cationic lipid mediated transient transfection  (Thermo Fisher)


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    Thermo Fisher cationic lipid mediated transient transfection
    Enzyme activity of iNOS FL and iNOS 8 − 9 − in transfected HEK 293 cells. ( A ) Nitrite accumulation (23 h) in the culture medium, after <t>transfection</t> with vector-only, iNOS FL or iNOS 8 − 9 − , is expressed as nanomoles per milligram of total cell protein. ( B ) NOS activity in lysates of transfected cells expressed as nanomoles of nitrite per milligram of total cell protein per 3 h. Data are means ± SD of three independent experiments, each done in duplicate. Invisible error bars are too small to be drawn.
    Cationic Lipid Mediated Transient Transfection, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cationic lipid mediated transient transfection - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Cloning and characterization of human inducible nitric oxide synthase splice variants: A domain, encoded by exons 8 and 9, is critical for dimerization"

    Article Title: Cloning and characterization of human inducible nitric oxide synthase splice variants: A domain, encoded by exons 8 and 9, is critical for dimerization

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Enzyme activity of iNOS FL and iNOS 8 − 9 − in transfected HEK 293 cells. ( A ) Nitrite accumulation (23 h) in the culture medium, after transfection with vector-only, iNOS FL or iNOS 8 − 9 − , is expressed as nanomoles per milligram of total cell protein. ( B ) NOS activity in lysates of transfected cells expressed as nanomoles of nitrite per milligram of total cell protein per 3 h. Data are means ± SD of three independent experiments, each done in duplicate. Invisible error bars are too small to be drawn.
    Figure Legend Snippet: Enzyme activity of iNOS FL and iNOS 8 − 9 − in transfected HEK 293 cells. ( A ) Nitrite accumulation (23 h) in the culture medium, after transfection with vector-only, iNOS FL or iNOS 8 − 9 − , is expressed as nanomoles per milligram of total cell protein. ( B ) NOS activity in lysates of transfected cells expressed as nanomoles of nitrite per milligram of total cell protein per 3 h. Data are means ± SD of three independent experiments, each done in duplicate. Invisible error bars are too small to be drawn.

    Techniques Used: Activity Assay, Transfection, Plasmid Preparation

    2) Product Images from "A Critical Role for CHIP in the Aggresome Pathway ▿A Critical Role for CHIP in the Aggresome Pathway ▿ †"

    Article Title: A Critical Role for CHIP in the Aggresome Pathway ▿A Critical Role for CHIP in the Aggresome Pathway ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00829-08

    CHIP colocalizes with iNOS aggresome. (A) HEK293 cells were cotransfected with a plasmid encoding the cDNA of iNOS-GFP and with a plasmid containing a vector as a control or Flag-tagged CHIP. The molar ratio of the iNOS plasmid to the coplasmid was 1:10. Forty eight hours after transfection, cells were fixed and examined by fluorescence microscopy. Exogenously expressed CHIP colocalized with cytosolic iNOS (row i) and translocated with iNOS to the aggresome (row ii). iNOS aggresomes in CHIP-transfected cells were ubiquitin enriched (row iii). (B) Endogenous CHIP colocalized with the iNOS aggresome in primary airway epithelial cells cultured at the air-liquid interphase (row i), in RT4 cells (row ii), and in HEK293 cells (row iii). Primary cells and RT4 cells expressed iNOS following cytokine stimulation, whereas HEK293 cells stably expressed iNOS-GFP. Scale bar, 10 μm.
    Figure Legend Snippet: CHIP colocalizes with iNOS aggresome. (A) HEK293 cells were cotransfected with a plasmid encoding the cDNA of iNOS-GFP and with a plasmid containing a vector as a control or Flag-tagged CHIP. The molar ratio of the iNOS plasmid to the coplasmid was 1:10. Forty eight hours after transfection, cells were fixed and examined by fluorescence microscopy. Exogenously expressed CHIP colocalized with cytosolic iNOS (row i) and translocated with iNOS to the aggresome (row ii). iNOS aggresomes in CHIP-transfected cells were ubiquitin enriched (row iii). (B) Endogenous CHIP colocalized with the iNOS aggresome in primary airway epithelial cells cultured at the air-liquid interphase (row i), in RT4 cells (row ii), and in HEK293 cells (row iii). Primary cells and RT4 cells expressed iNOS following cytokine stimulation, whereas HEK293 cells stably expressed iNOS-GFP. Scale bar, 10 μm.

