hek 293  (TaKaRa)


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    Structured Review

    TaKaRa hek 293
    EBOV VP24 protein interacts with emerin in a tag-independent manner. (A) <t>HEK-293</t> cells were cotransfected with ISG54-luciferase together with pcDNA or the indicated VP24 expression plasmids. At 24 h after transfection, cells were treated with IFN-α, and at 16 h after treatment, luciferase production was analyzed. Columns are representative of the mean, and error bars represent the standard deviation of three biological replicates (left). Cell lysates from the experiment were analyzed by Western blotting for VP24 expression (right). (B) Minigenome assay in cells cotransfected with HA-VP24. Columns are representative of the mean, and error bars represent the standard deviation of three biological replicates. (C) Coimmunoprecipitation between HA-VP24 and NP. Vero cells were cotransfected with 2.5 μg of HA-VP24 and 2.5 μg of pcDNA or NP expression plasmids in a 100-mm dish, and 36 h after transfection, protein extracts of transfected cells were immunoprecipitated using anti-HA antibody. Immunoprecipitated proteins were analyzed by Western blotting using the indicated antibodies. The experiments were repeated twice, and representative images of one experiment are shown; IP, immunoprecipitated samples; IPT, input cell extract. (D) Localization of GFP-VP24 protein in Vero cells. Chromosomes were stained with DAPI. (E) Localization of GFP-VP24 protein in cells cotransfected with pcDNA or karyopherin 5 (KPNA5) expression plasmid. Chromosomes were stained with DAPI. (F) Colocalization of emerin and GFP-VP24 in Vero cells cotransfected with GFP or GFP-VP24. Chromosomes are stained with DAPI. Arrowheads indicate colocalization of GFP-VP24 and emerin. (G) Colocalization of emerin and VP24 in Vero cells transfected with untagged VP24 expression plasmid. Chromosomes are stained with DAPI. Inset shows higher magnification of the boxed area.
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    Images

    1) Product Images from "Expression of the Ebola Virus VP24 Protein Compromises the Integrity of the Nuclear Envelope and Induces a Laminopathy-Like Cellular Phenotype"

    Article Title: Expression of the Ebola Virus VP24 Protein Compromises the Integrity of the Nuclear Envelope and Induces a Laminopathy-Like Cellular Phenotype

    Journal: mBio

    doi: 10.1128/mBio.00972-21

    EBOV VP24 protein interacts with emerin in a tag-independent manner. (A) HEK-293 cells were cotransfected with ISG54-luciferase together with pcDNA or the indicated VP24 expression plasmids. At 24 h after transfection, cells were treated with IFN-α, and at 16 h after treatment, luciferase production was analyzed. Columns are representative of the mean, and error bars represent the standard deviation of three biological replicates (left). Cell lysates from the experiment were analyzed by Western blotting for VP24 expression (right). (B) Minigenome assay in cells cotransfected with HA-VP24. Columns are representative of the mean, and error bars represent the standard deviation of three biological replicates. (C) Coimmunoprecipitation between HA-VP24 and NP. Vero cells were cotransfected with 2.5 μg of HA-VP24 and 2.5 μg of pcDNA or NP expression plasmids in a 100-mm dish, and 36 h after transfection, protein extracts of transfected cells were immunoprecipitated using anti-HA antibody. Immunoprecipitated proteins were analyzed by Western blotting using the indicated antibodies. The experiments were repeated twice, and representative images of one experiment are shown; IP, immunoprecipitated samples; IPT, input cell extract. (D) Localization of GFP-VP24 protein in Vero cells. Chromosomes were stained with DAPI. (E) Localization of GFP-VP24 protein in cells cotransfected with pcDNA or karyopherin 5 (KPNA5) expression plasmid. Chromosomes were stained with DAPI. (F) Colocalization of emerin and GFP-VP24 in Vero cells cotransfected with GFP or GFP-VP24. Chromosomes are stained with DAPI. Arrowheads indicate colocalization of GFP-VP24 and emerin. (G) Colocalization of emerin and VP24 in Vero cells transfected with untagged VP24 expression plasmid. Chromosomes are stained with DAPI. Inset shows higher magnification of the boxed area.
    Figure Legend Snippet: EBOV VP24 protein interacts with emerin in a tag-independent manner. (A) HEK-293 cells were cotransfected with ISG54-luciferase together with pcDNA or the indicated VP24 expression plasmids. At 24 h after transfection, cells were treated with IFN-α, and at 16 h after treatment, luciferase production was analyzed. Columns are representative of the mean, and error bars represent the standard deviation of three biological replicates (left). Cell lysates from the experiment were analyzed by Western blotting for VP24 expression (right). (B) Minigenome assay in cells cotransfected with HA-VP24. Columns are representative of the mean, and error bars represent the standard deviation of three biological replicates. (C) Coimmunoprecipitation between HA-VP24 and NP. Vero cells were cotransfected with 2.5 μg of HA-VP24 and 2.5 μg of pcDNA or NP expression plasmids in a 100-mm dish, and 36 h after transfection, protein extracts of transfected cells were immunoprecipitated using anti-HA antibody. Immunoprecipitated proteins were analyzed by Western blotting using the indicated antibodies. The experiments were repeated twice, and representative images of one experiment are shown; IP, immunoprecipitated samples; IPT, input cell extract. (D) Localization of GFP-VP24 protein in Vero cells. Chromosomes were stained with DAPI. (E) Localization of GFP-VP24 protein in cells cotransfected with pcDNA or karyopherin 5 (KPNA5) expression plasmid. Chromosomes were stained with DAPI. (F) Colocalization of emerin and GFP-VP24 in Vero cells cotransfected with GFP or GFP-VP24. Chromosomes are stained with DAPI. Arrowheads indicate colocalization of GFP-VP24 and emerin. (G) Colocalization of emerin and VP24 in Vero cells transfected with untagged VP24 expression plasmid. Chromosomes are stained with DAPI. Inset shows higher magnification of the boxed area.

