sf9 cell line  (Roche)


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    Structured Review

    Roche sf9 cell line
    Expression profile of Se-AQP . ( A ) Western blot analysis. Se-AQP was transfected into <t>Sf9</t> cells. Protein size of recombinant Se-AQP was ~32 kDa. It was captured by V5 antibody. ( B ) Immunofluorescence assay for the detection of transient expression of Se-AQP in Sf9 cells. F-actin was specifically detected with Alexa Fluor 555 phalloidin while nucleus was stained with DAPI. To check transient expression, anti-V5-FITC antibody was used. ( C ) Expression patterns of Se-AQP in different developmental stages, including egg, first to fifth instar larvae (‘L1–L5’), pupa, and adult. ( D ) Expression patterns in indicated tissues of L5 larvae, including hemocyte (‘HC’), fat body (‘FB’), and gut (‘Gut’). A ribosomal gene RL32 was used as reference gene. Each treatment was replicated three times with independent tissue preparations. Different letters indicate significant differences among means at Type I error = 0.05 (LSD test).
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    Images

    1) Product Images from "An aquaporin mediates cell shape change required for cellular immunity in the beet armyworm, Spodoptera exigua"

    Article Title: An aquaporin mediates cell shape change required for cellular immunity in the beet armyworm, Spodoptera exigua

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-41541-2

    Expression profile of Se-AQP . ( A ) Western blot analysis. Se-AQP was transfected into Sf9 cells. Protein size of recombinant Se-AQP was ~32 kDa. It was captured by V5 antibody. ( B ) Immunofluorescence assay for the detection of transient expression of Se-AQP in Sf9 cells. F-actin was specifically detected with Alexa Fluor 555 phalloidin while nucleus was stained with DAPI. To check transient expression, anti-V5-FITC antibody was used. ( C ) Expression patterns of Se-AQP in different developmental stages, including egg, first to fifth instar larvae (‘L1–L5’), pupa, and adult. ( D ) Expression patterns in indicated tissues of L5 larvae, including hemocyte (‘HC’), fat body (‘FB’), and gut (‘Gut’). A ribosomal gene RL32 was used as reference gene. Each treatment was replicated three times with independent tissue preparations. Different letters indicate significant differences among means at Type I error = 0.05 (LSD test).
    Figure Legend Snippet: Expression profile of Se-AQP . ( A ) Western blot analysis. Se-AQP was transfected into Sf9 cells. Protein size of recombinant Se-AQP was ~32 kDa. It was captured by V5 antibody. ( B ) Immunofluorescence assay for the detection of transient expression of Se-AQP in Sf9 cells. F-actin was specifically detected with Alexa Fluor 555 phalloidin while nucleus was stained with DAPI. To check transient expression, anti-V5-FITC antibody was used. ( C ) Expression patterns of Se-AQP in different developmental stages, including egg, first to fifth instar larvae (‘L1–L5’), pupa, and adult. ( D ) Expression patterns in indicated tissues of L5 larvae, including hemocyte (‘HC’), fat body (‘FB’), and gut (‘Gut’). A ribosomal gene RL32 was used as reference gene. Each treatment was replicated three times with independent tissue preparations. Different letters indicate significant differences among means at Type I error = 0.05 (LSD test).

    Techniques Used: Expressing, Western Blot, Transfection, Recombinant, Immunofluorescence, Staining

    2) Product Images from "Characterisation of Human Oestrogen Receptor beta (ERβ) Splice Variants in Neuronal Cells"

    Article Title: Characterisation of Human Oestrogen Receptor beta (ERβ) Splice Variants in Neuronal Cells

    Journal: Journal of neuroendocrinology

    doi: 10.1111/j.1365-2826.2012.02337.x

    Role of phosphoinositide 3-kinase (PI3K) and p38 kinase inhibition on human oestrogen receptor (hER) β splice variant-mediated repression of human AVP (hAVP) promoter activity. SK-N-SH cells were transiently transfected with 0.15 μ g of hAVP-luciferase reporter construct and 0.15 μ g of an expression vector containing hER β 1, hER β 2, hER β 4 or hER β 5. Twenty-four hours post transfection, cells were treated with vehicle (0.001% EtOH) or 100 nM 17 β -oestradiol (E 2 ), (A) 10 μ M LY 294002 or (B) 10 μ M SB202190 and 100 nM ICI 182 780 for 12 h. Data represent the percentage change in relative light units compared to empty vector, vehicle-treated controls. *P
    Figure Legend Snippet: Role of phosphoinositide 3-kinase (PI3K) and p38 kinase inhibition on human oestrogen receptor (hER) β splice variant-mediated repression of human AVP (hAVP) promoter activity. SK-N-SH cells were transiently transfected with 0.15 μ g of hAVP-luciferase reporter construct and 0.15 μ g of an expression vector containing hER β 1, hER β 2, hER β 4 or hER β 5. Twenty-four hours post transfection, cells were treated with vehicle (0.001% EtOH) or 100 nM 17 β -oestradiol (E 2 ), (A) 10 μ M LY 294002 or (B) 10 μ M SB202190 and 100 nM ICI 182 780 for 12 h. Data represent the percentage change in relative light units compared to empty vector, vehicle-treated controls. *P

    Techniques Used: Inhibition, Variant Assay, Activity Assay, Transfection, Luciferase, Construct, Expressing, Plasmid Preparation

