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Mirus Bio lipid based transfection reagent
RNAi is active in WNV infected cells . RNAi of influenza M2 gene in cells that replicate WNV RNA. Huh7.5, Huh7.5-Rep, and WNV infected Huh7.5 cells (8 hours post infection) were transfected with pCM2 and Cap or M2 siRNA as described in Materials and Methods. 24 hours later cells were processed by flow cytometry for M2 expression using antibody 14C2. The percentage of M2 inhibition was calculated according to the following formula: (1 – (% M2 expression of siRNA-transfected cells / % M2 expression in cells transfected with <t>transfection</t> vehicle only) × 100). The results are an average of three independent experiments and error bars indicate standard error of the mean.
Lipid Based Transfection Reagent, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/lipid based transfection reagent/product/Mirus Bio
Average 88 stars, based on 6 article reviews
Price from $9.99 to $1999.99
lipid based transfection reagent - by Bioz Stars, 2020-09
88/100 stars

Images

1) Product Images from "Actively replicating West Nile virus is resistant to cytoplasmic delivery of siRNA"

Article Title: Actively replicating West Nile virus is resistant to cytoplasmic delivery of siRNA

Journal: Virology Journal

doi: 10.1186/1743-422X-2-53

RNAi is active in WNV infected cells . RNAi of influenza M2 gene in cells that replicate WNV RNA. Huh7.5, Huh7.5-Rep, and WNV infected Huh7.5 cells (8 hours post infection) were transfected with pCM2 and Cap or M2 siRNA as described in Materials and Methods. 24 hours later cells were processed by flow cytometry for M2 expression using antibody 14C2. The percentage of M2 inhibition was calculated according to the following formula: (1 – (% M2 expression of siRNA-transfected cells / % M2 expression in cells transfected with transfection vehicle only) × 100). The results are an average of three independent experiments and error bars indicate standard error of the mean.
Figure Legend Snippet: RNAi is active in WNV infected cells . RNAi of influenza M2 gene in cells that replicate WNV RNA. Huh7.5, Huh7.5-Rep, and WNV infected Huh7.5 cells (8 hours post infection) were transfected with pCM2 and Cap or M2 siRNA as described in Materials and Methods. 24 hours later cells were processed by flow cytometry for M2 expression using antibody 14C2. The percentage of M2 inhibition was calculated according to the following formula: (1 – (% M2 expression of siRNA-transfected cells / % M2 expression in cells transfected with transfection vehicle only) × 100). The results are an average of three independent experiments and error bars indicate standard error of the mean.

Techniques Used: Infection, Transfection, Flow Cytometry, Cytometry, Expressing, Inhibition

2) Product Images from "In vivo trafficking and immunostimulatory potential of an intranasally-administered primary dendritic cell-based vaccine"

Article Title: In vivo trafficking and immunostimulatory potential of an intranasally-administered primary dendritic cell-based vaccine

Journal: BMC Immunology

doi: 10.1186/1471-2172-11-60

Immunophenotype of primary DCs derived from (A) BALB/c and (B) C57BL6 mice, does not change after nonviral genetic transfection . The 2dDC were transfected with pVR1012-Ag2/PRA plasmid DNA. After 24 h of transfection, the cells were stained with fluorochrome-conjugated antibodies specific to MHC class II, CD11c, CD40, CD80, CD86 cell-surface antigens. The cell debris was gated-out and histogram charts were plotted. The filled histogram charts are of cells stained with isotype-control antibody. The mean fluorescent intensity (MFI) values were determined for the cells under the bar (⊓) region. The values within the charts indicate the MFI values for the nontransfected (NT, black line) and transfected (T, green line) cells stained with antibodies specific to cell-surface antigens. The results are from one representative experiment conducted thrice.
Figure Legend Snippet: Immunophenotype of primary DCs derived from (A) BALB/c and (B) C57BL6 mice, does not change after nonviral genetic transfection . The 2dDC were transfected with pVR1012-Ag2/PRA plasmid DNA. After 24 h of transfection, the cells were stained with fluorochrome-conjugated antibodies specific to MHC class II, CD11c, CD40, CD80, CD86 cell-surface antigens. The cell debris was gated-out and histogram charts were plotted. The filled histogram charts are of cells stained with isotype-control antibody. The mean fluorescent intensity (MFI) values were determined for the cells under the bar (⊓) region. The values within the charts indicate the MFI values for the nontransfected (NT, black line) and transfected (T, green line) cells stained with antibodies specific to cell-surface antigens. The results are from one representative experiment conducted thrice.