    Techniques Used: Luciferase, Expressing, Transfection, Standard Deviation, Western Blot, Immunoprecipitation, Staining, Plasmid Preparation

    2) Product Images from "Transfection of bovine fetal fibroblast with polyethylenimine (PEI) nanoparticles: effect of particle size and presence of fetal bovine serum on transgene delivery and cytotoxicity"

    Article Title: Transfection of bovine fetal fibroblast with polyethylenimine (PEI) nanoparticles: effect of particle size and presence of fetal bovine serum on transgene delivery and cytotoxicity

    Journal: Cytotechnology

    doi: 10.1007/s10616-017-0075-6

    a – c Effect of preincubation of different sized polyplexes (polymer/pDNA at ratio 2:1) in DMEM alone or supplemented with 10% FBS (DMEM + FBS) on transfection efficiency (DMEM, black bars ; DMEM + FBS, white bars ) in Hep G2 and HEK 293 cell lines and BFF. Asterisk denote significant differences (p
    Figure Legend Snippet: a – c Effect of preincubation of different sized polyplexes (polymer/pDNA at ratio 2:1) in DMEM alone or supplemented with 10% FBS (DMEM + FBS) on transfection efficiency (DMEM, black bars ; DMEM + FBS, white bars ) in Hep G2 and HEK 293 cell lines and BFF. Asterisk denote significant differences (p

    Techniques Used: Transfection

    a PCR analysis of genomic DNA from SB-transgenic monoclonal cell lines. Venus amplicon (280 pb product) originated from genomic DNA from four BFF ( left panel ), two HEK 293 and two Hep G2 ( right panel ) randomly selected clonal lines. C+ = positive controls. C− = negative controls. MW markers: GeneRuler 50 bp DNA Ladder (Thermo Scientific; left panel ) and 100 bp DNA Ladder (Invitrogen™, right panel ). Fluorescence microscopy images of four monoclonal BFF colonies ( b – e) transfected with the Sleeping Beauty system and selected during 21 days with G418. Fluorescence microscopy images of two HEK 293 ( f , g ) and two Hep G2 monoclonal colonies ( h , i ) transfected with the Sleeping Beauty system and selected during 21 days with G418. Bar = 100 μm
    Figure Legend Snippet: a PCR analysis of genomic DNA from SB-transgenic monoclonal cell lines. Venus amplicon (280 pb product) originated from genomic DNA from four BFF ( left panel ), two HEK 293 and two Hep G2 ( right panel ) randomly selected clonal lines. C+ = positive controls. C− = negative controls. MW markers: GeneRuler 50 bp DNA Ladder (Thermo Scientific; left panel ) and 100 bp DNA Ladder (Invitrogen™, right panel ). Fluorescence microscopy images of four monoclonal BFF colonies ( b – e) transfected with the Sleeping Beauty system and selected during 21 days with G418. Fluorescence microscopy images of two HEK 293 ( f , g ) and two Hep G2 monoclonal colonies ( h , i ) transfected with the Sleeping Beauty system and selected during 21 days with G418. Bar = 100 μm

    Techniques Used: Polymerase Chain Reaction, Transgenic Assay, Amplification, Fluorescence, Microscopy, Transfection

    3) Product Images from "Circular non-coding RNA ANRIL modulates ribosomal RNA maturation and atherosclerosis in humans"

    Article Title: Circular non-coding RNA ANRIL modulates ribosomal RNA maturation and atherosclerosis in humans

    Journal: Nature Communications

    doi: 10.1038/ncomms12429

    Role of endogenous circANRIL on apoptosis and cell proliferation. ( a ) Schematic of CRISPR/Cas9-mediated knockout of ANRIL in HEK-293 cells. Genomic regions encompassing exons (Ex) 5–20 were deleted. Arrowheads indicate location and orientation of primers used for genotyping. ( b ) PCR genotyping. Sequencing of PCR products from Ex5–20 assay validated successful deletion in mutant cell lines ( Supplementary Table 2 ). ( c ) Apoptosis and ( d ) proliferation in heterozygous and homozygous ANRIL knockout or control cell lines. As control, Cas9 was overexpressed without guide RNAs (gRNAs). ( e ) Apoptosis and ( f ) proliferation in homozygous knockout cells following transient re-expression of circANRIL . * P
    Figure Legend Snippet: Role of endogenous circANRIL on apoptosis and cell proliferation. ( a ) Schematic of CRISPR/Cas9-mediated knockout of ANRIL in HEK-293 cells. Genomic regions encompassing exons (Ex) 5–20 were deleted. Arrowheads indicate location and orientation of primers used for genotyping. ( b ) PCR genotyping. Sequencing of PCR products from Ex5–20 assay validated successful deletion in mutant cell lines ( Supplementary Table 2 ). ( c ) Apoptosis and ( d ) proliferation in heterozygous and homozygous ANRIL knockout or control cell lines. As control, Cas9 was overexpressed without guide RNAs (gRNAs). ( e ) Apoptosis and ( f ) proliferation in homozygous knockout cells following transient re-expression of circANRIL . * P