    Techniques Used: Chromatin Immunoprecipitation, Plasmid Preparation, Transfection, Fluorescence, Microscopy, Cell Culture, Stable Transfection

    Critical role for CHIP in iNOS aggresome formation. (A and B) HEK293 cells stably expressing iNOS-GFP were transfected for 72 h with plasmids expressing either control shRNA or shRNA specific for CHIP. Cells were incubated for 3 h in the presence of vehicle only (DMSO) (A) or a 10 μM concentration of the proteasome inhibitor MG132 (B). Cells were then fixed, stained with DAPI to visualize the nuclei (blue), immunostained with CHIP antibody, and examined by fluorescence microscopy. Arrows in panel B point to the mature iNOS aggresome. Graphs show quantitation of the percentage of cells expressing iNOS aggresome or preaggresome structures. (C to E) HEK293 cells were cotransfected with a plasmid encoding cDNA of human iNOS-GFP and with a plasmid containing vector as a control or Flag-tagged CHIP. The molar ratio of the iNOS plasmid to the coplasmid was 1:10. At 24 (C) or 48 (D and E) h after transfection, cells were examined by fluorescence microscopy. Quantitation of the percentage of cells expressing the iNOS aggresome is shown (C and D). CHIP-transfected cells exhibited larger iNOS aggresomes (E). Data represent mean ± standard deviation ( n = 3). *, P
    Figure Legend Snippet: Critical role for CHIP in iNOS aggresome formation. (A and B) HEK293 cells stably expressing iNOS-GFP were transfected for 72 h with plasmids expressing either control shRNA or shRNA specific for CHIP. Cells were incubated for 3 h in the presence of vehicle only (DMSO) (A) or a 10 μM concentration of the proteasome inhibitor MG132 (B). Cells were then fixed, stained with DAPI to visualize the nuclei (blue), immunostained with CHIP antibody, and examined by fluorescence microscopy. Arrows in panel B point to the mature iNOS aggresome. Graphs show quantitation of the percentage of cells expressing iNOS aggresome or preaggresome structures. (C to E) HEK293 cells were cotransfected with a plasmid encoding cDNA of human iNOS-GFP and with a plasmid containing vector as a control or Flag-tagged CHIP. The molar ratio of the iNOS plasmid to the coplasmid was 1:10. At 24 (C) or 48 (D and E) h after transfection, cells were examined by fluorescence microscopy. Quantitation of the percentage of cells expressing the iNOS aggresome is shown (C and D). CHIP-transfected cells exhibited larger iNOS aggresomes (E). Data represent mean ± standard deviation ( n = 3). *, P

    Techniques Used: Chromatin Immunoprecipitation, Stable Transfection, Expressing, Transfection, shRNA, Incubation, Concentration Assay, Staining, Fluorescence, Microscopy, Quantitation Assay, Plasmid Preparation, Standard Deviation

    Disruption of dynein-dependent microtubular transport leads to accumulation of preaggresomal structures and prevents iNOS aggresome formation. HEK293 cells stably expressing iNOS-GFP were fixed and analyzed by fluorescence microscopy. (A) Cells were pretreated with DMSO (vehicle) or 1 μM nocodazole for 1 h, followed by either vehicle or 1 μM nocodazole plus 10 μM MG132 for 3 h. (B) Experiments were done as in panel A, except that 1 mM EHNA was used instead of nocodazole. (C) Cells were transfected with control vector or Flag-tagged p50. Twenty-four hours after transfection, cells were treated with 10 μM MG132 for 4 h, fixed, and immunostained with flag antibody.
    Figure Legend Snippet: Disruption of dynein-dependent microtubular transport leads to accumulation of preaggresomal structures and prevents iNOS aggresome formation. HEK293 cells stably expressing iNOS-GFP were fixed and analyzed by fluorescence microscopy. (A) Cells were pretreated with DMSO (vehicle) or 1 μM nocodazole for 1 h, followed by either vehicle or 1 μM nocodazole plus 10 μM MG132 for 3 h. (B) Experiments were done as in panel A, except that 1 mM EHNA was used instead of nocodazole. (C) Cells were transfected with control vector or Flag-tagged p50. Twenty-four hours after transfection, cells were treated with 10 μM MG132 for 4 h, fixed, and immunostained with flag antibody.

