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Generation and characterization of novel human IRAS monoclonal antibodies.

Wang B, Liu Y, Shan Y, Yao Z, Liu X, Su R, Sun Q, Cong Y, Li J - J. Biomed. Biotechnol. (2009)

Bottom Line: Imidazoline receptors were first proposed by Bousquet et al., when they studied antihypertensive effect of clonidine.Monoclonal antibodies stained in a predominantly punctate cytoplasmic pattern when applied to IRAS-transfected HEK293 cells by indirect immunofluorescence assays and demonstrated excellent reactivity in flow immunocytometry.These monoclonal antibodies will provide powerful reagents for the further investigation of hIRAS protein functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China.

ABSTRACT
Imidazoline receptors were first proposed by Bousquet et al., when they studied antihypertensive effect of clonidine. A strong candidate for I1R, known as imidazoline receptor antisera-selected protein (IRAS), has been cloned from human hippocampus. We reported that IRAS mediated agmatine-induced inhibition of opioid dependence in morphine-dependent cells. To elucidate the functional and structure properties of I1R, we developed the newly monoclonal antibody against the N-terminal hIRAS region including the PX domain (10-120aa) through immunization of BALB/c mice with the NusA-IRAS fusion protein containing an IRAS N-terminal (10-120aa). Stable hybridoma cell lines were established and monoclonal antibodies specifically recognized full-length IRAS proteins in their native state by immunoblotting and immunoprecipitation. Monoclonal antibodies stained in a predominantly punctate cytoplasmic pattern when applied to IRAS-transfected HEK293 cells by indirect immunofluorescence assays and demonstrated excellent reactivity in flow immunocytometry. These monoclonal antibodies will provide powerful reagents for the further investigation of hIRAS protein functions.

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Related in: MedlinePlus

Analysis of immunoprecipitates revealed that the IRAS mAbs specifically recognized the native state of the IRAS protein. The HEK293 cells were transiently transfected with the empty vector PCMV-myc (negative control) and PCMV-myc-IRAS. Whole cell lysates were prepared and immunoprecipitated with the c-myc mAb or the mAbs DA041, DD015, BE073, BA022, and AH021. Immunoprecipitation of the same lysates using mouse normal IgG did not result in the detection of any protein species. Immunocomplexes were analyzed by western blot with indicated antibodies.
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fig4: Analysis of immunoprecipitates revealed that the IRAS mAbs specifically recognized the native state of the IRAS protein. The HEK293 cells were transiently transfected with the empty vector PCMV-myc (negative control) and PCMV-myc-IRAS. Whole cell lysates were prepared and immunoprecipitated with the c-myc mAb or the mAbs DA041, DD015, BE073, BA022, and AH021. Immunoprecipitation of the same lysates using mouse normal IgG did not result in the detection of any protein species. Immunocomplexes were analyzed by western blot with indicated antibodies.

Mentions: We next characterized IRAS mAb ability to recognize IRAS proteins in their native states by immunoprecipitation. Cell extracts were immunoprecipitated with the mAb DA041 and analyzed with mAb DA041 immunoblotting after 48-hour transfection with PCMV-myc-IRAS. IRAS was selectively immunoprecipitated from cell lysates that expressed the myc-IRAS protein (approximately 175 kDa), and those which corresponded to the predicted size of the full-length IRAS (Figure 4). Immunoprecipitation of lysates with mouse normal IgG did not result in detection of protein species. Lysates were immunoprecipitated with the c-myc mAb to confirm mAb specificity, and similarly sized bands were detected. No corresponding band was visualized when the negative control c-myc mAb was used to immunoprecipitate cell lysate of the transfectant expressing the empty PCMV-myc vector. Similar results were obtained with the mAbs DD015 and BE073 under the same conditions, and the remaining mAbs BA022 and AH021 scored negatively. Therefore, the 3 isolated antibodies specifically recognized native full-length IRAS protein products.