    Techniques Used: CRISPR, Knock-Out, Polymerase Chain Reaction, Sequencing, Mutagenesis, Expressing

    Atherosclerosis-related cell functions in c ircANRIL -overexpressing cells. ( a ) Schematic of the vector construct for circANRIL stable and transient overexpression. RNA polymerase II (RNAP II). ( b ) ANRIL isoform expression in HEK-293 cells that stably express circANRIL , linANRIL or empty vector (three biological replicates each). PCR of reverse-transcribed RNA, glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ), house-keeping gene; OdT, oligo(d)T primers; RH, random hexamer primers. ( c ) Apoptosis and ( d ) proliferation in HEK-293 cell line stably overexpressing circANRIL (pool of 3 biological replicates, and 8 and 12 technical replicates per condition, respectively), compared with overexpression of an unrelated circular RNA ( circHPRT1 ). Apoptosis ( e ) and proliferation ( f ) after RNAi against circANRIL or non- circANRIL or scrambled (SCR) siRNA control. Quadruplicate measurements per condition. * P
    Figure Legend Snippet: Atherosclerosis-related cell functions in c ircANRIL -overexpressing cells. ( a ) Schematic of the vector construct for circANRIL stable and transient overexpression. RNA polymerase II (RNAP II). ( b ) ANRIL isoform expression in HEK-293 cells that stably express circANRIL , linANRIL or empty vector (three biological replicates each). PCR of reverse-transcribed RNA, glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ), house-keeping gene; OdT, oligo(d)T primers; RH, random hexamer primers. ( c ) Apoptosis and ( d ) proliferation in HEK-293 cell line stably overexpressing circANRIL (pool of 3 biological replicates, and 8 and 12 technical replicates per condition, respectively), compared with overexpression of an unrelated circular RNA ( circHPRT1 ). Apoptosis ( e ) and proliferation ( f ) after RNAi against circANRIL or non- circANRIL or scrambled (SCR) siRNA control. Quadruplicate measurements per condition. * P

    Techniques Used: Plasmid Preparation, Construct, Over Expression, Expressing, Stable Transfection, Polymerase Chain Reaction, Random Hexamer Labeling

    Identification of circANRIL- binding proteins. ( a ) Schematic of λN-peptide-mediated capture of circANRIL -BoxB from cellular lysates of stably overexpressing HEK-293 cell lines, and label-free mass spectrometric quantification. Experiments were performed in a pool of three biological replicates (quadruplicate measurements). ( b ) Quantification of RNAs by qPCR after λN-peptide capture (quadruplicate measurements). ( c ) Summary of circANRIL -BoxB-bound proteins according to annotated (KEGG, GO) and published functions. ( d ) Volcano plot of label-free quantification (LFQ) of circANRIL -BoxB-bound proteins compared with circANRIL input control. ( e ) RIP of PES1, NOLC1, NOP14 and mouse IgG (mIgG) control (RIP was performed in a pool of three biological replicates, quadruplicate measurements). ( f ) Western blot of PES1 after λN-peptide-mediated circANRIL-BoxB capture using nuclear extracts from indicated cell lines.
    Figure Legend Snippet: Identification of circANRIL- binding proteins. ( a ) Schematic of λN-peptide-mediated capture of circANRIL -BoxB from cellular lysates of stably overexpressing HEK-293 cell lines, and label-free mass spectrometric quantification. Experiments were performed in a pool of three biological replicates (quadruplicate measurements). ( b ) Quantification of RNAs by qPCR after λN-peptide capture (quadruplicate measurements). ( c ) Summary of circANRIL -BoxB-bound proteins according to annotated (KEGG, GO) and published functions. ( d ) Volcano plot of label-free quantification (LFQ) of circANRIL -BoxB-bound proteins compared with circANRIL input control. ( e ) RIP of PES1, NOLC1, NOP14 and mouse IgG (mIgG) control (RIP was performed in a pool of three biological replicates, quadruplicate measurements). ( f ) Western blot of PES1 after λN-peptide-mediated circANRIL-BoxB capture using nuclear extracts from indicated cell lines.

    Techniques Used: Binding Assay, Stable Transfection, Real-time Polymerase Chain Reaction, Western Blot

    CircANRIL does not regulate 9p21 protein-coding genes and lacks miRNA sponging activity. ( a ) Overexpression of circANRIL in HEK-293 cells does not modulate endogenous linANRIL RNA abundance as measured by qPCR (left panel). Expression of p16 INK4a and p14 ARF , encoded by CDKN2A , and p15 INK4b , encoded by CDKN2B , in circANRIL -overexpressing cells (right panel) (pool of three biological replicates, quadruplicate qPCR measurements). ( b ) Prediction of miRNA-binding sites in circANRIL using miRanda and TargetSpy algorithms. ( c ) miRNA expression normalized to miR-27b as measured by qPCR (four biological replicates, quadruplicate measurements). ( d ) RNA immunoprecipitation (RIP) of AGO2. Analysis of precipitated RNAs: CDR1as , MALAT1 , positive controls; U1 , negative control (RIP was performed in a pool of three biological replicates, quadruplicate qPCR measurements). *** P
    Figure Legend Snippet: CircANRIL does not regulate 9p21 protein-coding genes and lacks miRNA sponging activity. ( a ) Overexpression of circANRIL in HEK-293 cells does not modulate endogenous linANRIL RNA abundance as measured by qPCR (left panel). Expression of p16 INK4a and p14 ARF , encoded by CDKN2A , and p15 INK4b , encoded by CDKN2B , in circANRIL -overexpressing cells (right panel) (pool of three biological replicates, quadruplicate qPCR measurements). ( b ) Prediction of miRNA-binding sites in circANRIL using miRanda and TargetSpy algorithms. ( c ) miRNA expression normalized to miR-27b as measured by qPCR (four biological replicates, quadruplicate measurements). ( d ) RNA immunoprecipitation (RIP) of AGO2. Analysis of precipitated RNAs: CDR1as , MALAT1 , positive controls; U1 , negative control (RIP was performed in a pool of three biological replicates, quadruplicate qPCR measurements). *** P