    Techniques Used: Stable Transfection, Expressing, Fluorescence, Microscopy, Transfection, Plasmid Preparation

    CHIP promotes HDAC6-mediated iNOS aggresome formation. (A) CHIP increases the association of iNOS with HDAC6. HEK293 cells were cotransfected for 24 h with a plasmid encoding iNOS and with a plasmid containing control vector, WT CHIP, the CHIP-H260Q mutant, or the CHIP-K30A mutant. The molar ratio of the iNOS plasmid to the coplasmid was 1:1. Coimmunoprecipitation was carried out on cell lysates using antibodies against iNOS or HDAC6. Immunoprecipitated proteins were analyzed by Western blotting for the presence of iNOS or HDAC6. Immunoprecipitation (IP) with irrelevant antibody (IκB-β) was used as a negative control. Western analysis (lower panel) of cell lysates was done prior to immunoprecipitation using equal aliquots corresponding to 2.5% of amount of proteins used for immunoprecipitation. (B) Knockdown of CHIP decreases the interaction of iNOS and HDAC6. HEK293 cells were transduced for 48 h with lentiviral vector expressing either control shRNA or CHIP shRNA and then transfected with iNOS cDNA for 24 h. Coimmunoprecipitation (IP) and Western analysis were done as described in panel A. (C) HDAC6 colocalizes with the iNOS aggresome but not with cytosolic iNOS or preaggresomal structures caused by CHIP-H260Q. (i) HEK293 cells stably expressing iNOS-GFP without aggresome formation. (ii) Cells exhibiting iNOS aggresome following MG132 treatment. (iii) Cells with iNOS aggresome promoted by overexpression of WT-CHIP. (iv) Cells with iNOS preaggresomal structures promoted by overexpression of CHIP-H260Q. (D) Knockdown of HDAC6 prevents iNOS aggresome formation. HEK293 cells stably expressing iNOS-GFP were transfected with vector encoding control shRNA or shRNA specific for HDAC6 for 3 days followed by another 3-day transfection with the same vectors to ensure knockdown of HDAC6. Western analysis of cell lysates was performed with antibodies against HDAC6, iNOS, acetylated tubulin, or tubulin (upper panel). Cells were treated with 10 μM MG132 for 10 h, fixed, and analyzed by immunofluorescence with HDAC6 antibody (middle panel). The graph shows quantitation of the percentage of cells expressing iNOS aggresome or preaggresome structures. Data represent means ± standard deviations ( n = 3). *, P
    Figure Legend Snippet: CHIP promotes HDAC6-mediated iNOS aggresome formation. (A) CHIP increases the association of iNOS with HDAC6. HEK293 cells were cotransfected for 24 h with a plasmid encoding iNOS and with a plasmid containing control vector, WT CHIP, the CHIP-H260Q mutant, or the CHIP-K30A mutant. The molar ratio of the iNOS plasmid to the coplasmid was 1:1. Coimmunoprecipitation was carried out on cell lysates using antibodies against iNOS or HDAC6. Immunoprecipitated proteins were analyzed by Western blotting for the presence of iNOS or HDAC6. Immunoprecipitation (IP) with irrelevant antibody (IκB-β) was used as a negative control. Western analysis (lower panel) of cell lysates was done prior to immunoprecipitation using equal aliquots corresponding to 2.5% of amount of proteins used for immunoprecipitation. (B) Knockdown of CHIP decreases the interaction of iNOS and HDAC6. HEK293 cells were transduced for 48 h with lentiviral vector expressing either control shRNA or CHIP shRNA and then transfected with iNOS cDNA for 24 h. Coimmunoprecipitation (IP) and Western analysis were done as described in panel A. (C) HDAC6 colocalizes with the iNOS aggresome but not with cytosolic iNOS or preaggresomal structures caused by CHIP-H260Q. (i) HEK293 cells stably expressing iNOS-GFP without aggresome formation. (ii) Cells exhibiting iNOS aggresome following MG132 treatment. (iii) Cells with iNOS aggresome promoted by overexpression of WT-CHIP. (iv) Cells with iNOS preaggresomal structures promoted by overexpression of CHIP-H260Q. (D) Knockdown of HDAC6 prevents iNOS aggresome formation. HEK293 cells stably expressing iNOS-GFP were transfected with vector encoding control shRNA or shRNA specific for HDAC6 for 3 days followed by another 3-day transfection with the same vectors to ensure knockdown of HDAC6. Western analysis of cell lysates was performed with antibodies against HDAC6, iNOS, acetylated tubulin, or tubulin (upper panel). Cells were treated with 10 μM MG132 for 10 h, fixed, and analyzed by immunofluorescence with HDAC6 antibody (middle panel). The graph shows quantitation of the percentage of cells expressing iNOS aggresome or preaggresome structures. Data represent means ± standard deviations ( n = 3). *, P