    Effects of 17 β -oestradiol (E 2 ), 17 β -diol (3 β -diol) and ICI 182 780 on human oestrogen receptor (hER) β splice variant-mediated arginine vasopressin (AVP) promoter activity before and after deletion of an activator protein-1 (AP-1) site. SK-N-SH cells were transiently transfected with 0.15 μ g of (A) human AVP (hAVP)-luciferase or (B) hAVPΔ611 – 604-luciferase reporter constructs and 0.15 μ g of expression vectors containing hER β 1, hER β 2, hER β 4 or hER β 5. Twenty-four hours post transfection, cells were treated with vehicle (0.001% EtOH), 100 nM E 2 , 3 β -diol or ICI 182 780 for 12 h. Data represent the percentage change in relative light units compared to empty vector, vehicle-treated controls. *P
    Figure Legend Snippet: Effects of 17 β -oestradiol (E 2 ), 17 β -diol (3 β -diol) and ICI 182 780 on human oestrogen receptor (hER) β splice variant-mediated arginine vasopressin (AVP) promoter activity before and after deletion of an activator protein-1 (AP-1) site. SK-N-SH cells were transiently transfected with 0.15 μ g of (A) human AVP (hAVP)-luciferase or (B) hAVPΔ611 – 604-luciferase reporter constructs and 0.15 μ g of expression vectors containing hER β 1, hER β 2, hER β 4 or hER β 5. Twenty-four hours post transfection, cells were treated with vehicle (0.001% EtOH), 100 nM E 2 , 3 β -diol or ICI 182 780 for 12 h. Data represent the percentage change in relative light units compared to empty vector, vehicle-treated controls. *P

    Techniques Used: Variant Assay, Activity Assay, Transfection, Luciferase, Construct, Expressing, Plasmid Preparation

    Effects of 17 β -oestradiol (E 2 ), 17 β -diol (3 β -diol) and ICI 182 780 on human oestrogen receptor (hER) β splice variant-mediated oestrogen response element (ERE) and activator protein-1 (AP-1) promoter activity. HT-22 cells were transiently transfected with 0.15 μ g of (A) 2x-ERE- or (B) AP-1-luciferase reporter constructs and 0.15 μ g of expression vectors containing hER β 1, hER β 2, hER β 4 or hER β 5. Twenty-four hours post transfection, cells were treated with vehicle (0.001% EtOH), 100 nM of E 2 , 3 β -diol or ICI 182 780 for 12 h. Data represent the percentage change in relative light units compared to empty vector controls. *P
    Figure Legend Snippet: Effects of 17 β -oestradiol (E 2 ), 17 β -diol (3 β -diol) and ICI 182 780 on human oestrogen receptor (hER) β splice variant-mediated oestrogen response element (ERE) and activator protein-1 (AP-1) promoter activity. HT-22 cells were transiently transfected with 0.15 μ g of (A) 2x-ERE- or (B) AP-1-luciferase reporter constructs and 0.15 μ g of expression vectors containing hER β 1, hER β 2, hER β 4 or hER β 5. Twenty-four hours post transfection, cells were treated with vehicle (0.001% EtOH), 100 nM of E 2 , 3 β -diol or ICI 182 780 for 12 h. Data represent the percentage change in relative light units compared to empty vector controls. *P

    Techniques Used: Variant Assay, Activity Assay, Transfection, Luciferase, Construct, Expressing, Plasmid Preparation

    3) Product Images from "Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response"

    Article Title: Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200408003

    Interplay of repressive and stimulatory uORFs regulates ATF4 translation. (A) mRNA expressed by the reporter genes used in these experiments. Where indicated, the initiating AUG codons of the uORFs were converted to AUA and the stop codons between the colinear uORF1 and uORF2 converted to UCG and UAC, respectively. (B) Autoradiograms of SDS-PAGE of radiolabeled proteins from untreated and AP20187-treated Fv2E-PERK (+) CHO cells transfected with the 5′ATF4-GFP reporter, or reporters with a mutation in the start codon of uORF1, uORF2 or both uORFs, or the parental GFP reporter. Cytoplasmic RNA from a parallel sample of untreated transfected cells was resolved by Northern blot and hybridized to GFP and GAPDH probes, as indicated. (C) As in B except cells were transfected with reporters in which the stop codons between the two uORFs were converted to Ser or Tyr codons.
    Figure Legend Snippet: Interplay of repressive and stimulatory uORFs regulates ATF4 translation. (A) mRNA expressed by the reporter genes used in these experiments. Where indicated, the initiating AUG codons of the uORFs were converted to AUA and the stop codons between the colinear uORF1 and uORF2 converted to UCG and UAC, respectively. (B) Autoradiograms of SDS-PAGE of radiolabeled proteins from untreated and AP20187-treated Fv2E-PERK (+) CHO cells transfected with the 5′ATF4-GFP reporter, or reporters with a mutation in the start codon of uORF1, uORF2 or both uORFs, or the parental GFP reporter. Cytoplasmic RNA from a parallel sample of untreated transfected cells was resolved by Northern blot and hybridized to GFP and GAPDH probes, as indicated. (C) As in B except cells were transfected with reporters in which the stop codons between the two uORFs were converted to Ser or Tyr codons.

    Techniques Used: SDS Page, Transfection, Mutagenesis, Northern Blot

    Both ATF4 uORFs are translated under basal conditions. (A) Predicted structure of the mRNA expressed by the reporter genes used in these experiments. (B) Autoradiograms of SDS-PAGE of radiolabeled proteins from untreated and AP20187-treated Fv2E-PERK (+) CHO cells transfected with the ATF4-GFP reporter, or reporters fusing uORF1 or uORF2 to GFP. Cytoplasmic RNA from a parallel sample of untreated, transfected cells was resolved by Northern blot and hybridized to GFP and GAPDH probes. The low basal expression of NPTII in cells transfected with the uORF1-GFP is a reproducible if unexplained finding.
    Figure Legend Snippet: Both ATF4 uORFs are translated under basal conditions. (A) Predicted structure of the mRNA expressed by the reporter genes used in these experiments. (B) Autoradiograms of SDS-PAGE of radiolabeled proteins from untreated and AP20187-treated Fv2E-PERK (+) CHO cells transfected with the ATF4-GFP reporter, or reporters fusing uORF1 or uORF2 to GFP. Cytoplasmic RNA from a parallel sample of untreated, transfected cells was resolved by Northern blot and hybridized to GFP and GAPDH probes. The low basal expression of NPTII in cells transfected with the uORF1-GFP is a reproducible if unexplained finding.

