected to ChIP-qPCR analysis. The bar graphs are the average and SEM from three independent experiments. Statistical significance was assessed using t -test: * P

    Journal: Nar Cancer

    Article Title: DDX5 resolves R-loops at DNA double-strand breaks to promote DNA repair and avoid chromosomal deletions

    doi: 10.1093/narcan/zcaa028

    Figure Lengend Snippet: The CRISPR–Cas LMNA HDR system accumulates R-loops in a DDX5-dependent manner. ( A ) Illustration of the Cas9-directed knock-in of the Clover in the LMNA coding sequence. The red and the blue arrows represent both arms used for HDR. Cells were transfected with plasmids for CRISPR–Cas LMNA HDR analysis. B, E and H denote the location of the Bsr GI, Eco RI and Hind III restriction sites. qPCR amplification region is shown at the top of the red homology arm of the LMNA used for the DRIP-qPCR. ( B ) HEK293 cells were transfected with pcDNA or RNAse H1 (RNH1) expressing vector along with the siRNAs. Cells were then transfected with the CRISPR–Cas LMNA HDR system and iRFP plasmids and subjected to DRIP-qPCR analysis at both LMNA homology arms and the EGR1 control locus. ( C ) HEK293 cells were transfected with Flag-DDX5 and subjected to ChIP-qPCR analysis. The bar graphs are the average and SEM from three independent experiments. Statistical significance was assessed using t -test: * P

    Article Snippet: For the inducible NHEJ reporter system, the tetracycline-on puro-GFP reporter plasmid (pRetroX-Tight-Pur-GFP) was transfected in HEK293 cells in which pRetroX-Tet-On advanced (Clontech) vector had been integrated and stable cell lines were generated by selection with puromycin in the presence of doxycycline (Dox).

    Techniques: CRISPR, Knock-In, Sequencing, Transfection, Real-time Polymerase Chain Reaction, Amplification, Expressing, Plasmid Preparation, Chromatin Immunoprecipitation

    Increased DNA end deletions in DDX5-deficient cells are associated with local gene transcription. ( A ) Illustration of a tetracycline-induced (tetO) reporter system for NHEJ repair analysis in HEK293. The reporter construct is similar to the one described in Figure 4A except that the reporter expression is controlled by a tet-on promoter. ( B ) RT-qPCR analysis of the expression of puromycin in the absence (−Dox) or presence (+Dox) of 1 μg/ml Dox. ( C ) The HEK293-tetO-puro-GFP reporter cells were transfected with indicated siLuc control siRNA (siCTL) and siDDX5 #3 (siDDX5), respectively, in the absence (−Dox) or presence (+Dox) of 1 μg/ml Dox. Forty to forty-four hours after the siRNA transfection, the cells were then transfected with I-SceI-expressing vector (pCAG-I-SceI). Seventy hours after the plasmid transfection, the cells were harvested and the genomic DNA was extracted for PCR analysis using the primers shown in ( A ). The PCR primers amplify a DNA fragment with a size of 733 bp if the two ends are accurately repaired. Under the PCR reaction condition, the DNA (3573–1581 equals 1992 bp) without cleavage cannot be amplified. A representative agarose gel was shown for the analysis of the PCR products. ( D ) The PCR products amplified with the primers as in ( A ) were subjected to qPCR analysis targeting different regions surrounding the I-SceI sites. The ratio of F1/F2 that was normalized to the one in the siCTL sample. ( E ) The graph shows the average and SEM from three independent experiments performed in triplicates. ( F , G ) The HEK293-tetO-puro-GFP reporter cells were co-transfected with Flag-DDX5 and I-SceI-expressing plasmids in the absence (−Dox) or presence (+Dox) of 1 μg/ml Dox. ChIP-qPCR was performed to determine DDX5 occupancy near the I-SceI-cleaved DNA breaks (P1 and P2). MDM2 promoter region was used as a positive control. The results were normalized to IgG control at each condition. The graph shows the average and SEM from four independent experiments. Statistical significance was assessed using Student’s t -test: * P

    Journal: Nar Cancer

    Article Title: DDX5 resolves R-loops at DNA double-strand breaks to promote DNA repair and avoid chromosomal deletions