    Techniques Used: Chromatin Immunoprecipitation, Plasmid Preparation, Mutagenesis, Immunoprecipitation, Western Blot, Negative Control, Expressing, shRNA, Transfection, Stable Transfection, Over Expression, Immunofluorescence, Quantitation Assay

    Contributions of the chaperone binding domain and ubiquitin ligase domain to CHIP's role in cellular triage of iNOS. (A) Diagram and domain organization of CHIP. TPR-containing domain mediating interaction with chaperones (violet) and E3 ubiquitin ligase domain (green; U-box) are highlighted. The mutation sites of the chaperone binding-deficient mutant CHIP-K30A and the ubiquitin ligase-deficient mutant CHIP-H260Q are shown. HEK293 cells were transfected for 24 h with WT CHIP or CHIP mutants. Cells were lysed, and Western analysis was carried out to evaluate CHIP or GAPDH using His-tagged antibody or GAPDH antibody, respectively. (B) CHIP-H260Q mutant inactivates iNOS. HEK293 cells were cotransfected for 24 h with a plasmid encoding cDNA of human iNOS and with a plasmid containing control vector, WT His-CHIP, the His-CHIP-H260Q mutant, or the His-CHIP-K30A mutant. The molar ratio of the iNOS plasmid to the coplasmid was 1:5. iNOS activity was evaluated by measuring nitrite accumulation in culture medium (mean ± standard deviation; n = 6). (C) CHIP-H260Q mutant exhibits avid interaction with iNOS. HEK293 cells were cotransfected as in panel B except that the molar ratio of the iNOS plasmid to the coplasmid was 1:1. Twenty-four hours after transfection, cells were incubated with a 10 μM concentration of the proteasome inhibitor MG132 for 6 h and then lysed in NETN buffer. Cell lysates were subjected to immunoprecipitation (IP) with iNOS antibody or with His-tagged antibody. An aliquot of the immunoprecipitate was subjected to Western blotting with iNOS antibody or with His-tagged antibody. Immunoprecipitation with IκB-β antibody was used as a negative control. Western analysis (lower panel) was done on cell lysates prior to immunoprecipitation. Protein loading was done in equal aliquots corresponding to 2.5% of the amount of proteins used for immunoprecipitation. (D) CHIP-H260Q mutant does not ubiquitinate iNOS. HEK293 cells were cotransfected as in panel B. At 24 h posttransfection, cells were incubated with 10 μM MG132 for 3 h and then lysed. Cell lysates were subjected to immunoprecipitation (IP) with an iNOS antibody. An aliquot of the immunoprecipitate was subjected to Western blotting (W) with ubiquitin (Ub) antibody. (E and F) HEK293 cells were cotransfected as in panel B. At 24 (E) or 48 (F) h after transfection, cells were lysed using 1% Triton X-100 as a detergent, and lysates were divided into Triton X-100-soluble (S) and Triton X-100-insoluble (IS) fractions. Equal aliquots of cell lysates (50 μg) were evaluated by Western blotting using antibodies against iNOS, CHIP, or GAPDH. *, P
    Figure Legend Snippet: Contributions of the chaperone binding domain and ubiquitin ligase domain to CHIP's role in cellular triage of iNOS. (A) Diagram and domain organization of CHIP. TPR-containing domain mediating interaction with chaperones (violet) and E3 ubiquitin ligase domain (green; U-box) are highlighted. The mutation sites of the chaperone binding-deficient mutant CHIP-K30A and the ubiquitin ligase-deficient mutant CHIP-H260Q are shown. HEK293 cells were transfected for 24 h with WT CHIP or CHIP mutants. Cells were lysed, and Western analysis was carried out to evaluate CHIP or GAPDH using His-tagged antibody or GAPDH antibody, respectively. (B) CHIP-H260Q mutant inactivates iNOS. HEK293 cells were cotransfected for 24 h with a plasmid encoding cDNA of human iNOS and with a plasmid containing control vector, WT His-CHIP, the His-CHIP-H260Q mutant, or the His-CHIP-K30A mutant. The molar ratio of the iNOS plasmid to the coplasmid was 1:5. iNOS activity was evaluated by measuring nitrite accumulation in culture medium (mean ± standard deviation; n = 6). (C) CHIP-H260Q mutant exhibits avid interaction with iNOS. HEK293 cells were cotransfected as in panel B except that the molar ratio of the iNOS plasmid to the coplasmid was 1:1. Twenty-four hours after transfection, cells were incubated with a 10 μM concentration of the proteasome inhibitor MG132 for 6 h and then lysed in NETN buffer. Cell lysates were subjected to immunoprecipitation (IP) with iNOS antibody or with His-tagged antibody. An aliquot of the immunoprecipitate was subjected to Western blotting with iNOS antibody or with His-tagged antibody. Immunoprecipitation with IκB-β antibody was used as a negative control. Western analysis (lower panel) was done on cell lysates prior to immunoprecipitation. Protein loading was done in equal aliquots corresponding to 2.5% of the amount of proteins used for immunoprecipitation. (D) CHIP-H260Q mutant does not ubiquitinate iNOS. HEK293 cells were cotransfected as in panel B. At 24 h posttransfection, cells were incubated with 10 μM MG132 for 3 h and then lysed. Cell lysates were subjected to immunoprecipitation (IP) with an iNOS antibody. An aliquot of the immunoprecipitate was subjected to Western blotting (W) with ubiquitin (Ub) antibody. (E and F) HEK293 cells were cotransfected as in panel B. At 24 (E) or 48 (F) h after transfection, cells were lysed using 1% Triton X-100 as a detergent, and lysates were divided into Triton X-100-soluble (S) and Triton X-100-insoluble (IS) fractions. Equal aliquots of cell lysates (50 μg) were evaluated by Western blotting using antibodies against iNOS, CHIP, or GAPDH. *, P