    Techniques Used: SDS Page, Transfection, Northern Blot, Expressing

    ATF4 mRNA translation requires ribosomes scanning. (A) Predicted structure of the mRNA expressed by the reporter genes. Depicted is the stem loop introduced at the 5′ end of the mRNA and replacement of vector derived viral termination and poly-adenylation sequences (thin line) with ATF4 derived termination sequences (white rectangle). (B) Autoradiogram of SDS-PAGE of radiolabeled proteins from untreated and AP20187-treated CHO cells expressing Fv2E-PERK. The cells were transfected with ATF4-GFP reporters with or without the stable stem-loop structure at the 5′ end of the mRNA. Radiolabeled GFP, NPTII, and endogenous ATF4 were immunoprecipitated with a specific antisera. Cytoplasmic RNA from a parallel sample of untreated, transfected cells was resolved by Northern blot and hybridized to GFP and GAPDH probes, as indicated. The ratio of the labeled GFP to RNA signal in each lane is provided. (C) As in B, cells were transfected with ATF4.GFP reporters with a viral 3′ termination sequences (5′ATF4.GFP), or the genomic ATF4 3′ termination sequences (5′3′ATF4.GFP) or the parental GFP reporter.
    Figure Legend Snippet: ATF4 mRNA translation requires ribosomes scanning. (A) Predicted structure of the mRNA expressed by the reporter genes. Depicted is the stem loop introduced at the 5′ end of the mRNA and replacement of vector derived viral termination and poly-adenylation sequences (thin line) with ATF4 derived termination sequences (white rectangle). (B) Autoradiogram of SDS-PAGE of radiolabeled proteins from untreated and AP20187-treated CHO cells expressing Fv2E-PERK. The cells were transfected with ATF4-GFP reporters with or without the stable stem-loop structure at the 5′ end of the mRNA. Radiolabeled GFP, NPTII, and endogenous ATF4 were immunoprecipitated with a specific antisera. Cytoplasmic RNA from a parallel sample of untreated, transfected cells was resolved by Northern blot and hybridized to GFP and GAPDH probes, as indicated. The ratio of the labeled GFP to RNA signal in each lane is provided. (C) As in B, cells were transfected with ATF4.GFP reporters with a viral 3′ termination sequences (5′ATF4.GFP), or the genomic ATF4 3′ termination sequences (5′3′ATF4.GFP) or the parental GFP reporter.

    Techniques Used: Plasmid Preparation, Derivative Assay, SDS Page, Expressing, Transfection, Immunoprecipitation, Northern Blot, Labeling

    The 5′ end of the ATF4 mRNA mediates translational regulation of the gene. (A) Organization of the 5′ end of the mouse ATF4 mRNA, the derivative 5′ATF4-GFP reporter and the parental GFP reporter. (B) Autoradiogram of SDS-PAGE of radiolabeled proteins after a brief labeling pulse of wild-type CHO cells or cells stably overexpressing the COOH terminus of GADD34 (which blocks eIF2α phosphorylation) transfected with the indicated reporter plasmids. The cells were treated with the endoplasmic reticulum stress-inducing agent thapsigargin (TG) for the indicated time and the encoded GFP fusion proteins (top) and endogenous ATF4 (bottom) were immunoprecipitated with specific antibodies. (C) Autoradiogram of SDS-PAGE of radiolabeled proteins after a brief labeling pulse of CHO cells transfected with the ATF4-GFP reporter and pretreated for 30 min with the indicated concentration of arsenite (ARS), the electrophile methyl-methanesulfonate (MMS), histidinol (HIS, which activates GCN2), thapsigargin (TG), and the encoded GFP fusion proteins (top), the NPTII, encoded by a different gene on the same plasmid (second panel) and endogenous ATF4 (third panel) were immunoprecipitated with specific antisera. The GFP/NPTII ratio provides an estimate of the translational inducibility of the reporter in the treated cells. Immunoblots of phosphorylated (eIF2α-P) and total eIF2α from parallel lysates are shown in the bottom panels. (D) Autoradiogram of radiolabeled proteins from Fv2E-PERK expressing CHO cells treated with the indicated concentration of the activating AP20187 ligand for 30′ before and during the 20′ labeling pulse. The cells were transfected with the ATF4 reporter (5′ATF4-GFP), a cricket paralysis virus internal ribosome entry site reporter (IGR IRE5-YFP) or a GFP translational reporter derived from the 5′ end of mouse CD36 gene (mCD36-GFP). GFP, endogenous ATF4 and NPTII were immunoprecipitated as in C with specific antisera.
    Figure Legend Snippet: The 5′ end of the ATF4 mRNA mediates translational regulation of the gene. (A) Organization of the 5′ end of the mouse ATF4 mRNA, the derivative 5′ATF4-GFP reporter and the parental GFP reporter. (B) Autoradiogram of SDS-PAGE of radiolabeled proteins after a brief labeling pulse of wild-type CHO cells or cells stably overexpressing the COOH terminus of GADD34 (which blocks eIF2α phosphorylation) transfected with the indicated reporter plasmids. The cells were treated with the endoplasmic reticulum stress-inducing agent thapsigargin (TG) for the indicated time and the encoded GFP fusion proteins (top) and endogenous ATF4 (bottom) were immunoprecipitated with specific antibodies. (C) Autoradiogram of SDS-PAGE of radiolabeled proteins after a brief labeling pulse of CHO cells transfected with the ATF4-GFP reporter and pretreated for 30 min with the indicated concentration of arsenite (ARS), the electrophile methyl-methanesulfonate (MMS), histidinol (HIS, which activates GCN2), thapsigargin (TG), and the encoded GFP fusion proteins (top), the NPTII, encoded by a different gene on the same plasmid (second panel) and endogenous ATF4 (third panel) were immunoprecipitated with specific antisera. The GFP/NPTII ratio provides an estimate of the translational inducibility of the reporter in the treated cells. Immunoblots of phosphorylated (eIF2α-P) and total eIF2α from parallel lysates are shown in the bottom panels. (D) Autoradiogram of radiolabeled proteins from Fv2E-PERK expressing CHO cells treated with the indicated concentration of the activating AP20187 ligand for 30′ before and during the 20′ labeling pulse. The cells were transfected with the ATF4 reporter (5′ATF4-GFP), a cricket paralysis virus internal ribosome entry site reporter (IGR IRE5-YFP) or a GFP translational reporter derived from the 5′ end of mouse CD36 gene (mCD36-GFP). GFP, endogenous ATF4 and NPTII were immunoprecipitated as in C with specific antisera.