    doi: 10.1093/narcan/zcaa028

    Figure Lengend Snippet: Increased DNA end deletions in DDX5-deficient cells are associated with local gene transcription. ( A ) Illustration of a tetracycline-induced (tetO) reporter system for NHEJ repair analysis in HEK293. The reporter construct is similar to the one described in Figure 4A except that the reporter expression is controlled by a tet-on promoter. ( B ) RT-qPCR analysis of the expression of puromycin in the absence (−Dox) or presence (+Dox) of 1 μg/ml Dox. ( C ) The HEK293-tetO-puro-GFP reporter cells were transfected with indicated siLuc control siRNA (siCTL) and siDDX5 #3 (siDDX5), respectively, in the absence (−Dox) or presence (+Dox) of 1 μg/ml Dox. Forty to forty-four hours after the siRNA transfection, the cells were then transfected with I-SceI-expressing vector (pCAG-I-SceI). Seventy hours after the plasmid transfection, the cells were harvested and the genomic DNA was extracted for PCR analysis using the primers shown in ( A ). The PCR primers amplify a DNA fragment with a size of 733 bp if the two ends are accurately repaired. Under the PCR reaction condition, the DNA (3573–1581 equals 1992 bp) without cleavage cannot be amplified. A representative agarose gel was shown for the analysis of the PCR products. ( D ) The PCR products amplified with the primers as in ( A ) were subjected to qPCR analysis targeting different regions surrounding the I-SceI sites. The ratio of F1/F2 that was normalized to the one in the siCTL sample. ( E ) The graph shows the average and SEM from three independent experiments performed in triplicates. ( F , G ) The HEK293-tetO-puro-GFP reporter cells were co-transfected with Flag-DDX5 and I-SceI-expressing plasmids in the absence (−Dox) or presence (+Dox) of 1 μg/ml Dox. ChIP-qPCR was performed to determine DDX5 occupancy near the I-SceI-cleaved DNA breaks (P1 and P2). MDM2 promoter region was used as a positive control. The results were normalized to IgG control at each condition. The graph shows the average and SEM from four independent experiments. Statistical significance was assessed using Student’s t -test: * P

    Article Snippet: For the inducible NHEJ reporter system, the tetracycline-on puro-GFP reporter plasmid (pRetroX-Tight-Pur-GFP) was transfected in HEK293 cells in which pRetroX-Tet-On advanced (Clontech) vector had been integrated and stable cell lines were generated by selection with puromycin in the presence of doxycycline (Dox).

    Techniques: Non-Homologous End Joining, Construct, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Positive Control

    A population of NSP4-EGFP fusion protein localizes in the ER or ERGIC compartment in HEK 293 cells. Cells were induced for 24 h, fixed, and stained with rabbit anti-calnexin and mouse monoclonal anti-ERGIC-53 antibodies, followed with Alexa 568-conjugated

    Journal: Journal of Virology

    Article Title: Rotavirus NSP4 Induces a Novel Vesicular Compartment Regulated by Calcium and Associated with Viroplasms

    doi: 10.1128/JVI.02167-05

    Figure Lengend Snippet: A population of NSP4-EGFP fusion protein localizes in the ER or ERGIC compartment in HEK 293 cells. Cells were induced for 24 h, fixed, and stained with rabbit anti-calnexin and mouse monoclonal anti-ERGIC-53 antibodies, followed with Alexa 568-conjugated

    Article Snippet: The pTRE NSP4-EGFP vector was transfected into HEK 293 “Tet-on” cells (Clontech Laboratories, Inc., Palo Alto, CA) together with the selection marker vector pTK-Hyg (Clontech Laboratories, Inc., Palo Alto, CA) in a 20:1 ratio using Lipofectamine Plus (Gibco BRL, Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer's recommendations.

    Techniques: Staining

    NSP4-EGFP vesicles do not align along microtubules in HEK 293 cells. Cells were induced for 24 h, and 1 h prior to fixation and permeabilization the cells were kept at 4°C to induce coalescence of microtubules into MTOC. Cells were stained with

    Journal: Journal of Virology

    Article Title: Rotavirus NSP4 Induces a Novel Vesicular Compartment Regulated by Calcium and Associated with Viroplasms

    doi: 10.1128/JVI.02167-05

    Figure Lengend Snippet: NSP4-EGFP vesicles do not align along microtubules in HEK 293 cells. Cells were induced for 24 h, and 1 h prior to fixation and permeabilization the cells were kept at 4°C to induce coalescence of microtubules into MTOC. Cells were stained with

    Article Snippet: The pTRE NSP4-EGFP vector was transfected into HEK 293 “Tet-on” cells (Clontech Laboratories, Inc., Palo Alto, CA) together with the selection marker vector pTK-Hyg (Clontech Laboratories, Inc., Palo Alto, CA) in a 20:1 ratio using Lipofectamine Plus (Gibco BRL, Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer's recommendations.