Techniques Used: Derivative Assay, Mouse Assay, Transfection, Plasmid Preparation, Staining

Cytokine (TNF-α, IL-12p70, IL-6 and IL10) levels (pg/ml) in cell-free supernatants of JAWS II and BALB/c mice derived primary DCs after 24 h of genetic transfection with pVR1012-Ag2/PRA plasmid DNA . Results are from two experiments performed in triplicate.
Figure Legend Snippet: Cytokine (TNF-α, IL-12p70, IL-6 and IL10) levels (pg/ml) in cell-free supernatants of JAWS II and BALB/c mice derived primary DCs after 24 h of genetic transfection with pVR1012-Ag2/PRA plasmid DNA . Results are from two experiments performed in triplicate.

Techniques Used: Mouse Assay, Derivative Assay, Transfection, Plasmid Preparation

(A) Photomicrographs of primary DCs after nonviral genetic transfection . The top panel is a fluorescence photomicrograph of primary DCs showing GFP-expression (in green) after 24 h of transfection. The bottom panel is a photomicrograph of cells in the same field under bright-field filter. (B) Flow cytometric histograms of transfected primary DCs showing the effect of transfection on their viability. The transfected (dotted line) and nontransfected (solid line) primary DCs after two days of culture were stained with propidium iodide. The values indicate % viable nontransfected (NT) and transfected (T) cells under the marked region. The results are from one representative experiment conducted more than three times.
Figure Legend Snippet: (A) Photomicrographs of primary DCs after nonviral genetic transfection . The top panel is a fluorescence photomicrograph of primary DCs showing GFP-expression (in green) after 24 h of transfection. The bottom panel is a photomicrograph of cells in the same field under bright-field filter. (B) Flow cytometric histograms of transfected primary DCs showing the effect of transfection on their viability. The transfected (dotted line) and nontransfected (solid line) primary DCs after two days of culture were stained with propidium iodide. The values indicate % viable nontransfected (NT) and transfected (T) cells under the marked region. The results are from one representative experiment conducted more than three times.

Techniques Used: Transfection, Fluorescence, Expressing, Flow Cytometry, Staining

The expression of transgenes-encoded Coccidioides -Ag2/PRA and TK antigens by genetically-transfected primary DCs . The cell-lysates were prepared after 24 h of transfection, and dot-immunoblotting was performed with Ag2/PRA and TK-specific antibodies. Left Panel : An Ag2/PRA dot-immunoblot of 5 μg cell-lysate protein from nontransfected (A), transfected primary DCs (B), and 0.2 μg recombinant Ag2/PRA protein (C). Right panel : A TK Dot-immunoblot of 1 μg cell-lysate protein from nontransfected (A) and transfected DCs (B). Results are from one experiment representative of more than three experiments.
Figure Legend Snippet: The expression of transgenes-encoded Coccidioides -Ag2/PRA and TK antigens by genetically-transfected primary DCs . The cell-lysates were prepared after 24 h of transfection, and dot-immunoblotting was performed with Ag2/PRA and TK-specific antibodies. Left Panel : An Ag2/PRA dot-immunoblot of 5 μg cell-lysate protein from nontransfected (A), transfected primary DCs (B), and 0.2 μg recombinant Ag2/PRA protein (C). Right panel : A TK Dot-immunoblot of 1 μg cell-lysate protein from nontransfected (A) and transfected DCs (B). Results are from one experiment representative of more than three experiments.