    Techniques Used: SDS Page, Labeling, Stable Transfection, Transfection, Immunoprecipitation, Concentration Assay, Plasmid Preparation, Western Blot, Expressing, Derivative Assay

    4) Product Images from "Comparison of Ectopic Gene Expression Methods in Rat Neural Stem Cells"

    Article Title: Comparison of Ectopic Gene Expression Methods in Rat Neural Stem Cells

    Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

    doi: 10.4196/kjpp.2013.17.1.23

    Comparison of transfection efficiency of three programs -DS112, DS113 and CA137- of nucleofection. (A~C) Representative merged images of NSCs nucleofected using program (A), (B) DS113 and (C) CA137 for 48 hours. GFP positive cells are shown in green and DAPI stained nuclei are shown in blue. Scale bar=100 µm. (D) Quantification of the transfected NSCs. The ratio of GFP-positive cells to the total cells was calculated and presented. Quantitative data are shown as mean±S.D. ( ** p
    Figure Legend Snippet: Comparison of transfection efficiency of three programs -DS112, DS113 and CA137- of nucleofection. (A~C) Representative merged images of NSCs nucleofected using program (A), (B) DS113 and (C) CA137 for 48 hours. GFP positive cells are shown in green and DAPI stained nuclei are shown in blue. Scale bar=100 µm. (D) Quantification of the transfected NSCs. The ratio of GFP-positive cells to the total cells was calculated and presented. Quantitative data are shown as mean±S.D. ( ** p

    Techniques Used: Transfection, Staining

    Comparison of transfection efficiency of lipid-mediated transfection, electroporation, nucleofection and retroviral transduction. (A~D) Representative merged images of NSCs transfected with GFP vector for 48 hours using (A) X-tremeGENE9 transfection reagent, (B) NEPA21 electroporator, (C) Amaxa 4D Nucleofector, and (D) retroviral transduction. By immunostaining GFP positive cells are shown in green and nuclei are shown in blue. Scale bar=100 µm. (E) Quantification data of the transfected cells. The ratio of GFP-positive cells to the total cells was calculated. Quantitative data are shown as mean±S.D. ( ** p
    Figure Legend Snippet: Comparison of transfection efficiency of lipid-mediated transfection, electroporation, nucleofection and retroviral transduction. (A~D) Representative merged images of NSCs transfected with GFP vector for 48 hours using (A) X-tremeGENE9 transfection reagent, (B) NEPA21 electroporator, (C) Amaxa 4D Nucleofector, and (D) retroviral transduction. By immunostaining GFP positive cells are shown in green and nuclei are shown in blue. Scale bar=100 µm. (E) Quantification data of the transfected cells. The ratio of GFP-positive cells to the total cells was calculated. Quantitative data are shown as mean±S.D. ( ** p

    Techniques Used: Transfection, Electroporation, Transduction, Plasmid Preparation, Immunostaining

    Transfection efficiency of rat NSCs 48 hours after lipid-mediated transfection. (A, B) Representative merged images of NSCs transfected with (A) lipid alone and (B) lipid with GFP vector. GFP was visualized by immunostaining with anti-GFP primary antibody and Alexa Fluor 488-conjugated secondary antibody (green). Nuclei were visualized by DAPI staining (blue). Scale bar=100 µm. (C) Quantification of the immunostaining data. The ratio of GFP-positive cells to the total cells was calculated and presented. The data are shown as mean±S.D. ( * p
    Figure Legend Snippet: Transfection efficiency of rat NSCs 48 hours after lipid-mediated transfection. (A, B) Representative merged images of NSCs transfected with (A) lipid alone and (B) lipid with GFP vector. GFP was visualized by immunostaining with anti-GFP primary antibody and Alexa Fluor 488-conjugated secondary antibody (green). Nuclei were visualized by DAPI staining (blue). Scale bar=100 µm. (C) Quantification of the immunostaining data. The ratio of GFP-positive cells to the total cells was calculated and presented. The data are shown as mean±S.D. ( * p

    Techniques Used: Transfection, Plasmid Preparation, Immunostaining, Staining

    5) Product Images from "Mouse Lymphoid Cell Line Selected To Have High Immunoglobulin Promoter Activity"

