pgfp n1 plasmid  (TaKaRa)

Journal: The Journal of Biological Chemistry

Article Title: Heme oxygenase-1 affects cytochrome P450 function through the formation of heteromeric complexes: Interactions between CYP1A2 and heme oxygenase-1

doi: 10.1074/jbc.RA120.015911

Figure Lengend Snippet: Determination of complex formation between CYP1A2 and HO-1—Effect of POR . A , HEK 293T/17 cells were transfected with plasmids coding for rabbit CYP1A2-GFP, Rluc-HO-1, in the absence and presence of untagged-POR. The CYP1A2-GFP–Rluc-HO-1 BRET pair was measured 24 h after transfection in the absence ( blue ) and presence ( red ) of 500 ng of cotransfected POR DNA. Error bars represent the standard deviation (SD) of triplicate measurements of cells from a single transfection and generally do not exceed the size of the points. The experiment was performed three times with small adjustments to transfection conditions for optimization of protein expression levels. Results from each transfection were consistent. B , total protein expression was estimated from the sum of the fluorescence of the GFP and luminescence of the Rluc tags, using a GFP–Rluc fusion protein as a standard. C , effect of changes in protein concentration at a fixed ratio of GFP-CYP1A2 to Rluc-HO-1. HEK 293T/17 cells were transfected with different amounts of DNA, while maintaining an excess of the CYP1A2-GFP–tagged protein. The relative levels of GFP–Rluc protein expression were in excess of 38:1. BRET, bioluminescence resonance energy transfer; GFP, green fluorescent protein; HO-1, heme oxygenase 1; POR, NADPH-cytochrome P450 reductase.

Article Snippet: The plasmids used to generate BRET vectors (pGFP 2 -N1, pGFP 2 -N2, pRluc-N2, pRluc-N3) and the pGFP 2 -Rluc vector were obtained from BioSignal Packard (Waltham, WA).

Techniques: Transfection, Standard Deviation, Expressing, Fluorescence, Protein Concentration, Bioluminescence Resonance Energy Transfer

Journal: The Journal of Biological Chemistry

Article Title: Heme oxygenase-1 affects cytochrome P450 function through the formation of heteromeric complexes: Interactions between CYP1A2 and heme oxygenase-1

doi: 10.1074/jbc.RA120.015911

Figure Lengend Snippet: Effect of HO-1 on the interaction between CYP1A2-GFP and POR-Rluc. A , HEK 293T/17 cells were transfected with plasmids coding for rabbit CYP1A2-GFP and POR-Rluc in the absence and presence of 500 ng of unlabeled HO-1. Twenty-four hours post transfection, cells were collected and BRET was measured. When cells were cotransfected with HO-1 DNA, the maximum BRET signal generated was significantly lower ( red ) than that of cells transfected with the CYP1A2-GFP•POR-Rluc pair alone ( blue ). Data points represent triplicate measurements of cells from a single transfection; error bars represent the standard deviation (SD) and generally did not exceed the size of the points. Each experiment was repeated with small adjustments to transfection conditions for optimization of protein expression. Results were both transfections were consistent. B , total protein expression was estimated from the sum of the fluorescence of the GFP and luminescence of the Rluc tags. BRET, bioluminescence resonance energy transfer; GFP, green fluorescent protein; HO-1, heme oxygenase 1; POR, NADPH-cytochrome P450 reductase.

Article Snippet: The plasmids used to generate BRET vectors (pGFP 2 -N1, pGFP 2 -N2, pRluc-N2, pRluc-N3) and the pGFP 2 -Rluc vector were obtained from BioSignal Packard (Waltham, WA).