    Techniques: Staining

    All expressed NSP4-EGFP is recognized by rabbit anti-NSP4 120-147 antibody, and there is no intracellular NSP4 protein not fused with EGFP. HEK 293/NSP4-EGFP cells were induced for 24 h, fixed, and stained with rabbit anti-NSP4 120-147 antibody and antirabbit

    Journal: Journal of Virology

    Article Title: Rotavirus NSP4 Induces a Novel Vesicular Compartment Regulated by Calcium and Associated with Viroplasms

    doi: 10.1128/JVI.02167-05

    Figure Lengend Snippet: All expressed NSP4-EGFP is recognized by rabbit anti-NSP4 120-147 antibody, and there is no intracellular NSP4 protein not fused with EGFP. HEK 293/NSP4-EGFP cells were induced for 24 h, fixed, and stained with rabbit anti-NSP4 120-147 antibody and antirabbit

    Article Snippet: The pTRE NSP4-EGFP vector was transfected into HEK 293 “Tet-on” cells (Clontech Laboratories, Inc., Palo Alto, CA) together with the selection marker vector pTK-Hyg (Clontech Laboratories, Inc., Palo Alto, CA) in a 20:1 ratio using Lipofectamine Plus (Gibco BRL, Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer's recommendations.

    Techniques: Staining

    NSP4-EGFP is localized in the vicinity of, but not in, the plasma membrane of HEK 293 cells. Twenty-four hours postinduction, cells were fixed with 4% formaldehyde, incubated with Texas Red-conjugated wheat germ agglutinin without permeabilization to

    Journal: Journal of Virology

    Article Title: Rotavirus NSP4 Induces a Novel Vesicular Compartment Regulated by Calcium and Associated with Viroplasms

    doi: 10.1128/JVI.02167-05

    Figure Lengend Snippet: NSP4-EGFP is localized in the vicinity of, but not in, the plasma membrane of HEK 293 cells. Twenty-four hours postinduction, cells were fixed with 4% formaldehyde, incubated with Texas Red-conjugated wheat germ agglutinin without permeabilization to

    Article Snippet: The pTRE NSP4-EGFP vector was transfected into HEK 293 “Tet-on” cells (Clontech Laboratories, Inc., Palo Alto, CA) together with the selection marker vector pTK-Hyg (Clontech Laboratories, Inc., Palo Alto, CA) in a 20:1 ratio using Lipofectamine Plus (Gibco BRL, Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer's recommendations.

    Techniques: Incubation

    NSP4-EGFP colocalizes with the autophagosomal marker LC3 in HEK 293 cells. Cells were fixed and permeabilized 24 h postinduction and stained with rabbit anti-LC3 antibody and the Alexa 594-conjugated anti-rabbit secondary antibody. (A) NSP4-EGFP fluorescence;

    Journal: Journal of Virology

    Article Title: Rotavirus NSP4 Induces a Novel Vesicular Compartment Regulated by Calcium and Associated with Viroplasms

    doi: 10.1128/JVI.02167-05

    Figure Lengend Snippet: NSP4-EGFP colocalizes with the autophagosomal marker LC3 in HEK 293 cells. Cells were fixed and permeabilized 24 h postinduction and stained with rabbit anti-LC3 antibody and the Alexa 594-conjugated anti-rabbit secondary antibody. (A) NSP4-EGFP fluorescence;

    Article Snippet: The pTRE NSP4-EGFP vector was transfected into HEK 293 “Tet-on” cells (Clontech Laboratories, Inc., Palo Alto, CA) together with the selection marker vector pTK-Hyg (Clontech Laboratories, Inc., Palo Alto, CA) in a 20:1 ratio using Lipofectamine Plus (Gibco BRL, Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer's recommendations.

    Techniques: Marker, Staining, Fluorescence

    Expression of NSP4-EGFP in inducible HEK 293/NSP4-EGFP cells. (A) Schematic diagram of NSP4-EGFP fusion protein; (B) autoradiograph of radiolabeled NSP4-EGFP immunoprecipitated from doxycycline-induced cells using anti-NSP4 120-147 and anti-GFP antibody;

    Journal: Journal of Virology

    Article Title: Rotavirus NSP4 Induces a Novel Vesicular Compartment Regulated by Calcium and Associated with Viroplasms

    doi: 10.1128/JVI.02167-05

    Figure Lengend Snippet: Expression of NSP4-EGFP in inducible HEK 293/NSP4-EGFP cells. (A) Schematic diagram of NSP4-EGFP fusion protein; (B) autoradiograph of radiolabeled NSP4-EGFP immunoprecipitated from doxycycline-induced cells using anti-NSP4 120-147 and anti-GFP antibody;

    Article Snippet: The pTRE NSP4-EGFP vector was transfected into HEK 293 “Tet-on” cells (Clontech Laboratories, Inc., Palo Alto, CA) together with the selection marker vector pTK-Hyg (Clontech Laboratories, Inc., Palo Alto, CA) in a 20:1 ratio using Lipofectamine Plus (Gibco BRL, Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer's recommendations.