    Article Title: Mouse Lymphoid Cell Line Selected To Have High Immunoglobulin Promoter Activity

    Journal: Molecular and Cellular Biology

    doi:

    Transfection characteristics of the selected cell lines. (A) High-GFP-expressing cells from four rounds of FACS were single-cell cloned and transiently transfected with pGL3 containing four different promoters (H2B-59 + 1, IgH-154 + 35, B29-164 + 32, and mb-1-252 + 48). Cell line 2017, the parental clone 2017-IgH-GFP no. 9, and 70Z/3 were also transfected. The histone H2B promoter was used as a control. pCMV-βGal was cotransfected, and the luciferase/β-Gal ratios for the H2B construct were normalized to 1. The dark bars depict Ig promoter activity. Shaded bars are B29 and mb-1. Lipid-mediated transfection reagents were used. (B) Selected high-GFP cell lines 70Z/3 and 2017 and parent line 2017-IgH-GFP no. 9 were transiently transfected with either the 17.2.25 promoter (gray bars) or the V H 186.2 promoter with the IgH intronic enhancer upstream (white bars). pCMV-βGal was cotransfected, and luciferase/β-Gal ratios are depicted on the y axis. Lipid-mediated transfection reagents were used. (C) Selected cell lines m4, h3, and 70Z/3, and the parental cell line 2017-IgH-GFP no. 9 were transiently transfected with either IgH-154 + 35 (gray bars) or the same construct in which the intronic enhancer had been inserted (white bars) (see Materials and Methods). Lipid-mediated transfection reagents were used. pCMV-βGal was cotransfected as an internal control. Experiments were performed in triplicate, and error bars denote standard deviations. (D) Scanning promoter mutants were transiently transfected into either the parent cell line 2017-IgH-GFP no. 9 (gray bars) or the high-GFP-selected cell line m4 (white bars). Lipid-mediated transfection reagents were used. pCMV-βGal was cotransfected, and luciferase/β-Gal ratios are depicted on the y axis.
    Figure Legend Snippet: Transfection characteristics of the selected cell lines. (A) High-GFP-expressing cells from four rounds of FACS were single-cell cloned and transiently transfected with pGL3 containing four different promoters (H2B-59 + 1, IgH-154 + 35, B29-164 + 32, and mb-1-252 + 48). Cell line 2017, the parental clone 2017-IgH-GFP no. 9, and 70Z/3 were also transfected. The histone H2B promoter was used as a control. pCMV-βGal was cotransfected, and the luciferase/β-Gal ratios for the H2B construct were normalized to 1. The dark bars depict Ig promoter activity. Shaded bars are B29 and mb-1. Lipid-mediated transfection reagents were used. (B) Selected high-GFP cell lines 70Z/3 and 2017 and parent line 2017-IgH-GFP no. 9 were transiently transfected with either the 17.2.25 promoter (gray bars) or the V H 186.2 promoter with the IgH intronic enhancer upstream (white bars). pCMV-βGal was cotransfected, and luciferase/β-Gal ratios are depicted on the y axis. Lipid-mediated transfection reagents were used. (C) Selected cell lines m4, h3, and 70Z/3, and the parental cell line 2017-IgH-GFP no. 9 were transiently transfected with either IgH-154 + 35 (gray bars) or the same construct in which the intronic enhancer had been inserted (white bars) (see Materials and Methods). Lipid-mediated transfection reagents were used. pCMV-βGal was cotransfected as an internal control. Experiments were performed in triplicate, and error bars denote standard deviations. (D) Scanning promoter mutants were transiently transfected into either the parent cell line 2017-IgH-GFP no. 9 (gray bars) or the high-GFP-selected cell line m4 (white bars). Lipid-mediated transfection reagents were used. pCMV-βGal was cotransfected, and luciferase/β-Gal ratios are depicted on the y axis.

    Techniques Used: Transfection, Expressing, FACS, Clone Assay, Luciferase, Construct, Activity Assay

    (A) Cell lines BJA-B, M12, 70Z/3, θ4b, p5424, 2017, and EL-4 were transfected with either the pGL3 backbone (white bars) or IgH-luciferase promoter constructs containing (IgH-154 + 35) (light gray bars) or lacking (IgH-154 + 1) (dark gray bars) the sequences from the transcription initiation site to +35. pCMV-βGal was cotransfected as an internal control. Transfections were performed by electroporation. The luciferase/β-Gal activity ratio is depicted on the y axis. Averages from three replicate experiments are shown. Error bars denote standard deviations. (B) Promoter constructs containing 3-bp mutants spanning the transcription initiation site were electroporated into either the B-cell line BJA-B (white bars) or the nonlymphoid cell line WERI-27 (dark gray bars). The construct name is shown to the right of the sequence for that construct. The full-length (+35) and deletion (+1) constructs were included as controls. pCMV-βGal was cotransfected, and the luciferase/β-Gal activity ratio is shown on the x ). pCMV-βGal was cotransfected as an internal control. pUC18 was used to equalize the mass of the amount of DNA transfected. Transfections were performed by electroporation. The luciferase/β-Gal activity ratio is depicted on the y axis. Error bars denote standard deviations.
    Figure Legend Snippet: (A) Cell lines BJA-B, M12, 70Z/3, θ4b, p5424, 2017, and EL-4 were transfected with either the pGL3 backbone (white bars) or IgH-luciferase promoter constructs containing (IgH-154 + 35) (light gray bars) or lacking (IgH-154 + 1) (dark gray bars) the sequences from the transcription initiation site to +35. pCMV-βGal was cotransfected as an internal control. Transfections were performed by electroporation. The luciferase/β-Gal activity ratio is depicted on the y axis. Averages from three replicate experiments are shown. Error bars denote standard deviations. (B) Promoter constructs containing 3-bp mutants spanning the transcription initiation site were electroporated into either the B-cell line BJA-B (white bars) or the nonlymphoid cell line WERI-27 (dark gray bars). The construct name is shown to the right of the sequence for that construct. The full-length (+35) and deletion (+1) constructs were included as controls. pCMV-βGal was cotransfected, and the luciferase/β-Gal activity ratio is shown on the x ). pCMV-βGal was cotransfected as an internal control. pUC18 was used to equalize the mass of the amount of DNA transfected. Transfections were performed by electroporation. The luciferase/β-Gal activity ratio is depicted on the y axis. Error bars denote standard deviations.