Techniques Used: Expressing, Transfection, Recombinant

3) Product Images from "TorsinA participates in endoplasmic reticulum-associated degradation"

Article Title: TorsinA participates in endoplasmic reticulum-associated degradation

Journal: Nature communications

doi: 10.1038/ncomms1383

Downregulation of torsinA inhibits retro-translocation of the cholera toxin A1-chain (a) Immunoblot showing > 95% suppression of torsinA expression in COS cells treated with siRNA 1958 or 1963 for torsinA, with scr. siRNA as control. (b c): (b) After siRNA transfection cells were intoxicated with wild-type CT for 45 min at 37°C and then fractionated into cytosolic and membrane components which were resolved by SDS-PAGE. Immunoblotting was carried out with antibodies to CTA1, CTB, BiP, β-actin and Hsp90. (c) Bar graph showing relative band densities of CTA1 in the cytosol normalized to Hsp90 in the cytosol, and CTA1 in the membrane fraction normalized to BiP. Results are shown as the mean of 3 experiments ± S.D.; * represents p
Figure Legend Snippet: Downregulation of torsinA inhibits retro-translocation of the cholera toxin A1-chain (a) Immunoblot showing > 95% suppression of torsinA expression in COS cells treated with siRNA 1958 or 1963 for torsinA, with scr. siRNA as control. (b c): (b) After siRNA transfection cells were intoxicated with wild-type CT for 45 min at 37°C and then fractionated into cytosolic and membrane components which were resolved by SDS-PAGE. Immunoblotting was carried out with antibodies to CTA1, CTB, BiP, β-actin and Hsp90. (c) Bar graph showing relative band densities of CTA1 in the cytosol normalized to Hsp90 in the cytosol, and CTA1 in the membrane fraction normalized to BiP. Results are shown as the mean of 3 experiments ± S.D.; * represents p

Techniques Used: Translocation Assay, Expressing, Transfection, SDS Page, CtB Assay

Involvement of torsinA in degradation of GFP-CFTRΔF508 (a) 293T cells transfected with an expression cassette for GFP-CFTRΔF508 and 48 h later cell lysates were immunoprecipitated with IgG or antibodies to CFTR or torsinA, followed by SDS-PAGE and immunoblotting for GFP and torsinA. (b c): (b) 293T cells were co-transfected with expression cassettes for GFP-CFTRΔF508 or torsinA or torsinAΔE or torsinAΔC or with the control cassette, pcDNA 3.1. Twenty-four h later samples were treated or not with MG132 for 16 h. Seventy-two h post-transfection, cell lysates were processed by SDS-PAGE and gels immunoblotted with antibodies to GFP, torsinA and β-actin. Representative immunoblots are shown with short (1 min) and long (3 min) exposures for GFP, and with 3 min exposure for torsinA and β-actin. (c) GFP-CFTRΔF508 band densities were normalized to those of torsinA and β-actin and represented as the mean of 3 experiments ± S.D. For statistical comparisons, * denotes the comparisons of control to torsinA or torsinAΔE or torsinAΔC transfections (p,
Figure Legend Snippet: Involvement of torsinA in degradation of GFP-CFTRΔF508 (a) 293T cells transfected with an expression cassette for GFP-CFTRΔF508 and 48 h later cell lysates were immunoprecipitated with IgG or antibodies to CFTR or torsinA, followed by SDS-PAGE and immunoblotting for GFP and torsinA. (b c): (b) 293T cells were co-transfected with expression cassettes for GFP-CFTRΔF508 or torsinA or torsinAΔE or torsinAΔC or with the control cassette, pcDNA 3.1. Twenty-four h later samples were treated or not with MG132 for 16 h. Seventy-two h post-transfection, cell lysates were processed by SDS-PAGE and gels immunoblotted with antibodies to GFP, torsinA and β-actin. Representative immunoblots are shown with short (1 min) and long (3 min) exposures for GFP, and with 3 min exposure for torsinA and β-actin. (c) GFP-CFTRΔF508 band densities were normalized to those of torsinA and β-actin and represented as the mean of 3 experiments ± S.D. For statistical comparisons, * denotes the comparisons of control to torsinA or torsinAΔE or torsinAΔC transfections (p,

Techniques Used: Transfection, Expressing, Immunoprecipitation, SDS Page, Western Blot

Related Articles

Transfection:

Article Title: In vivo trafficking and immunostimulatory potential of an intranasally-administered primary dendritic cell-based vaccine
Article Snippet: .. A non-viral, lipid-based transfection reagent: TransIT-TKO (Mirus Bio, WI) was used. .. The transfection reagent-DNA complex (4 μl:2 μg of each plasmid DNA) was prepared in DMEM medium by adding plasmid DNAs (2 μg pHYG-EGFP or 2 μg pVR1012-VP22 vector plasmid DNA or 2 μg pVR1012-Ag2/PRA-cDNA ± 2 μg pVR1012-TK) to the TransIT-TKO reagent.