    Techniques Used: Activity Assay, Over Expression, Real-time Polymerase Chain Reaction, Expressing, Binding Assay, Immunoprecipitation, Negative Control

    rRNA maturation defects and nucleolar stress in circANRIL -overexpressing cells. ( a ) Relative quantification of pre-rRNA in circANRIL - or in circHPRT1- overexpressing HEK-293 cells using isoform-specific qPCRs (RNA from a pool of three biological replicates, quadruplicate measurements). ( b ) Pre-rRNA levels after RNAi against circANRIL or using scrambled siRNA control (quadruplicate measurements per condition). ( c ) Immunofluorescent staining of PES1 in circANRIL - or in circHPRT1 -overexpressing HEK-293 or empty vector control cells. Quantification of ( d ) nucleoli and ( e ) nucleolar size in circANRIL - or circHPRT1 -overexpressing or empty vector control cells (*** P
    Figure Legend Snippet: rRNA maturation defects and nucleolar stress in circANRIL -overexpressing cells. ( a ) Relative quantification of pre-rRNA in circANRIL - or in circHPRT1- overexpressing HEK-293 cells using isoform-specific qPCRs (RNA from a pool of three biological replicates, quadruplicate measurements). ( b ) Pre-rRNA levels after RNAi against circANRIL or using scrambled siRNA control (quadruplicate measurements per condition). ( c ) Immunofluorescent staining of PES1 in circANRIL - or in circHPRT1 -overexpressing HEK-293 or empty vector control cells. Quantification of ( d ) nucleoli and ( e ) nucleolar size in circANRIL - or circHPRT1 -overexpressing or empty vector control cells (*** P

    Techniques Used: Staining, Plasmid Preparation

    CircANRIL expression in human vascular tissue and association with atheroprotection at 9p21. ( a ) Schematic of circANRIL at 9p21 and qPCR assays for isoform specific quantification. CircANRIL contains exons 5, 6 and 7. Exon 7 is non-canonically spliced to exon 5. ( b ) qPCR analysis of circANRIL expression in human tissues, primary cells and cell lines. Beta actin ( ACTB ), house-keeping gene; FB, adventitial fibroblasts; HEK, HEK-293 cell line; THP1 and MonoMac, human monocytic cell line. Analyses were done in RNA pools of at least three donors. ( c ) In situ hybridization of circANRIL in human atherosclerotic plaque and co-localization with smooth muscle actin (SMA)-positive cells and macrophages (CD68). Sense control (CTR)-negative control. Representative staining out of three biological replicates. ( d ) Association of circANRIL with 9p21 haplotypes in PBMC from CAD patients. Protective (A, n =498), heterozygote (H, n =979) and risk (G, n =456) haplotypes. ( e ) Association of circANRIL with 9p21 haplotypes A ( n =49), H ( n =114) and G ( n =55) in endarterectomy specimens. ( f ) Association of circ/linANRIL ratio in PBMC with severity of CAD (No, n =745;
    Figure Legend Snippet: CircANRIL expression in human vascular tissue and association with atheroprotection at 9p21. ( a ) Schematic of circANRIL at 9p21 and qPCR assays for isoform specific quantification. CircANRIL contains exons 5, 6 and 7. Exon 7 is non-canonically spliced to exon 5. ( b ) qPCR analysis of circANRIL expression in human tissues, primary cells and cell lines. Beta actin ( ACTB ), house-keeping gene; FB, adventitial fibroblasts; HEK, HEK-293 cell line; THP1 and MonoMac, human monocytic cell line. Analyses were done in RNA pools of at least three donors. ( c ) In situ hybridization of circANRIL in human atherosclerotic plaque and co-localization with smooth muscle actin (SMA)-positive cells and macrophages (CD68). Sense control (CTR)-negative control. Representative staining out of three biological replicates. ( d ) Association of circANRIL with 9p21 haplotypes in PBMC from CAD patients. Protective (A, n =498), heterozygote (H, n =979) and risk (G, n =456) haplotypes. ( e ) Association of circANRIL with 9p21 haplotypes A ( n =49), H ( n =114) and G ( n =55) in endarterectomy specimens. ( f ) Association of circ/linANRIL ratio in PBMC with severity of CAD (No, n =745;

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, In Situ Hybridization, Negative Control, Staining