    Techniques Used: Transfection, Luciferase, Construct, Electroporation, Activity Assay, Sequencing

    6) Product Images from "Differential Fibroblast Growth Factor 8 (FGF8)-Mediated Autoregulation of Its Cognate Receptors, Fgfr1 and Fgfr3, in Neuronal Cell Lines"

    Article Title: Differential Fibroblast Growth Factor 8 (FGF8)-Mediated Autoregulation of Its Cognate Receptors, Fgfr1 and Fgfr3, in Neuronal Cell Lines

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0010143

    Effects of FGF8b on GnRH promoter activity. Transient transfection of GT1-7 cells with 0.15 µg/well of mouse full-length GnRH-luciferase reporter construct. Following transfection, cells were treated with vehicle, FGF8b (50 ng/ml), PD173074 (100 nM), or FGF8b + PD173074 for 8 hours. Data are represented as mean percent change in RLU's from vehicle-treated controls ± SEM. Dissimilar letters indicate statistically significant difference among groups, P
    Figure Legend Snippet: Effects of FGF8b on GnRH promoter activity. Transient transfection of GT1-7 cells with 0.15 µg/well of mouse full-length GnRH-luciferase reporter construct. Following transfection, cells were treated with vehicle, FGF8b (50 ng/ml), PD173074 (100 nM), or FGF8b + PD173074 for 8 hours. Data are represented as mean percent change in RLU's from vehicle-treated controls ± SEM. Dissimilar letters indicate statistically significant difference among groups, P

    Techniques Used: Activity Assay, Transfection, Luciferase, Construct

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    Electroporation:

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    Transfection:

    Article Title: A classical NLS and the SUN domain contribute to the targeting of SUN2 to the inner nuclear membrane
    Article Snippet: .. Transient transfections were performed using FuGene transfection reagent (Roche). .. Cells were fixed 20–36 h after transfection with 4% PFA for 10 min. After washing with PBS, cover slips were mounted in VectaShield (VectorLabs) for microscopic analysis.

    Article Title: A Haplotype of Angiotensin Receptor Type 1 Associated with Human Hypertension Increases Blood Pressure in Transgenic Mice *
    Article Snippet: .. Transient transfections were performed with FuGENE 6 transfection reagent (Roche Applied Science) following the manufacturer's protocol. .. For co-transfection experiments, expression vector pSV-USF2 (100 ng) was added to the reporter constructs.

    Article Title: Human Parvovirus B19 Nonstructural Protein (NS1) Induces Cell Cycle Arrest at G1 Phase
    Article Snippet: .. Transient transfections to 293T cells were performed with FuGene 6 (Roche Diagnostics, Indianapolis, Ind.) and to UT7/Epo-S1 cells were performed with “amaxa” electroporation gene transfer tool (Amaxa GmbH, Berlin, Germany). ..

    Article Title: The Intraflagellar Transport Protein IFT27 Promotes BBSome Exit from Cilia through the GTPase ARL6/BBS3
    Article Snippet: .. Transient transfections used XtremeGENE9 (Roche). .. For IFT27 knockdown, IMCD3 cells were transfected with 30 nM of either IFT27 siRNA (Qiagen, #SI02743860) or AllStars Negative Control siRNA (Qiagen, #SI03650318) using Lipofectamine RNAiMAX (Life Technologies).

    Article Title: Microsatellite-encoded domain in rodent Sry functions as a genetic capacitor to enable the rapid evolution of biological novelty
    Article Snippet: .. Transient transfections were carried out by the Fugene HD protocol (Hoffmann LaRoche). .. PCR primers were in accordance with human genomic sequences.

    Article Title: An aquaporin mediates cell shape change required for cellular immunity in the beet armyworm, Spodoptera exigua
    Article Snippet: .. Heterologous expression of Se-AQP in Sf9 cells Using an eukaryotic expression vector pIB/V5-His (Invitrogen), a recombinant pIB/V5-His-Se-AQP was prepared and transiently expressed in Sf9 cell line by cationic lipid-mediated transfection using X-treme GENE 9 DNA transfection reagent (Roche, Mannheim, Germany). .. Transfection procedure and extraction of cellular proteins from Sf9 cells were performed according to the method described by Kumar and Kim and quantified using Bradford method .

    Article Title: Np9 Protein of Human Endogenous Retrovirus K Interacts with Ligand of Numb Protein X
    Article Snippet: .. Transient transfections of Cos-1 cell cultures with FuGene-6 (Roche) routinely produced 70 to 80% transfected cells. ..

    Article Title: The FAM Deubiquitylating Enzyme Localizes to Multiple Points of Protein Trafficking in Epithelia, where It Associates with E-cadherin and ?-catenin
    Article Snippet: .. Transient transfections were performed using DOTAP Liposomal Transfection Reagent (Roche) according to their instructions. .. Briefly, MCF-7 cells were grown to 60–70% confluence either in flasks or on glass coverslips and transfected with the amount of DNA indicated using the solutions provided.