Article Title: Phosphoproteomics Identified an NS5A Phosphorylation Site Involved in Hepatitis C Virus Replication *
Article Snippet: .. One μl of D4476 was dissolved in 3 μl of lipid-based transfection reagent (Mirus Bio, TransIT-LT1, catalogue no. MIR 2304) plus 6 μl of serum-free DMEM before being added to the cells. .. The cells were incubated with the inhibitor for 24 h before immunoblotting.

Article Title: TorsinA participates in endoplasmic reticulum-associated degradation
Article Snippet: .. After 24 h the cells were transfected with expression cassettes for GFP-CFTRΔF508 or pcDNA (negative control) and pcDNA-torsinAwt using a lipid-based transfection reagent: TransIT-2020, following the protocol of the manufacturer (Mirus Bio, WI). ..

Article Title: Actively replicating West Nile virus is resistant to cytoplasmic delivery of siRNA
Article Snippet: .. Because the lipid-based transfection reagent targets nucleic acids to the cytoplasm (Mirus Corp, personal communication), the presence of additional membranes between the viral RNA and the cytoplasm could prevent the siRNA from reaching the actively replicating WNV RNA complex. .. Electroporation, in contrast, transiently opens pores in cellular membranes [ ] allowing nucleic acids and cytoplasmic components to cross membranous structures such as the nucleus, endoplasmic reticulum, and potentially the membranous vesicles induced by WNV.

Negative Control:

Article Title: TorsinA participates in endoplasmic reticulum-associated degradation
Article Snippet: .. After 24 h the cells were transfected with expression cassettes for GFP-CFTRΔF508 or pcDNA (negative control) and pcDNA-torsinAwt using a lipid-based transfection reagent: TransIT-2020, following the protocol of the manufacturer (Mirus Bio, WI). ..

Expressing:

Article Title: TorsinA participates in endoplasmic reticulum-associated degradation
Article Snippet: .. After 24 h the cells were transfected with expression cassettes for GFP-CFTRΔF508 or pcDNA (negative control) and pcDNA-torsinAwt using a lipid-based transfection reagent: TransIT-2020, following the protocol of the manufacturer (Mirus Bio, WI). ..

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    Mirus Bio lipid based transfection reagent
    RNAi is active in WNV infected cells . RNAi of influenza M2 gene in cells that replicate WNV RNA. Huh7.5, Huh7.5-Rep, and WNV infected Huh7.5 cells (8 hours post infection) were transfected with pCM2 and Cap or M2 siRNA as described in Materials and Methods. 24 hours later cells were processed by flow cytometry for M2 expression using antibody 14C2. The percentage of M2 inhibition was calculated according to the following formula: (1 – (% M2 expression of siRNA-transfected cells / % M2 expression in cells transfected with <t>transfection</t> vehicle only) × 100). The results are an average of three independent experiments and error bars indicate standard error of the mean.
    Lipid Based Transfection Reagent, supplied by Mirus Bio, used in various techniques. Bioz Stars score: 88/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lipid based transfection reagent/product/Mirus Bio
    Average 88 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    lipid based transfection reagent - by Bioz Stars, 2020-09
    88/100 stars
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    RNAi is active in WNV infected cells . RNAi of influenza M2 gene in cells that replicate WNV RNA. Huh7.5, Huh7.5-Rep, and WNV infected Huh7.5 cells (8 hours post infection) were transfected with pCM2 and Cap or M2 siRNA as described in Materials and Methods. 24 hours later cells were processed by flow cytometry for M2 expression using antibody 14C2. The percentage of M2 inhibition was calculated according to the following formula: (1 – (% M2 expression of siRNA-transfected cells / % M2 expression in cells transfected with transfection vehicle only) × 100). The results are an average of three independent experiments and error bars indicate standard error of the mean.