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Mutagenesis, Transfection, Western Blot, Plasmid Preparation, Activity Assay, Standard Deviation, Incubation, Concentration Assay, Immunoprecipitation, Negative Control

    Ubiquitin ligase activity of CHIP is required for aggresome formation. HEK293 cells were cotransfected with a plasmid encoding the cDNA of human iNOS and with either a plasmid encoding WT CHIP (row i) or CHIP-H260Q. The molar ratio of the iNOS plasmid to the coplasmid was 1:5. Forty-eight hours after transfection, cells were fixed and immunostained with antibodies against His-tagged CHIP, ubiquitin, or γ-tubulin. Graphs show quantitation of the percentage of cells expressing iNOS aggresome and preaggresome structures. Data represent means ± standard deviations ( n = 3). *, P
    Figure Legend Snippet: Ubiquitin ligase activity of CHIP is required for aggresome formation. HEK293 cells were cotransfected with a plasmid encoding the cDNA of human iNOS and with either a plasmid encoding WT CHIP (row i) or CHIP-H260Q. The molar ratio of the iNOS plasmid to the coplasmid was 1:5. Forty-eight hours after transfection, cells were fixed and immunostained with antibodies against His-tagged CHIP, ubiquitin, or γ-tubulin. Graphs show quantitation of the percentage of cells expressing iNOS aggresome and preaggresome structures. Data represent means ± standard deviations ( n = 3). *, P

    Techniques Used: Activity Assay, Chromatin Immunoprecipitation, Plasmid Preparation, Transfection, Quantitation Assay, Expressing

    3) Product Images from "The physiologic aggresome mediates cellular inactivation of iNOS"

    Article Title: The physiologic aggresome mediates cellular inactivation of iNOS

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0810968106

    Cells with iNOS aggresome produce less NO. HEK293 cells were transfected with iNOS-GFP. ( A ) Twenty-four hours following transfection, cells were sorted according to GFP intensity. Cells not likely to be transfected and thus exhibiting lowest fluorescence intensity were not further used. Cells expressing moderate and the top 10% of fluorescence intensity were termed X 1 and X 2 , respectively. ( B ) Sorted cells were cultured for 24 h and then subjected to live cell imaging in the presence of DAR. Representative fluorescence microscopy images of sorted cells show marked enrichment of aggresome-containing cells in the X 2 population. ( C ) Nitrite accumulation in tissue culture media of sorted cells. *, P
    Figure Legend Snippet: Cells with iNOS aggresome produce less NO. HEK293 cells were transfected with iNOS-GFP. ( A ) Twenty-four hours following transfection, cells were sorted according to GFP intensity. Cells not likely to be transfected and thus exhibiting lowest fluorescence intensity were not further used. Cells expressing moderate and the top 10% of fluorescence intensity were termed X 1 and X 2 , respectively. ( B ) Sorted cells were cultured for 24 h and then subjected to live cell imaging in the presence of DAR. Representative fluorescence microscopy images of sorted cells show marked enrichment of aggresome-containing cells in the X 2 population. ( C ) Nitrite accumulation in tissue culture media of sorted cells. *, P