    Molecular mechanism of circANRIL controlling PES1 function. ( a ) Homology of circANRIL and precursor 47S rRNA (blue boxes) determined by BLASTn algorithm. ( b ) Prediction of RNA–protein interaction of circANRIL with PES1 using the catRAPID algorithm. Homology regions (HR1–HR5)—predicted RNA–protein interaction motifs in circANRIL . ( c ) Secondary structure prediction and HR1–5 of circANRIL using the Vienna RNA package 69 . ( d ) Schematic of PES1 with functional protein domains. Wild-type PES1 (PES1-WT) and two mutants lacking the 5′ (PES1Δ1–54) or the 3′ (PES1Δ446–588) lysine-rich NLSs. ( e ) Immunoprecipitation (IP) of PES1 isoforms from HEK-293 cells and quantification of RNA by qPCR (IP was performed in a pool of three biological replicates, quadruplicate measurements). ( f ) Pre-rRNA binding to PES1-WT and PES1Δ446–588 in circANRIL -overexpressing and control cells (IP was performed in a pool of three biological replicates, quadruplicate measurements). ( g ) Pre-rRNA and 7SL control RNA in circANRIL- expressing HEK-293 cells after transient PES1-WT or PES1Δ446–588 overexpression (pool of three biological replicates, quadruplicate measurements). ( h ) p53 western blot, ( i ), apoptosis and ( j ) cell proliferation in circANRIL -overexpressing HEK-293 cells with transient overexpression of PES1-WT or of PES1Δ446–588. Quadruplicate measurements per condition. * P
    Figure Legend Snippet: Molecular mechanism of circANRIL controlling PES1 function. ( a ) Homology of circANRIL and precursor 47S rRNA (blue boxes) determined by BLASTn algorithm. ( b ) Prediction of RNA–protein interaction of circANRIL with PES1 using the catRAPID algorithm. Homology regions (HR1–HR5)—predicted RNA–protein interaction motifs in circANRIL . ( c ) Secondary structure prediction and HR1–5 of circANRIL using the Vienna RNA package 69 . ( d ) Schematic of PES1 with functional protein domains. Wild-type PES1 (PES1-WT) and two mutants lacking the 5′ (PES1Δ1–54) or the 3′ (PES1Δ446–588) lysine-rich NLSs. ( e ) Immunoprecipitation (IP) of PES1 isoforms from HEK-293 cells and quantification of RNA by qPCR (IP was performed in a pool of three biological replicates, quadruplicate measurements). ( f ) Pre-rRNA binding to PES1-WT and PES1Δ446–588 in circANRIL -overexpressing and control cells (IP was performed in a pool of three biological replicates, quadruplicate measurements). ( g ) Pre-rRNA and 7SL control RNA in circANRIL- expressing HEK-293 cells after transient PES1-WT or PES1Δ446–588 overexpression (pool of three biological replicates, quadruplicate measurements). ( h ) p53 western blot, ( i ), apoptosis and ( j ) cell proliferation in circANRIL -overexpressing HEK-293 cells with transient overexpression of PES1-WT or of PES1Δ446–588. Quadruplicate measurements per condition. * P

    Techniques Used: Functional Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Expressing, Over Expression, Western Blot

    4) Product Images from "Induction of the Alternative NF-?B Pathway by Lymphotoxin ?? (LT??) Relies on Internalization of LT? Receptor ▿Induction of the Alternative NF-?B Pathway by Lymphotoxin ?? (LT??) Relies on Internalization of LT? Receptor ▿ †"

    Article Title: Induction of the Alternative NF-?B Pathway by Lymphotoxin ?? (LT??) Relies on Internalization of LT? Receptor ▿Induction of the Alternative NF-?B Pathway by Lymphotoxin ?? (LT??) Relies on Internalization of LT? Receptor ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.05033-11

    Intracellular LTβR activates the alternative NF-κB pathway independently of TRAF degradation. (A) HEK 293 cells were transiently transfected (+) or not (−) with the indicated expression vectors. Cells were lysed in 1% SDS and diluted up to 0.1% prior to the first round of immunoprecipitation (1st IP) with a control antibody (Ctrl Ab) or an anti-TRAF3 antibody. The immunoprecipitated material was analyzed by immunoblotting for TRAF3 and K48-linked TRAF3. The supernatants from the 1st IP were incubated with control beads or anti-Flag-coated beads. The immunoprecipitated materials were analyzed by immunoblotting for NIK and K48-linked ubiquitinated NIK. The asterisks represent the cross-reactivity with Ig heavy chains. (B) HeLa cells were stimulated with the agonist (Ago) to LTβR in the presence of DMSO (vehicle) or the proteasome inhibitor MG132, and cell extracts were analyzed by immunoblotting for the indicated proteins. (C) HeLa cells were treated with compound A (CmpA) or the Ago to LTβR alone or in combination as indicated, and the indicated proteins were analyzed by immunoblotting. (D) HeLa cells were stimulated as indicated with the Ago to LTβR in the absence (vehicle) or presence of bafilomycin A1. Total cell extracts were analyzed by Western blotting for the indicated proteins.
    Figure Legend Snippet: Intracellular LTβR activates the alternative NF-κB pathway independently of TRAF degradation. (A) HEK 293 cells were transiently transfected (+) or not (−) with the indicated expression vectors. Cells were lysed in 1% SDS and diluted up to 0.1% prior to the first round of immunoprecipitation (1st IP) with a control antibody (Ctrl Ab) or an anti-TRAF3 antibody. The immunoprecipitated material was analyzed by immunoblotting for TRAF3 and K48-linked TRAF3. The supernatants from the 1st IP were incubated with control beads or anti-Flag-coated beads. The immunoprecipitated materials were analyzed by immunoblotting for NIK and K48-linked ubiquitinated NIK. The asterisks represent the cross-reactivity with Ig heavy chains. (B) HeLa cells were stimulated with the agonist (Ago) to LTβR in the presence of DMSO (vehicle) or the proteasome inhibitor MG132, and cell extracts were analyzed by immunoblotting for the indicated proteins. (C) HeLa cells were treated with compound A (CmpA) or the Ago to LTβR alone or in combination as indicated, and the indicated proteins were analyzed by immunoblotting. (D) HeLa cells were stimulated as indicated with the Ago to LTβR in the absence (vehicle) or presence of bafilomycin A1. Total cell extracts were analyzed by Western blotting for the indicated proteins.