    Journal: Virology Journal

    Article Title: Actively replicating West Nile virus is resistant to cytoplasmic delivery of siRNA

    doi: 10.1186/1743-422X-2-53

    Figure Lengend Snippet: RNAi is active in WNV infected cells . RNAi of influenza M2 gene in cells that replicate WNV RNA. Huh7.5, Huh7.5-Rep, and WNV infected Huh7.5 cells (8 hours post infection) were transfected with pCM2 and Cap or M2 siRNA as described in Materials and Methods. 24 hours later cells were processed by flow cytometry for M2 expression using antibody 14C2. The percentage of M2 inhibition was calculated according to the following formula: (1 – (% M2 expression of siRNA-transfected cells / % M2 expression in cells transfected with transfection vehicle only) × 100). The results are an average of three independent experiments and error bars indicate standard error of the mean.

    Article Snippet: Because the lipid-based transfection reagent targets nucleic acids to the cytoplasm (Mirus Corp, personal communication), the presence of additional membranes between the viral RNA and the cytoplasm could prevent the siRNA from reaching the actively replicating WNV RNA complex.

    Techniques: Infection, Transfection, Flow Cytometry, Cytometry, Expressing, Inhibition

    Immunophenotype of primary DCs derived from (A) BALB/c and (B) C57BL6 mice, does not change after nonviral genetic transfection . The 2dDC were transfected with pVR1012-Ag2/PRA plasmid DNA. After 24 h of transfection, the cells were stained with fluorochrome-conjugated antibodies specific to MHC class II, CD11c, CD40, CD80, CD86 cell-surface antigens. The cell debris was gated-out and histogram charts were plotted. The filled histogram charts are of cells stained with isotype-control antibody. The mean fluorescent intensity (MFI) values were determined for the cells under the bar (⊓) region. The values within the charts indicate the MFI values for the nontransfected (NT, black line) and transfected (T, green line) cells stained with antibodies specific to cell-surface antigens. The results are from one representative experiment conducted thrice.

    Journal: BMC Immunology

    Article Title: In vivo trafficking and immunostimulatory potential of an intranasally-administered primary dendritic cell-based vaccine

    doi: 10.1186/1471-2172-11-60

    Figure Lengend Snippet: Immunophenotype of primary DCs derived from (A) BALB/c and (B) C57BL6 mice, does not change after nonviral genetic transfection . The 2dDC were transfected with pVR1012-Ag2/PRA plasmid DNA. After 24 h of transfection, the cells were stained with fluorochrome-conjugated antibodies specific to MHC class II, CD11c, CD40, CD80, CD86 cell-surface antigens. The cell debris was gated-out and histogram charts were plotted. The filled histogram charts are of cells stained with isotype-control antibody. The mean fluorescent intensity (MFI) values were determined for the cells under the bar (⊓) region. The values within the charts indicate the MFI values for the nontransfected (NT, black line) and transfected (T, green line) cells stained with antibodies specific to cell-surface antigens. The results are from one representative experiment conducted thrice.

    Article Snippet: A non-viral, lipid-based transfection reagent: TransIT-TKO (Mirus Bio, WI) was used.

    Techniques: Derivative Assay, Mouse Assay, Transfection, Plasmid Preparation, Staining

    Cytokine (TNF-α, IL-12p70, IL-6 and IL10) levels (pg/ml) in cell-free supernatants of JAWS II and BALB/c mice derived primary DCs after 24 h of genetic transfection with pVR1012-Ag2/PRA plasmid DNA . Results are from two experiments performed in triplicate.

    Journal: BMC Immunology

    Article Title: In vivo trafficking and immunostimulatory potential of an intranasally-administered primary dendritic cell-based vaccine

    doi: 10.1186/1471-2172-11-60

    Figure Lengend Snippet: Cytokine (TNF-α, IL-12p70, IL-6 and IL10) levels (pg/ml) in cell-free supernatants of JAWS II and BALB/c mice derived primary DCs after 24 h of genetic transfection with pVR1012-Ag2/PRA plasmid DNA . Results are from two experiments performed in triplicate.