    Produced:

    Article Title: Np9 Protein of Human Endogenous Retrovirus K Interacts with Ligand of Numb Protein X
    Article Snippet: .. Transient transfections of Cos-1 cell cultures with FuGene-6 (Roche) routinely produced 70 to 80% transfected cells. ..

    Expressing:

    Article Title: An aquaporin mediates cell shape change required for cellular immunity in the beet armyworm, Spodoptera exigua
    Article Snippet: .. Heterologous expression of Se-AQP in Sf9 cells Using an eukaryotic expression vector pIB/V5-His (Invitrogen), a recombinant pIB/V5-His-Se-AQP was prepared and transiently expressed in Sf9 cell line by cationic lipid-mediated transfection using X-treme GENE 9 DNA transfection reagent (Roche, Mannheim, Germany). .. Transfection procedure and extraction of cellular proteins from Sf9 cells were performed according to the method described by Kumar and Kim and quantified using Bradford method .

    Recombinant:

    Article Title: An aquaporin mediates cell shape change required for cellular immunity in the beet armyworm, Spodoptera exigua
    Article Snippet: .. Heterologous expression of Se-AQP in Sf9 cells Using an eukaryotic expression vector pIB/V5-His (Invitrogen), a recombinant pIB/V5-His-Se-AQP was prepared and transiently expressed in Sf9 cell line by cationic lipid-mediated transfection using X-treme GENE 9 DNA transfection reagent (Roche, Mannheim, Germany). .. Transfection procedure and extraction of cellular proteins from Sf9 cells were performed according to the method described by Kumar and Kim and quantified using Bradford method .

    Plasmid Preparation:

    Article Title: An aquaporin mediates cell shape change required for cellular immunity in the beet armyworm, Spodoptera exigua
    Article Snippet: .. Heterologous expression of Se-AQP in Sf9 cells Using an eukaryotic expression vector pIB/V5-His (Invitrogen), a recombinant pIB/V5-His-Se-AQP was prepared and transiently expressed in Sf9 cell line by cationic lipid-mediated transfection using X-treme GENE 9 DNA transfection reagent (Roche, Mannheim, Germany). .. Transfection procedure and extraction of cellular proteins from Sf9 cells were performed according to the method described by Kumar and Kim and quantified using Bradford method .

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    Roche lipid mediated transfection
    <t>Transfection</t> characteristics of the selected cell lines. (A) High-GFP-expressing cells from four rounds of FACS were single-cell cloned and transiently transfected with pGL3 containing four different promoters (H2B-59 + 1, IgH-154 + 35, B29-164 + 32, and mb-1-252 + 48). Cell line 2017, the parental clone 2017-IgH-GFP no. 9, and 70Z/3 were also transfected. The histone H2B promoter was used as a control. pCMV-βGal was cotransfected, and the luciferase/β-Gal ratios for the H2B construct were normalized to 1. The dark bars depict Ig promoter activity. Shaded bars are B29 and mb-1. Lipid-mediated transfection reagents were used. (B) Selected high-GFP cell lines 70Z/3 and 2017 and parent line 2017-IgH-GFP no. 9 were transiently transfected with either the 17.2.25 promoter (gray bars) or the V H 186.2 promoter with the IgH intronic enhancer upstream (white bars). pCMV-βGal was cotransfected, and luciferase/β-Gal ratios are depicted on the y axis. Lipid-mediated transfection reagents were used. (C) Selected cell lines m4, h3, and 70Z/3, and the parental cell line 2017-IgH-GFP no. 9 were transiently transfected with either IgH-154 + 35 (gray bars) or the same construct in which the intronic enhancer had been inserted (white bars) (see Materials and Methods). Lipid-mediated transfection reagents were used. pCMV-βGal was cotransfected as an internal control. Experiments were performed in triplicate, and error bars denote standard deviations. (D) Scanning promoter mutants were transiently transfected into either the parent cell line 2017-IgH-GFP no. 9 (gray bars) or the high-GFP-selected cell line m4 (white bars). Lipid-mediated transfection reagents were used. pCMV-βGal was cotransfected, and luciferase/β-Gal ratios are depicted on the y axis.
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    Transfection characteristics of the selected cell lines. (A) High-GFP-expressing cells from four rounds of FACS were single-cell cloned and transiently transfected with pGL3 containing four different promoters (H2B-59 + 1, IgH-154 + 35, B29-164 + 32, and mb-1-252 + 48). Cell line 2017, the parental clone 2017-IgH-GFP no. 9, and 70Z/3 were also transfected. The histone H2B promoter was used as a control. pCMV-βGal was cotransfected, and the luciferase/β-Gal ratios for the H2B construct were normalized to 1. The dark bars depict Ig promoter activity. Shaded bars are B29 and mb-1. Lipid-mediated transfection reagents were used. (B) Selected high-GFP cell lines 70Z/3 and 2017 and parent line 2017-IgH-GFP no. 9 were transiently transfected with either the 17.2.25 promoter (gray bars) or the V H 186.2 promoter with the IgH intronic enhancer upstream (white bars). pCMV-βGal was cotransfected, and luciferase/β-Gal ratios are depicted on the y axis. Lipid-mediated transfection reagents were used. (C) Selected cell lines m4, h3, and 70Z/3, and the parental cell line 2017-IgH-GFP no. 9 were transiently transfected with either IgH-154 + 35 (gray bars) or the same construct in which the intronic enhancer had been inserted (white bars) (see Materials and Methods). Lipid-mediated transfection reagents were used. pCMV-βGal was cotransfected as an internal control. Experiments were performed in triplicate, and error bars denote standard deviations. (D) Scanning promoter mutants were transiently transfected into either the parent cell line 2017-IgH-GFP no. 9 (gray bars) or the high-GFP-selected cell line m4 (white bars). Lipid-mediated transfection reagents were used. pCMV-βGal was cotransfected, and luciferase/β-Gal ratios are depicted on the y axis.