    Techniques Used: Transfection, Expressing, Immunoprecipitation, Incubation, Western Blot

    Identification of the cytoplasmic domain involved in LTβR-mediated p100 processing. (A) Schematic representation of human full-length and deletion mutant LTβR. The black bar, the gray bar, and TM represent the extracellular domain (amino acids 1 to 121), the cytosolic tail (amino acids 242 to 425), and the transmembrane domain (amino acids 222 to 241), respectively. (B to D) HEK 293 cells were transiently transfected with different sets of LTβR deletion mutants of the cytosolic tail and with the triple D 390 D 391 E 393 /AAA mutant. (E) 293T cells were stably infected with retrovirus encoding the indicated Myc-tagged LTβR, and expression was analyzed with anti-Myc immunoblotting. (F) Stable clones were stimulated for 6 h with an agonistic (Ago) anti-LTβR antibody prior to analysis by Western blotting of the processing of p100 into p52.
    Figure Legend Snippet: Identification of the cytoplasmic domain involved in LTβR-mediated p100 processing. (A) Schematic representation of human full-length and deletion mutant LTβR. The black bar, the gray bar, and TM represent the extracellular domain (amino acids 1 to 121), the cytosolic tail (amino acids 242 to 425), and the transmembrane domain (amino acids 222 to 241), respectively. (B to D) HEK 293 cells were transiently transfected with different sets of LTβR deletion mutants of the cytosolic tail and with the triple D 390 D 391 E 393 /AAA mutant. (E) 293T cells were stably infected with retrovirus encoding the indicated Myc-tagged LTβR, and expression was analyzed with anti-Myc immunoblotting. (F) Stable clones were stimulated for 6 h with an agonistic (Ago) anti-LTβR antibody prior to analysis by Western blotting of the processing of p100 into p52.

    Techniques Used: Mutagenesis, Transfection, Stable Transfection, Infection, Expressing, Clone Assay, Western Blot

    LTβR defective for p100 processing is sequestered into the plasma membrane. (A) HEK 293T cells were transfected with three differently tagged LTβRs (HA, Flag, and Myc tagged), and double immunoprecipitations were performed to analyze the trimerization of wt LTβR (see the supplemental material for details). (B) The same procedure as in panel A was applied for internal deletion mutants ΔI 345–358 and ΔI 359–368. (C) HEK 293T cells were transfected with wt LTβR, ΔI 345–358, ΔI 359–368, and Δ389. The cross-linker DSP was used prior to immunoprecipitation and immunoblotting of LTβR under nonreduced (−DTT) and reduced (+DTT) conditions. (D) Flow cytometry analysis of HEK 293 cells mock transfected or transfected with expression vector for wt LTβR, LTβR ΔI 345–358, or LTβR ΔI 359–368 and stained for cell surface LTβR (nonpermeabilized [NP]) or cell surface and intracellular LTβR (permeabilized [P]). MFI (mean fluorescence intensity) represents the value of one measurement out of three independent experiments. (E) Localization of wt LTβR, LTβR ΔI 345–358, and LTβR ΔI 359–368 in HEK 293 cells. Arrows indicate the perinuclear compartment. (F) Cell fractionation of LTβR into Triton X-100-soluble and -insoluble fractions from HEK 293 cells transfected with the indicated LTβR constructs.
    Figure Legend Snippet: LTβR defective for p100 processing is sequestered into the plasma membrane. (A) HEK 293T cells were transfected with three differently tagged LTβRs (HA, Flag, and Myc tagged), and double immunoprecipitations were performed to analyze the trimerization of wt LTβR (see the supplemental material for details). (B) The same procedure as in panel A was applied for internal deletion mutants ΔI 345–358 and ΔI 359–368. (C) HEK 293T cells were transfected with wt LTβR, ΔI 345–358, ΔI 359–368, and Δ389. The cross-linker DSP was used prior to immunoprecipitation and immunoblotting of LTβR under nonreduced (−DTT) and reduced (+DTT) conditions. (D) Flow cytometry analysis of HEK 293 cells mock transfected or transfected with expression vector for wt LTβR, LTβR ΔI 345–358, or LTβR ΔI 359–368 and stained for cell surface LTβR (nonpermeabilized [NP]) or cell surface and intracellular LTβR (permeabilized [P]). MFI (mean fluorescence intensity) represents the value of one measurement out of three independent experiments. (E) Localization of wt LTβR, LTβR ΔI 345–358, and LTβR ΔI 359–368 in HEK 293 cells. Arrows indicate the perinuclear compartment. (F) Cell fractionation of LTβR into Triton X-100-soluble and -insoluble fractions from HEK 293 cells transfected with the indicated LTβR constructs.