    Article Snippet: A non-viral, lipid-based transfection reagent: TransIT-TKO (Mirus Bio, WI) was used.

    Techniques: Mouse Assay, Derivative Assay, Transfection, Plasmid Preparation

    (A) Photomicrographs of primary DCs after nonviral genetic transfection . The top panel is a fluorescence photomicrograph of primary DCs showing GFP-expression (in green) after 24 h of transfection. The bottom panel is a photomicrograph of cells in the same field under bright-field filter. (B) Flow cytometric histograms of transfected primary DCs showing the effect of transfection on their viability. The transfected (dotted line) and nontransfected (solid line) primary DCs after two days of culture were stained with propidium iodide. The values indicate % viable nontransfected (NT) and transfected (T) cells under the marked region. The results are from one representative experiment conducted more than three times.

    Journal: BMC Immunology

    Article Title: In vivo trafficking and immunostimulatory potential of an intranasally-administered primary dendritic cell-based vaccine

    doi: 10.1186/1471-2172-11-60

    Figure Lengend Snippet: (A) Photomicrographs of primary DCs after nonviral genetic transfection . The top panel is a fluorescence photomicrograph of primary DCs showing GFP-expression (in green) after 24 h of transfection. The bottom panel is a photomicrograph of cells in the same field under bright-field filter. (B) Flow cytometric histograms of transfected primary DCs showing the effect of transfection on their viability. The transfected (dotted line) and nontransfected (solid line) primary DCs after two days of culture were stained with propidium iodide. The values indicate % viable nontransfected (NT) and transfected (T) cells under the marked region. The results are from one representative experiment conducted more than three times.

    Article Snippet: A non-viral, lipid-based transfection reagent: TransIT-TKO (Mirus Bio, WI) was used.

    Techniques: Transfection, Fluorescence, Expressing, Flow Cytometry, Staining

    The expression of transgenes-encoded Coccidioides -Ag2/PRA and TK antigens by genetically-transfected primary DCs . The cell-lysates were prepared after 24 h of transfection, and dot-immunoblotting was performed with Ag2/PRA and TK-specific antibodies. Left Panel : An Ag2/PRA dot-immunoblot of 5 μg cell-lysate protein from nontransfected (A), transfected primary DCs (B), and 0.2 μg recombinant Ag2/PRA protein (C). Right panel : A TK Dot-immunoblot of 1 μg cell-lysate protein from nontransfected (A) and transfected DCs (B). Results are from one experiment representative of more than three experiments.

    Journal: BMC Immunology

    Article Title: In vivo trafficking and immunostimulatory potential of an intranasally-administered primary dendritic cell-based vaccine

    doi: 10.1186/1471-2172-11-60

    Figure Lengend Snippet: The expression of transgenes-encoded Coccidioides -Ag2/PRA and TK antigens by genetically-transfected primary DCs . The cell-lysates were prepared after 24 h of transfection, and dot-immunoblotting was performed with Ag2/PRA and TK-specific antibodies. Left Panel : An Ag2/PRA dot-immunoblot of 5 μg cell-lysate protein from nontransfected (A), transfected primary DCs (B), and 0.2 μg recombinant Ag2/PRA protein (C). Right panel : A TK Dot-immunoblot of 1 μg cell-lysate protein from nontransfected (A) and transfected DCs (B). Results are from one experiment representative of more than three experiments.

    Article Snippet: A non-viral, lipid-based transfection reagent: TransIT-TKO (Mirus Bio, WI) was used.

    Techniques: Expressing, Transfection, Recombinant

    Downregulation of torsinA inhibits retro-translocation of the cholera toxin A1-chain (a) Immunoblot showing > 95% suppression of torsinA expression in COS cells treated with siRNA 1958 or 1963 for torsinA, with scr. siRNA as control. (b c): (b) After siRNA transfection cells were intoxicated with wild-type CT for 45 min at 37°C and then fractionated into cytosolic and membrane components which were resolved by SDS-PAGE. Immunoblotting was carried out with antibodies to CTA1, CTB, BiP, β-actin and Hsp90. (c) Bar graph showing relative band densities of CTA1 in the cytosol normalized to Hsp90 in the cytosol, and CTA1 in the membrane fraction normalized to BiP. Results are shown as the mean of 3 experiments ± S.D.; * represents p