    Techniques Used: Transfection, Fluorescence, Expressing, Cell Culture, Live Cell Imaging, Microscopy

    4) Product Images from "Characterization of key residues in the subdomain encoded by exons 8 and 9 of human inducible nitric oxide synthase: A critical role for Asp-280 in substrate binding and subunit interactions"

    Article Title: Characterization of key residues in the subdomain encoded by exons 8 and 9 of human inducible nitric oxide synthase: A critical role for Asp-280 in substrate binding and subunit interactions

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.181251298

    NO production of wild-type human iNOS and iNOS mutants. HEK 293 cells were transfected with plasmids containing vector only, iNOS, or iNOS mutant. ( A ) iNOS mutants with Phe replacing Trp-260 (W260F), Gln replacing Asn-261 (N261Q), or Phe replacing Tyr-267 (Y267F). ( B ) iNOS mutants with Glu (E), Asn (N), Ser (S), or Ala (A) replacing Asp-280 (D280). Nitrite accumulation in the culture medium was assessed 72 h ( A ) or 36 h ( B ) after transfection. Data are means ± SD of three experiments. Invisible error bars are too small to be drawn.
    Figure Legend Snippet: NO production of wild-type human iNOS and iNOS mutants. HEK 293 cells were transfected with plasmids containing vector only, iNOS, or iNOS mutant. ( A ) iNOS mutants with Phe replacing Trp-260 (W260F), Gln replacing Asn-261 (N261Q), or Phe replacing Tyr-267 (Y267F). ( B ) iNOS mutants with Glu (E), Asn (N), Ser (S), or Ala (A) replacing Asp-280 (D280). Nitrite accumulation in the culture medium was assessed 72 h ( A ) or 36 h ( B ) after transfection. Data are means ± SD of three experiments. Invisible error bars are too small to be drawn.

    Techniques Used: Transfection, Plasmid Preparation, Mutagenesis

    Related Articles

    Transfection:

    Article Title: Mutations in DCHS1 Cause Mitral Valve Prolapse
    Article Snippet: .. These constructs were expressed in mycoplasma-free HEK293 cells (ATCC) using cationic lipid-mediated transient transfection (Lipofectamine LTX, Invitrogen). .. Protein expression of transfected HEK cells was measured by quantifying Western blots using an antibody to the V5 epitope tag (Invitrogen).

    Article Title: Characterization of key residues in the subdomain encoded by exons 8 and 9 of human inducible nitric oxide synthase: A critical role for Asp-280 in substrate binding and subunit interactions
    Article Snippet: .. Cationic lipid-mediated transient transfection was performed by using the desired DNA, Lipofectamine, and a transfection-enhancing Plus reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions. ..

    Article Title: Src Kinase is a Novel Therapeutic Target in Lymphangio-leiomyomatosis
    Article Snippet: .. Cationic lipid-mediated transient transfection of plasmids was done using Lipofectamine 2000 (Invitrogen). ..

    Article Title: A Critical Role for CHIP in the Aggresome Pathway ▿A Critical Role for CHIP in the Aggresome Pathway ▿ †
    Article Snippet: .. Cationic lipid-mediated transient transfection of plasmids was done using Lipofectamine 2000 (Invitrogen) into 70 to 80% confluent cells. ..

    Article Title: Ubiquitination of inducible nitric oxide synthase is required for its degradation
    Article Snippet: .. Cationic lipid-mediated transient transfection was done by using LipofectAMINE 2000 (Invitrogen) following the manufacturer's instructions. .. Stable cell lines of HEK293, ts20, and E36 cells, expressing iNOS, were produced by using transfection of iNOS cDNA followed by positive colony selection using G418 (Invitrogen) at concentration of 600 μg/ml (HEK293) or 450 μg/ml (ts20 and E36) ( , ).

    Article Title: Cloning and characterization of human inducible nitric oxide synthase splice variants: A domain, encoded by exons 8 and 9, is critical for dimerization
    Article Snippet: .. Cationic lipid-mediated transient transfection was done in 100-mm diameter tissue culture plates by using 8 μg of the desired DNA and 48 μl of Lipofectamine (Life Technologies) following the manufacturer’s instructions. ..

    Article Title: The physiologic aggresome mediates cellular inactivation of iNOS
    Article Snippet: .. Cationic lipid-mediated transient transfection was done using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. .. All experiments on HEK-293 cells were done 24 h after transfection with iNOS-GFP fusion protein.