    Techniques Used: Transfection, Immunoprecipitation, Flow Cytometry, Cytometry, Expressing, Plasmid Preparation, Staining, Fluorescence, Cell Fractionation, Construct

    Perinuclear location of LTβR is a prerequisite for the recruitment of endogenous TRAF proteins and induction of p100 processing. (A) Flow cytometry analysis of HEK 293 cells mock transfected or transfected with expression vector for LTβR ΔS wt, LTβR ΔS/ΔI 345–358, or LTβR ΔS/ΔI 359–368 and stained for cell surface LTβR (nonpermeabilized) or cell surface and intracellular LTβR (permeabilized). MFI (mean fluorescence intensity) represents the value of one measurement out of three independent experiments. (B) HeLa cells transiently transfected with the indicated HA-tagged construct were stained for LTβR (in red) and nuclei (DAPI). Arrows indicate the punctate perinuclear staining of LTβR. (C) HEK 293 cells were mock transfected or transfected with LTβR expression vectors encoding either wt, ΔI 345–358, ΔI 359–368, or their signal sequence (ΔS)-defective counterpart. Immunoprecipitated LTβR was analyzed by Western blotting for the recruitment of endogenous TRAF2 and TRAF3. The asterisks represent the cross-reactivity with Ig heavy chains. (D) Extracts from cells transfected with signal sequence (ΔS)-defective mutants were used to analyze the processing of p100 by Western blotting.
    Figure Legend Snippet: Perinuclear location of LTβR is a prerequisite for the recruitment of endogenous TRAF proteins and induction of p100 processing. (A) Flow cytometry analysis of HEK 293 cells mock transfected or transfected with expression vector for LTβR ΔS wt, LTβR ΔS/ΔI 345–358, or LTβR ΔS/ΔI 359–368 and stained for cell surface LTβR (nonpermeabilized) or cell surface and intracellular LTβR (permeabilized). MFI (mean fluorescence intensity) represents the value of one measurement out of three independent experiments. (B) HeLa cells transiently transfected with the indicated HA-tagged construct were stained for LTβR (in red) and nuclei (DAPI). Arrows indicate the punctate perinuclear staining of LTβR. (C) HEK 293 cells were mock transfected or transfected with LTβR expression vectors encoding either wt, ΔI 345–358, ΔI 359–368, or their signal sequence (ΔS)-defective counterpart. Immunoprecipitated LTβR was analyzed by Western blotting for the recruitment of endogenous TRAF2 and TRAF3. The asterisks represent the cross-reactivity with Ig heavy chains. (D) Extracts from cells transfected with signal sequence (ΔS)-defective mutants were used to analyze the processing of p100 by Western blotting.

    Techniques Used: Flow Cytometry, Cytometry, Transfection, Expressing, Plasmid Preparation, Staining, Fluorescence, Construct, Sequencing, Immunoprecipitation, Western Blot

    5) Product Images from "PDX-1: Demonstration of Oncogenic Properties in Pancreatic Cancer"

    Article Title: PDX-1: Demonstration of Oncogenic Properties in Pancreatic Cancer

    Journal: Cancer

    doi: 10.1002/cncr.25629

    HEK 293, MIA PaCa2 and HPDE cells stably expressing PDX-1 with and without human PDX-1 shRNA had increased cell proliferation. PDX-1 overexpression caused significant increase of cell proliferation in all cell lines as shown in A, B and C. PDX-1 shRNA
    Figure Legend Snippet: HEK 293, MIA PaCa2 and HPDE cells stably expressing PDX-1 with and without human PDX-1 shRNA had increased cell proliferation. PDX-1 overexpression caused significant increase of cell proliferation in all cell lines as shown in A, B and C. PDX-1 shRNA

    Techniques Used: Stable Transfection, Expressing, shRNA, Over Expression

    PDX-1 overexpression promotes tumor formation and growth in SCID mice. HEK 293 or MIA PaCa2 cells overexpressing PDX-1 vs empty vector were implanted in SCID. Gross tumor volume at 30 days was was evaluated compared using x2 test (A) and tumor size was
    Figure Legend Snippet: PDX-1 overexpression promotes tumor formation and growth in SCID mice. HEK 293 or MIA PaCa2 cells overexpressing PDX-1 vs empty vector were implanted in SCID. Gross tumor volume at 30 days was was evaluated compared using x2 test (A) and tumor size was

    Techniques Used: Over Expression, Mouse Assay, Plasmid Preparation

    HEK 293, MIA PaCa2 and HPDE cells stably overexpressing PDX-1 were cultured with and without human PDX-1 shRNA, then transferred into invasion inserts for 48h (Fig 3A). Increased quantatative invasiveness iss shown in 3B, 3C and 3D.
    Figure Legend Snippet: HEK 293, MIA PaCa2 and HPDE cells stably overexpressing PDX-1 were cultured with and without human PDX-1 shRNA, then transferred into invasion inserts for 48h (Fig 3A). Increased quantatative invasiveness iss shown in 3B, 3C and 3D.

    Techniques Used: Stable Transfection, Cell Culture, shRNA

    Transient expression of PDX-1 increases cell proliferation. HEK 293, MIA PaCa2 and HPDE cells were transfected with human PDX-1 with and without human PDX-1 shRNA. Transient burst expression of PDX-1 results in increased cell proliferation in HEK 293
    Figure Legend Snippet: Transient expression of PDX-1 increases cell proliferation. HEK 293, MIA PaCa2 and HPDE cells were transfected with human PDX-1 with and without human PDX-1 shRNA. Transient burst expression of PDX-1 results in increased cell proliferation in HEK 293

    Techniques Used: Expressing, Transfection, shRNA

    PDX-1 overexpression results in cell cycle disruption. Western blot was performed on PDX-1 stably trasnfected HEK 293 cells with or without PDX-1 shRNA. Protein levels of PDX-1, cyclin E, cdk2 P21, p27 and P53 were determined by bands profiles (A) and
    Figure Legend Snippet: PDX-1 overexpression results in cell cycle disruption. Western blot was performed on PDX-1 stably trasnfected HEK 293 cells with or without PDX-1 shRNA. Protein levels of PDX-1, cyclin E, cdk2 P21, p27 and P53 were determined by bands profiles (A) and