    Journal: Molecular and Cellular Biology

    Article Title: Mouse Lymphoid Cell Line Selected To Have High Immunoglobulin Promoter Activity

    doi:

    Figure Lengend Snippet: Transfection characteristics of the selected cell lines. (A) High-GFP-expressing cells from four rounds of FACS were single-cell cloned and transiently transfected with pGL3 containing four different promoters (H2B-59 + 1, IgH-154 + 35, B29-164 + 32, and mb-1-252 + 48). Cell line 2017, the parental clone 2017-IgH-GFP no. 9, and 70Z/3 were also transfected. The histone H2B promoter was used as a control. pCMV-βGal was cotransfected, and the luciferase/β-Gal ratios for the H2B construct were normalized to 1. The dark bars depict Ig promoter activity. Shaded bars are B29 and mb-1. Lipid-mediated transfection reagents were used. (B) Selected high-GFP cell lines 70Z/3 and 2017 and parent line 2017-IgH-GFP no. 9 were transiently transfected with either the 17.2.25 promoter (gray bars) or the V H 186.2 promoter with the IgH intronic enhancer upstream (white bars). pCMV-βGal was cotransfected, and luciferase/β-Gal ratios are depicted on the y axis. Lipid-mediated transfection reagents were used. (C) Selected cell lines m4, h3, and 70Z/3, and the parental cell line 2017-IgH-GFP no. 9 were transiently transfected with either IgH-154 + 35 (gray bars) or the same construct in which the intronic enhancer had been inserted (white bars) (see Materials and Methods). Lipid-mediated transfection reagents were used. pCMV-βGal was cotransfected as an internal control. Experiments were performed in triplicate, and error bars denote standard deviations. (D) Scanning promoter mutants were transiently transfected into either the parent cell line 2017-IgH-GFP no. 9 (gray bars) or the high-GFP-selected cell line m4 (white bars). Lipid-mediated transfection reagents were used. pCMV-βGal was cotransfected, and luciferase/β-Gal ratios are depicted on the y axis.

    Article Snippet: Cells were harvested and assayed after 24 h. Lipid-mediated transfection experiments used the FuGene6 transfection reagent (Roche) according to the manufacturer's instructions.

    Techniques: Transfection, Expressing, FACS, Clone Assay, Luciferase, Construct, Activity Assay

    (A) Cell lines BJA-B, M12, 70Z/3, θ4b, p5424, 2017, and EL-4 were transfected with either the pGL3 backbone (white bars) or IgH-luciferase promoter constructs containing (IgH-154 + 35) (light gray bars) or lacking (IgH-154 + 1) (dark gray bars) the sequences from the transcription initiation site to +35. pCMV-βGal was cotransfected as an internal control. Transfections were performed by electroporation. The luciferase/β-Gal activity ratio is depicted on the y axis. Averages from three replicate experiments are shown. Error bars denote standard deviations. (B) Promoter constructs containing 3-bp mutants spanning the transcription initiation site were electroporated into either the B-cell line BJA-B (white bars) or the nonlymphoid cell line WERI-27 (dark gray bars). The construct name is shown to the right of the sequence for that construct. The full-length (+35) and deletion (+1) constructs were included as controls. pCMV-βGal was cotransfected, and the luciferase/β-Gal activity ratio is shown on the x ). pCMV-βGal was cotransfected as an internal control. pUC18 was used to equalize the mass of the amount of DNA transfected. Transfections were performed by electroporation. The luciferase/β-Gal activity ratio is depicted on the y axis. Error bars denote standard deviations.

    Journal: Molecular and Cellular Biology

    Article Title: Mouse Lymphoid Cell Line Selected To Have High Immunoglobulin Promoter Activity

    doi:

    Figure Lengend Snippet: (A) Cell lines BJA-B, M12, 70Z/3, θ4b, p5424, 2017, and EL-4 were transfected with either the pGL3 backbone (white bars) or IgH-luciferase promoter constructs containing (IgH-154 + 35) (light gray bars) or lacking (IgH-154 + 1) (dark gray bars) the sequences from the transcription initiation site to +35. pCMV-βGal was cotransfected as an internal control. Transfections were performed by electroporation. The luciferase/β-Gal activity ratio is depicted on the y axis. Averages from three replicate experiments are shown. Error bars denote standard deviations. (B) Promoter constructs containing 3-bp mutants spanning the transcription initiation site were electroporated into either the B-cell line BJA-B (white bars) or the nonlymphoid cell line WERI-27 (dark gray bars). The construct name is shown to the right of the sequence for that construct. The full-length (+35) and deletion (+1) constructs were included as controls. pCMV-βGal was cotransfected, and the luciferase/β-Gal activity ratio is shown on the x ). pCMV-βGal was cotransfected as an internal control. pUC18 was used to equalize the mass of the amount of DNA transfected. Transfections were performed by electroporation. The luciferase/β-Gal activity ratio is depicted on the y axis. Error bars denote standard deviations.

    Article Snippet: Cells were harvested and assayed after 24 h. Lipid-mediated transfection experiments used the FuGene6 transfection reagent (Roche) according to the manufacturer's instructions.

    Techniques: Transfection, Luciferase, Construct, Electroporation, Activity Assay, Sequencing