    Techniques: Expressing, Autoradiography, Immunoprecipitation

    NSP4-EGFP presence in vesicular structures in HEK 293 cells requires elevated levels of intracellular calcium. Cells were induced and grown for 24 h in calcium-free medium (see Materials and Methods). To normalize extracellular calcium, 2 mM calcium chloride

    Journal: Journal of Virology

    Article Title: Rotavirus NSP4 Induces a Novel Vesicular Compartment Regulated by Calcium and Associated with Viroplasms

    doi: 10.1128/JVI.02167-05

    Figure Lengend Snippet: NSP4-EGFP presence in vesicular structures in HEK 293 cells requires elevated levels of intracellular calcium. Cells were induced and grown for 24 h in calcium-free medium (see Materials and Methods). To normalize extracellular calcium, 2 mM calcium chloride

    Article Snippet: The pTRE NSP4-EGFP vector was transfected into HEK 293 “Tet-on” cells (Clontech Laboratories, Inc., Palo Alto, CA) together with the selection marker vector pTK-Hyg (Clontech Laboratories, Inc., Palo Alto, CA) in a 20:1 ratio using Lipofectamine Plus (Gibco BRL, Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer's recommendations.

    Techniques:

    Expressed NSP4-EGFP fusion protein is initially localized in the ER of HEK 293 cells. Cells were induced for 24 h, fixed, and stained with mouse monoclonal anti-PDI antibody (B, C) or rabbit anti-calnexin antibody (E, F) and corresponding Alexa 594-conjugated

    Journal: Journal of Virology

    Article Title: Rotavirus NSP4 Induces a Novel Vesicular Compartment Regulated by Calcium and Associated with Viroplasms

    doi: 10.1128/JVI.02167-05

    Figure Lengend Snippet: Expressed NSP4-EGFP fusion protein is initially localized in the ER of HEK 293 cells. Cells were induced for 24 h, fixed, and stained with mouse monoclonal anti-PDI antibody (B, C) or rabbit anti-calnexin antibody (E, F) and corresponding Alexa 594-conjugated

    Article Snippet: The pTRE NSP4-EGFP vector was transfected into HEK 293 “Tet-on” cells (Clontech Laboratories, Inc., Palo Alto, CA) together with the selection marker vector pTK-Hyg (Clontech Laboratories, Inc., Palo Alto, CA) in a 20:1 ratio using Lipofectamine Plus (Gibco BRL, Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer's recommendations.

    Techniques: Staining

    Only a portion of vesicular NSP4-EGFP is localized within the ER-Golgi intermediate compartment, and no NSP4-EGFP enters the Golgi apparatus in HEK 293 cells. Cells were fixed 24 h postinduction and stained with mouse monoclonal antibody to ERGIC-53 or

    Journal: Journal of Virology

    Article Title: Rotavirus NSP4 Induces a Novel Vesicular Compartment Regulated by Calcium and Associated with Viroplasms

    doi: 10.1128/JVI.02167-05

    Figure Lengend Snippet: Only a portion of vesicular NSP4-EGFP is localized within the ER-Golgi intermediate compartment, and no NSP4-EGFP enters the Golgi apparatus in HEK 293 cells. Cells were fixed 24 h postinduction and stained with mouse monoclonal antibody to ERGIC-53 or

    Article Snippet: The pTRE NSP4-EGFP vector was transfected into HEK 293 “Tet-on” cells (Clontech Laboratories, Inc., Palo Alto, CA) together with the selection marker vector pTK-Hyg (Clontech Laboratories, Inc., Palo Alto, CA) in a 20:1 ratio using Lipofectamine Plus (Gibco BRL, Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer's recommendations.

    Techniques: Staining

    NSP4-EGFP does not localize in endosomes or lysosomes of HEK 293 cells. Fixed and permeabilized cells, 24 h postinduction, were stained with mouse anti-Rab9 or rabbit anti-β-galactosidase antibody and the corresponding Alexa 594-conjugated secondary

    Journal: Journal of Virology

    Article Title: Rotavirus NSP4 Induces a Novel Vesicular Compartment Regulated by Calcium and Associated with Viroplasms

    doi: 10.1128/JVI.02167-05

    Figure Lengend Snippet: NSP4-EGFP does not localize in endosomes or lysosomes of HEK 293 cells. Fixed and permeabilized cells, 24 h postinduction, were stained with mouse anti-Rab9 or rabbit anti-β-galactosidase antibody and the corresponding Alexa 594-conjugated secondary

    Article Snippet: The pTRE NSP4-EGFP vector was transfected into HEK 293 “Tet-on” cells (Clontech Laboratories, Inc., Palo Alto, CA) together with the selection marker vector pTK-Hyg (Clontech Laboratories, Inc., Palo Alto, CA) in a 20:1 ratio using Lipofectamine Plus (Gibco BRL, Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer's recommendations.

    Techniques: Staining