Techniques: Transfection, Generated, Standard Deviation, Expressing, Fluorescence, Bioluminescence Resonance Energy Transfer

Journal: The Journal of Biological Chemistry

Article Title: Heme oxygenase-1 affects cytochrome P450 function through the formation of heteromeric complexes: Interactions between CYP1A2 and heme oxygenase-1

doi: 10.1074/jbc.RA120.015911

Figure Lengend Snippet: Effect of CYP1A2 on the interaction between GFP-HO-1 and POR-Rluc. A , HEK 293T/17 cells were transfected with plasmids coding for GFP-HO-1 and POR-Rluc in the absence and presence of untagged CYP1A2. BRET generated by the GFP-HO-1•POR-Rluc pair was measured 24 h after transfection in the presence ( red ) and absence ( blue ) of 2 μg of vector coding for untagged CYP1A2. Cotransfected CYP1A2 DNA did not significantly alter the BRET signal generated by the GFP-HO-1•POR-Rluc pair. Error bars represent the SD of triplicate measurements of cells from a single transfection and generally do not exceed the size of the points. Each experiment was repeated with small adjustments to transfection conditions for optimization of protein expression, generating similar results. B , total protein expression was estimated from the sum of the fluorescence of the GFP and luminescence of the Rluc tags. BRET, bioluminescence resonance energy transfer; GFP, green fluorescent protein; HO-1, heme oxygenase 1; POR, NADPH-cytochrome P450 reductase.

Article Snippet: The plasmids used to generate BRET vectors (pGFP 2 -N1, pGFP 2 -N2, pRluc-N2, pRluc-N3) and the pGFP 2 -Rluc vector were obtained from BioSignal Packard (Waltham, WA).

Techniques: Transfection, Generated, Plasmid Preparation, Expressing, Fluorescence, Bioluminescence Resonance Energy Transfer

Journal: The Journal of Biological Chemistry

Article Title: Heme oxygenase-1 affects cytochrome P450 function through the formation of heteromeric complexes: Interactions between CYP1A2 and heme oxygenase-1

doi: 10.1074/jbc.RA120.015911

Figure Lengend Snippet: Effect of HO-1 on formation of the homomeric CYP1A2 BRET complex. A , HEK 293T/17 cells were transfected with plasmids coding for CYP1A2-GFP and CYP1A2-Rluc in the absence and presence of untagged HO-1. BRET generated by the CYP1A2-GFP•CYP1A2-Rluc pair was measured 24 h after transfection with ( red ) and without ( blue ) 2000 ng of a vector coding for untagged HO-1. Cotransfected HO-1 DNA led to a significant disruption of the BRET signal generated by the CYP1A2-GFP•CYP1A2-Rluc pair. Error bars represent the standard deviation (SD) of triplicate measurements of cells from a single transfection and do not exceed the size of the data points. Each experiment was repeated with small adjustments to transfection conditions for optimization of protein expression, generating similar results. B , total protein expression was estimated from the sum of the fluorescence of the GFP and luminescence of the Rluc tags. BRET, bioluminescence resonance energy transfer; GFP, green fluorescent protein; HO-1, heme oxygenase 1.

Article Snippet: The plasmids used to generate BRET vectors (pGFP 2 -N1, pGFP 2 -N2, pRluc-N2, pRluc-N3) and the pGFP 2 -Rluc vector were obtained from BioSignal Packard (Waltham, WA).

Techniques: Transfection, Generated, Plasmid Preparation, Disruption, Standard Deviation, Expressing, Fluorescence, Bioluminescence Resonance Energy Transfer

Journal: The Journal of Biological Chemistry

Article Title: Heme oxygenase-1 affects cytochrome P450 function through the formation of heteromeric complexes: Interactions between CYP1A2 and heme oxygenase-1

doi: 10.1074/jbc.RA120.015911

Figure Lengend Snippet: Description of the BRET constructs and their restriction sites

Article Snippet: The plasmids used to generate BRET vectors (pGFP 2 -N1, pGFP 2 -N2, pRluc-N2, pRluc-N3) and the pGFP 2 -Rluc vector were obtained from BioSignal Packard (Waltham, WA).

Techniques: Construct, Plasmid Preparation, Sequencing