    Techniques Used: Over Expression, Western Blot, Stable Transfection, shRNA

    PDX-1 overexpression in HEK 293 (A, G) and MIA PaCa2 (D, H) cells causes cell transformation in anchorage-independent growth assay. Colonies were photographed at 21 days under a phase contrast fluorescent microscope. The number and size of colonies were
    Figure Legend Snippet: PDX-1 overexpression in HEK 293 (A, G) and MIA PaCa2 (D, H) cells causes cell transformation in anchorage-independent growth assay. Colonies were photographed at 21 days under a phase contrast fluorescent microscope. The number and size of colonies were

    Techniques Used: Over Expression, Transformation Assay, Growth Assay, Microscopy

    6) Product Images from "Rotavirus NSP4 Induces a Novel Vesicular Compartment Regulated by Calcium and Associated with Viroplasms"

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

    Journal: Journal of Virology

    doi: 10.1128/JVI.02167-05

    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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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;
    Figure Legend 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;

    Techniques Used: 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;
    Figure Legend 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;

    Techniques Used: 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
    Figure Legend 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

    Techniques Used:

    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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: Staining

    7) Product Images from "Rotavirus NSP4 Induces a Novel Vesicular Compartment Regulated by Calcium and Associated with Viroplasms"

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

    Journal: Journal of Virology

    doi: 10.1128/JVI.02167-05

    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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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;
    Figure Legend 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;

    Techniques Used: 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;
    Figure Legend 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;

    Techniques Used: 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
    Figure Legend 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

    Techniques Used:

    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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: Staining

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    TaKaRa lenti x 293t cell line
    Lenti X 293t Cell Line, supplied by TaKaRa, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lenti x 293t cell line/product/TaKaRa
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    90
    TaKaRa virus maintenance lenti x 293t cells
    Combination of a pseudovirus assay and protein-based assays enabled identification of novel inhibitors targeting the early stage of coronavirus infection. a) Schematic representation of a compound screen using a pseudovirus presenting the spike of MERS-CoV. To generate the pseudovirus, a lentivirus backbone, luciferase gene, and the spike were transfected onto <t>293T</t> <t>cells.</t> In the compound screen, Huh-7 cells were treated with compounds and infected with the pseudovirus, after which the accumulated luciferase signal was measured. b ) Schematic representation of the alpha test. In the alpha test, the receptor binding domain (RBD) of the spike of MERS-CoV was immobilized on the donor bead while its cellular receptor, hDPP4 was attached to an acceptor bead. c ) Summary of classification of hits from the pseudovirus screen and subsequent alpha test: ∼16% entry-specific (spike-specific) compounds among 1126 hits. d ) Structure of three identified hits: thiosemicarbazide, amiodarone, and raloxifene.
    Virus Maintenance Lenti X 293t Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/virus maintenance lenti x 293t cells/product/TaKaRa
    Average 90 stars, based on 1 article reviews
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    Combination of a pseudovirus assay and protein-based assays enabled identification of novel inhibitors targeting the early stage of coronavirus infection. a) Schematic representation of a compound screen using a pseudovirus presenting the spike of MERS-CoV. To generate the pseudovirus, a lentivirus backbone, luciferase gene, and the spike were transfected onto 293T cells. In the compound screen, Huh-7 cells were treated with compounds and infected with the pseudovirus, after which the accumulated luciferase signal was measured. b ) Schematic representation of the alpha test. In the alpha test, the receptor binding domain (RBD) of the spike of MERS-CoV was immobilized on the donor bead while its cellular receptor, hDPP4 was attached to an acceptor bead. c ) Summary of classification of hits from the pseudovirus screen and subsequent alpha test: ∼16% entry-specific (spike-specific) compounds among 1126 hits. d ) Structure of three identified hits: thiosemicarbazide, amiodarone, and raloxifene.

    Journal: Antiviral Research

    Article Title: Rapid discovery and classification of inhibitors of coronavirus infection by pseudovirus screen and amplified luminescence proximity homogeneous assay

    doi: 10.1016/j.antiviral.2022.105473

    Figure Lengend Snippet: Combination of a pseudovirus assay and protein-based assays enabled identification of novel inhibitors targeting the early stage of coronavirus infection. a) Schematic representation of a compound screen using a pseudovirus presenting the spike of MERS-CoV. To generate the pseudovirus, a lentivirus backbone, luciferase gene, and the spike were transfected onto 293T cells. In the compound screen, Huh-7 cells were treated with compounds and infected with the pseudovirus, after which the accumulated luciferase signal was measured. b ) Schematic representation of the alpha test. In the alpha test, the receptor binding domain (RBD) of the spike of MERS-CoV was immobilized on the donor bead while its cellular receptor, hDPP4 was attached to an acceptor bead. c ) Summary of classification of hits from the pseudovirus screen and subsequent alpha test: ∼16% entry-specific (spike-specific) compounds among 1126 hits. d ) Structure of three identified hits: thiosemicarbazide, amiodarone, and raloxifene.

    Article Snippet: 2.1 Cell line and virus maintenance Lenti-X™ 293T cells (Clontech, Mountain View, CA) were cultivated in Dulbecco's Modified Eagle's Medium (DMEM; Sigma-Aldrich, St. Louis, MO) supplemented with 4.5 g/L glucose, 4 mM L-glutamine, 3.7 g/L NaHCO3 , 10% fetal bovine serum (FBS; Gibco, Billings, MT), 1 mM sodium pyruvate (Sigma-Aldrich), and 1% penicillin/streptomycin (Sigma-Aldrich).

    Techniques: Infection, Luciferase, Transfection, Binding Assay