    Journal: Nature communications

    Article Title: TorsinA participates in endoplasmic reticulum-associated degradation

    doi: 10.1038/ncomms1383

    Figure Lengend Snippet: Downregulation of torsinA inhibits retro-translocation of the cholera toxin A1-chain (a) Immunoblot showing > 95% suppression of torsinA expression in COS cells treated with siRNA 1958 or 1963 for torsinA, with scr. siRNA as control. (b c): (b) After siRNA transfection cells were intoxicated with wild-type CT for 45 min at 37°C and then fractionated into cytosolic and membrane components which were resolved by SDS-PAGE. Immunoblotting was carried out with antibodies to CTA1, CTB, BiP, β-actin and Hsp90. (c) Bar graph showing relative band densities of CTA1 in the cytosol normalized to Hsp90 in the cytosol, and CTA1 in the membrane fraction normalized to BiP. Results are shown as the mean of 3 experiments ± S.D.; * represents p

    Article Snippet: After 24 h the cells were transfected with expression cassettes for GFP-CFTRΔF508 or pcDNA (negative control) and pcDNA-torsinAwt using a lipid-based transfection reagent: TransIT-2020, following the protocol of the manufacturer (Mirus Bio, WI).

    Techniques: Translocation Assay, Expressing, Transfection, SDS Page, CtB Assay

    Involvement of torsinA in degradation of GFP-CFTRΔF508 (a) 293T cells transfected with an expression cassette for GFP-CFTRΔF508 and 48 h later cell lysates were immunoprecipitated with IgG or antibodies to CFTR or torsinA, followed by SDS-PAGE and immunoblotting for GFP and torsinA. (b c): (b) 293T cells were co-transfected with expression cassettes for GFP-CFTRΔF508 or torsinA or torsinAΔE or torsinAΔC or with the control cassette, pcDNA 3.1. Twenty-four h later samples were treated or not with MG132 for 16 h. Seventy-two h post-transfection, cell lysates were processed by SDS-PAGE and gels immunoblotted with antibodies to GFP, torsinA and β-actin. Representative immunoblots are shown with short (1 min) and long (3 min) exposures for GFP, and with 3 min exposure for torsinA and β-actin. (c) GFP-CFTRΔF508 band densities were normalized to those of torsinA and β-actin and represented as the mean of 3 experiments ± S.D. For statistical comparisons, * denotes the comparisons of control to torsinA or torsinAΔE or torsinAΔC transfections (p,

    Journal: Nature communications

    Article Title: TorsinA participates in endoplasmic reticulum-associated degradation

    doi: 10.1038/ncomms1383

    Figure Lengend Snippet: Involvement of torsinA in degradation of GFP-CFTRΔF508 (a) 293T cells transfected with an expression cassette for GFP-CFTRΔF508 and 48 h later cell lysates were immunoprecipitated with IgG or antibodies to CFTR or torsinA, followed by SDS-PAGE and immunoblotting for GFP and torsinA. (b c): (b) 293T cells were co-transfected with expression cassettes for GFP-CFTRΔF508 or torsinA or torsinAΔE or torsinAΔC or with the control cassette, pcDNA 3.1. Twenty-four h later samples were treated or not with MG132 for 16 h. Seventy-two h post-transfection, cell lysates were processed by SDS-PAGE and gels immunoblotted with antibodies to GFP, torsinA and β-actin. Representative immunoblots are shown with short (1 min) and long (3 min) exposures for GFP, and with 3 min exposure for torsinA and β-actin. (c) GFP-CFTRΔF508 band densities were normalized to those of torsinA and β-actin and represented as the mean of 3 experiments ± S.D. For statistical comparisons, * denotes the comparisons of control to torsinA or torsinAΔE or torsinAΔC transfections (p,

    Article Snippet: After 24 h the cells were transfected with expression cassettes for GFP-CFTRΔF508 or pcDNA (negative control) and pcDNA-torsinAwt using a lipid-based transfection reagent: TransIT-2020, following the protocol of the manufacturer (Mirus Bio, WI).