Generation and characterization of novel human IRAS monoclonal antibodies.

Wang B, Liu Y, Shan Y, Yao Z, Liu X, Su R, Sun Q, Cong Y, Li J - J. Biomed. Biotechnol. (2009)

Analysis of immunoprecipitates revealed that the IRAS mAbs specifically recognized the native state of the IRAS protein. The HEK293 cells were transiently transfected with the empty vector PCMV-myc (negative control) and PCMV-myc-IRAS. Whole cell lysates were prepared and immunoprecipitated with the c-myc mAb or the mAbs DA041, DD015, BE073, BA022, and AH021. Immunoprecipitation of the same lysates using mouse normal IgG did not result in the detection of any protein species. Immunocomplexes were analyzed by western blot with indicated antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2723995&req=5

fig4: Analysis of immunoprecipitates revealed that the IRAS mAbs specifically recognized the native state of the IRAS protein. The HEK293 cells were transiently transfected with the empty vector PCMV-myc (negative control) and PCMV-myc-IRAS. Whole cell lysates were prepared and immunoprecipitated with the c-myc mAb or the mAbs DA041, DD015, BE073, BA022, and AH021. Immunoprecipitation of the same lysates using mouse normal IgG did not result in the detection of any protein species. Immunocomplexes were analyzed by western blot with indicated antibodies.
Mentions: We next characterized IRAS mAb ability to recognize IRAS proteins in their native states by immunoprecipitation. Cell extracts were immunoprecipitated with the mAb DA041 and analyzed with mAb DA041 immunoblotting after 48-hour transfection with PCMV-myc-IRAS. IRAS was selectively immunoprecipitated from cell lysates that expressed the myc-IRAS protein (approximately 175 kDa), and those which corresponded to the predicted size of the full-length IRAS (Figure 4). Immunoprecipitation of lysates with mouse normal IgG did not result in detection of protein species. Lysates were immunoprecipitated with the c-myc mAb to confirm mAb specificity, and similarly sized bands were detected. No corresponding band was visualized when the negative control c-myc mAb was used to immunoprecipitate cell lysate of the transfectant expressing the empty PCMV-myc vector. Similar results were obtained with the mAbs DD015 and BE073 under the same conditions, and the remaining mAbs BA022 and AH021 scored negatively. Therefore, the 3 isolated antibodies specifically recognized native full-length IRAS protein products.

Bottom Line: Imidazoline receptors were first proposed by Bousquet et al., when they studied antihypertensive effect of clonidine.Monoclonal antibodies stained in a predominantly punctate cytoplasmic pattern when applied to IRAS-transfected HEK293 cells by indirect immunofluorescence assays and demonstrated excellent reactivity in flow immunocytometry.These monoclonal antibodies will provide powerful reagents for the further investigation of hIRAS protein functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China.

ABSTRACT
Imidazoline receptors were first proposed by Bousquet et al., when they studied antihypertensive effect of clonidine. A strong candidate for I1R, known as imidazoline receptor antisera-selected protein (IRAS), has been cloned from human hippocampus. We reported that IRAS mediated agmatine-induced inhibition of opioid dependence in morphine-dependent cells. To elucidate the functional and structure properties of I1R, we developed the newly monoclonal antibody against the N-terminal hIRAS region including the PX domain (10-120aa) through immunization of BALB/c mice with the NusA-IRAS fusion protein containing an IRAS N-terminal (10-120aa). Stable hybridoma cell lines were established and monoclonal antibodies specifically recognized full-length IRAS proteins in their native state by immunoblotting and immunoprecipitation. Monoclonal antibodies stained in a predominantly punctate cytoplasmic pattern when applied to IRAS-transfected HEK293 cells by indirect immunofluorescence assays and demonstrated excellent reactivity in flow immunocytometry. These monoclonal antibodies will provide powerful reagents for the further investigation of hIRAS protein functions.

Show MeSH
Related in: MedlinePlus