    Construct:

    Article Title: Mutations in DCHS1 Cause Mitral Valve Prolapse
    Article Snippet: .. These constructs were expressed in mycoplasma-free HEK293 cells (ATCC) using cationic lipid-mediated transient transfection (Lipofectamine LTX, Invitrogen). .. Protein expression of transfected HEK cells was measured by quantifying Western blots using an antibody to the V5 epitope tag (Invitrogen).

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    Thermo Fisher cationic lipid transfections
    SPC25 knockdown inhibits the proliferation of PC-3 cells. (A) Expression of SPC25 mRNA following <t>transfection</t> with the indicated shRNAs in PC-3 cells. (B) An MTT assay demonstrated that knockdown of SPC25 inhibited cell proliferation in PC-3 cells. (C) The Celigo ® system revealed that knockdown of SPC25 inhibited cell proliferation in PC-3 cells. (D) Cell number of shCtrl or shSPC25 group in each day was calculated using the Celigo ® system. P
    Cationic Lipid Transfections, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Price from $9.99 to $1999.99
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    85
    Thermo Fisher lipid based transient transfection
    Measurements of PKA activation using the AKAR FRET biosensor for RI α −/− cells expressing the various RI α constructs. (A, B) Examples of RI α −/− cells expressing the indicated constructs stimulated with 8-CPT-cAMP or 8-CPT-cGMP at t = 0 (minutes). Cell images as shown were pseudocolored according to the calculated YFP/CFP FRET ratios, with the range displayed indicated on the color bars at right. Left panels show the tracings of the FRET ratios as % change over time. Scale bars: 10 μ M. (C) Box plots of the data showing all 8-CPT-cAMP and 8-CPT-cGMP cell stimulations plotted as % change of the normalized FRET ratios. Total number of individual cells analyzed for each condition is between 20 and 26, encompassing multiple independently conducted stimulations and <t>transfections.</t>
    Lipid Based Transient Transfection, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SPC25 knockdown inhibits the proliferation of PC-3 cells. (A) Expression of SPC25 mRNA following transfection with the indicated shRNAs in PC-3 cells. (B) An MTT assay demonstrated that knockdown of SPC25 inhibited cell proliferation in PC-3 cells. (C) The Celigo ® system revealed that knockdown of SPC25 inhibited cell proliferation in PC-3 cells. (D) Cell number of shCtrl or shSPC25 group in each day was calculated using the Celigo ® system. P

    Journal: Oncology Letters

    Article Title: Knockdown of spindle pole body component 25 homolog inhibits cell proliferation and cycle progression in prostate cancer

    doi: 10.3892/ol.2018.8003

    Figure Lengend Snippet: SPC25 knockdown inhibits the proliferation of PC-3 cells. (A) Expression of SPC25 mRNA following transfection with the indicated shRNAs in PC-3 cells. (B) An MTT assay demonstrated that knockdown of SPC25 inhibited cell proliferation in PC-3 cells. (C) The Celigo ® system revealed that knockdown of SPC25 inhibited cell proliferation in PC-3 cells. (D) Cell number of shCtrl or shSPC25 group in each day was calculated using the Celigo ® system. P

    Article Snippet: Opti-modified Eagle's medium (Opti-MEM) which was ideal for use during cationic lipid transfections especially Lipofectamine 2000 transfection reagents was purchased from Thermo Fisher Scientific, Inc (cat. no. 31985062).

    Techniques: Expressing, Transfection, MTT Assay

    MCM10 knockdown inhibits its expression levels in DU145 and PC‐3 cells. A‐C, Real‐time reverse transcription PCR analysis showed expression of MCM10 mRNA was reduced after transfection with the indicated shRNAs in DU145 (A) PC‐3 (B) and LNCaP (C) cells. D‐F, Western blot analysis showed protein expression of MCM10 was reduced after transfection with the indicated shRNAs in DU145 (D), PC‐3 (E), and LNCaP (F) cells. Data are represented as the mean ± SD (* P

    Journal: The Prostate

    Article Title: Overexpression of MCM10 promotes cell proliferation and predicts poor prognosis in prostate cancer. Overexpression of MCM10 promotes cell proliferation and predicts poor prognosis in prostate cancer

    doi: 10.1002/pros.23703

    Figure Lengend Snippet: MCM10 knockdown inhibits its expression levels in DU145 and PC‐3 cells. A‐C, Real‐time reverse transcription PCR analysis showed expression of MCM10 mRNA was reduced after transfection with the indicated shRNAs in DU145 (A) PC‐3 (B) and LNCaP (C) cells. D‐F, Western blot analysis showed protein expression of MCM10 was reduced after transfection with the indicated shRNAs in DU145 (D), PC‐3 (E), and LNCaP (F) cells. Data are represented as the mean ± SD (* P

    Article Snippet: Opti‐modified Eagle's medium (Opti‐MEM) which was ideal for use during cationic lipid transfections especially Lipofectamine™ transfection reagents was purchased from Thermo Fisher Scientific, Inc. (Catalog number: 31985062).