    Techniques: Transfection, Expressing, Immunoprecipitation, SDS Page, Western Blot

    Specific depletion of NFκB p65 attenuates CLA’s suppression of PPARγ and activation of MEK/ERK signaling in cultures of primary human adipocytes A: Experimental design of transfection protocol with siRNA NFκB p65 in primary cultures of human adipocytes. B: Transfection efficiency was evaluated by transfection with siGLO, fluorescence (Cy3)-tagged non-targeting siRNA. C: Knockdown specificity was analyzed using immunoblotting. Total cell extracts from either untreated (Unt) samples or samples transfected with non-targeting control siRNA (siCon), NFκB p65 siRNA (siP65), and positive control Lamin siRNA (siLa) were immunoblotted with the antibodies targeting NFκB p65, Lamin C, NFκB p50, GAPDH, actin and aP2. D: The impact of CLA on NFκB p65-depleted cultures were examined using immunoblotting. 72 h post-transfection with either siCon or siP65, cultures were treated with BSA vehicle or a 30 μM trans -10, cis -12 CLA (CLA) for additional 24 h. Total cell extracts were immunoblotted with antibodies targeting NFκB p65, PPARγ, Glut4, p- signal transducer and activators of transcription 3 (STAT3), p-ERK1/2, total-STAT3, and GAPDH. The blots for PPARγ and GAPDH were quantified by densitometry and the amount of PPARγ relative to GAPDH was expressed as a bar graph under the blot. E: Cultures were transfected, 72 h later treated with either BSA or 30 μM trans -10, cis -12 CLA (CLA) for 24 h, and then immunostained for PPARγ. Hoechst staining was conducted to identify nuclei.

    Journal: The Journal of biological chemistry

    Article Title: CONJUGATED LINOLEIC ACID PROMOTES HUMAN ADIPOCYTE INSULIN RESISTANCE THROUGH NF?B-DEPENDENT CYTOKINE PRODUCTION

    doi: 10.1074/jbc.M508159200

    Figure Lengend Snippet: Specific depletion of NFκB p65 attenuates CLA’s suppression of PPARγ and activation of MEK/ERK signaling in cultures of primary human adipocytes A: Experimental design of transfection protocol with siRNA NFκB p65 in primary cultures of human adipocytes. B: Transfection efficiency was evaluated by transfection with siGLO, fluorescence (Cy3)-tagged non-targeting siRNA. C: Knockdown specificity was analyzed using immunoblotting. Total cell extracts from either untreated (Unt) samples or samples transfected with non-targeting control siRNA (siCon), NFκB p65 siRNA (siP65), and positive control Lamin siRNA (siLa) were immunoblotted with the antibodies targeting NFκB p65, Lamin C, NFκB p50, GAPDH, actin and aP2. D: The impact of CLA on NFκB p65-depleted cultures were examined using immunoblotting. 72 h post-transfection with either siCon or siP65, cultures were treated with BSA vehicle or a 30 μM trans -10, cis -12 CLA (CLA) for additional 24 h. Total cell extracts were immunoblotted with antibodies targeting NFκB p65, PPARγ, Glut4, p- signal transducer and activators of transcription 3 (STAT3), p-ERK1/2, total-STAT3, and GAPDH. The blots for PPARγ and GAPDH were quantified by densitometry and the amount of PPARγ relative to GAPDH was expressed as a bar graph under the blot. E: Cultures were transfected, 72 h later treated with either BSA or 30 μM trans -10, cis -12 CLA (CLA) for 24 h, and then immunostained for PPARγ. Hoechst staining was conducted to identify nuclei.

    Article Snippet: On ~day 6 of differentiation, cultures containing fresh adipocyte media (AM-1, Zen Bio Inc, RTP, NC) were supplemented with either 25 nM NFκB p65 siRNA or non-targeting siRNA complexed with Trans IT-TKO (6 ul/ml), a non-lipid based transfection reagent from Mirus Corp. Transfection reagent and undelivered siRNA were removed 24 h post-transfection by removing the media and washing the cells twice with HBSS.

    Techniques: Activation Assay, Transfection, Fluorescence, Positive Control, Staining