    Techniques: Expressing, Polymerase Chain Reaction, Transfection, Western Blot

    MCM10 knockdown inhibits its expression levels in DU145 and PC‐3 cells. A‐C, Real‐time reverse transcription PCR analysis showed expression of MCM10 mRNA was reduced after transfection with the indicated shRNAs in DU145 (A) PC‐3 (B) and LNCaP (C) cells. D‐F, Western blot analysis showed protein expression of MCM10 was reduced after transfection with the indicated shRNAs in DU145 (D), PC‐3 (E), and LNCaP (F) cells. Data are represented as the mean ± SD (* P

    Journal: The Prostate

    Article Title: Overexpression of MCM10 promotes cell proliferation and predicts poor prognosis in prostate cancer. Overexpression of MCM10 promotes cell proliferation and predicts poor prognosis in prostate cancer

    doi: 10.1002/pros.23703

    Figure Lengend Snippet: MCM10 knockdown inhibits its expression levels in DU145 and PC‐3 cells. A‐C, Real‐time reverse transcription PCR analysis showed expression of MCM10 mRNA was reduced after transfection with the indicated shRNAs in DU145 (A) PC‐3 (B) and LNCaP (C) cells. D‐F, Western blot analysis showed protein expression of MCM10 was reduced after transfection with the indicated shRNAs in DU145 (D), PC‐3 (E), and LNCaP (F) cells. Data are represented as the mean ± SD (* P

    Article Snippet: Opti‐modified Eagle's medium (Opti‐MEM) which was ideal for use during cationic lipid transfections especially Lipofectamine™ transfection reagents was purchased from Thermo Fisher Scientific, Inc. (Catalog number: 31985062).

    Techniques: Expressing, Polymerase Chain Reaction, Transfection, Western Blot

    Measurements of PKA activation using the AKAR FRET biosensor for RI α −/− cells expressing the various RI α constructs. (A, B) Examples of RI α −/− cells expressing the indicated constructs stimulated with 8-CPT-cAMP or 8-CPT-cGMP at t = 0 (minutes). Cell images as shown were pseudocolored according to the calculated YFP/CFP FRET ratios, with the range displayed indicated on the color bars at right. Left panels show the tracings of the FRET ratios as % change over time. Scale bars: 10 μ M. (C) Box plots of the data showing all 8-CPT-cAMP and 8-CPT-cGMP cell stimulations plotted as % change of the normalized FRET ratios. Total number of individual cells analyzed for each condition is between 20 and 26, encompassing multiple independently conducted stimulations and transfections.

    Journal: ACS chemical biology

    Article Title: Switching Cyclic Nucleotide-Selective Activation of Cyclic Adenosine Monophosphate-Dependent Protein Kinase Holoenzyme Reveals Distinct Roles of Tandem Cyclic Nucleotide-Binding Domains

    doi: 10.1021/acschembio.7b00732

    Figure Lengend Snippet: Measurements of PKA activation using the AKAR FRET biosensor for RI α −/− cells expressing the various RI α constructs. (A, B) Examples of RI α −/− cells expressing the indicated constructs stimulated with 8-CPT-cAMP or 8-CPT-cGMP at t = 0 (minutes). Cell images as shown were pseudocolored according to the calculated YFP/CFP FRET ratios, with the range displayed indicated on the color bars at right. Left panels show the tracings of the FRET ratios as % change over time. Scale bars: 10 μ M. (C) Box plots of the data showing all 8-CPT-cAMP and 8-CPT-cGMP cell stimulations plotted as % change of the normalized FRET ratios. Total number of individual cells analyzed for each condition is between 20 and 26, encompassing multiple independently conducted stimulations and transfections.

    Article Snippet: In additional experiments, lipid-based transient transfection was carried out using Lipofectamine 2000 (Thermo-Fisher).

    Techniques: Activation Assay, Expressing, Construct, Cycling Probe Technology, Transfection