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The Gα s -Lys58Gln variant enhances constitutive activity of the ACTH receptor. (A) ACTH-mediated cAMP responses measured by GloSensor in Gα s wild-type and Gα s -Lys58Gln variant cell lines transiently expressing <t>MC2R</t> and MRAP1. (B) Area under the curve was used to generate cAMP concentration–response curves. (C) Potency of ACTH in cells expressing either WT or Lys58Gln Gα s transfected with the ACTH receptor. n = 5 biological replicates. (D) ACTH-mediated phospho-CREB (pCREB) responses measured by AlphaLISA in Gα s wild-type and Gα s -Lys58Gln variant cell lines transiently expressing MC2R and MRAP1, with (E) pEC 50 . n = 4 biological replicates. (F) ACTH-mediated CRE luciferase activity in Gα s wild-type and Gα s -Lys58Gln variant cell lines transiently expressing MC2R and MRAP1, with (G) pEC 50 . n = 4 biological replicates. Statistical analyses were performed by two-way ANOVA with Bonferroni’s multiple-comparisons test in B and D, two-way ANOVA with Sidak’s multiple-comparisons test in F and unpaired t -test in C, E, G. **** P < 0.0001, *** P < 0.001, ns = not significant.
Mc2r Tango, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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The Gα s -Lys58Gln variant enhances constitutive activity of the ACTH receptor. (A) ACTH-mediated cAMP responses measured by GloSensor in Gα s wild-type and Gα s -Lys58Gln variant cell lines transiently expressing MC2R and MRAP1. (B) Area under the curve was used to generate cAMP concentration–response curves. (C) Potency of ACTH in cells expressing either WT or Lys58Gln Gα s transfected with the ACTH receptor. n = 5 biological replicates. (D) ACTH-mediated phospho-CREB (pCREB) responses measured by AlphaLISA in Gα s wild-type and Gα s -Lys58Gln variant cell lines transiently expressing MC2R and MRAP1, with (E) pEC 50 . n = 4 biological replicates. (F) ACTH-mediated CRE luciferase activity in Gα s wild-type and Gα s -Lys58Gln variant cell lines transiently expressing MC2R and MRAP1, with (G) pEC 50 . n = 4 biological replicates. Statistical analyses were performed by two-way ANOVA with Bonferroni’s multiple-comparisons test in B and D, two-way ANOVA with Sidak’s multiple-comparisons test in F and unpaired t -test in C, E, G. **** P < 0.0001, *** P < 0.001, ns = not significant.

Journal: Endocrine Oncology

Article Title: Characterisation of a GNAS variant linked to cortisol-producing adrenocortical adenoma

doi: 10.1530/EO-25-0009

Figure Lengend Snippet: The Gα s -Lys58Gln variant enhances constitutive activity of the ACTH receptor. (A) ACTH-mediated cAMP responses measured by GloSensor in Gα s wild-type and Gα s -Lys58Gln variant cell lines transiently expressing MC2R and MRAP1. (B) Area under the curve was used to generate cAMP concentration–response curves. (C) Potency of ACTH in cells expressing either WT or Lys58Gln Gα s transfected with the ACTH receptor. n = 5 biological replicates. (D) ACTH-mediated phospho-CREB (pCREB) responses measured by AlphaLISA in Gα s wild-type and Gα s -Lys58Gln variant cell lines transiently expressing MC2R and MRAP1, with (E) pEC 50 . n = 4 biological replicates. (F) ACTH-mediated CRE luciferase activity in Gα s wild-type and Gα s -Lys58Gln variant cell lines transiently expressing MC2R and MRAP1, with (G) pEC 50 . n = 4 biological replicates. Statistical analyses were performed by two-way ANOVA with Bonferroni’s multiple-comparisons test in B and D, two-way ANOVA with Sidak’s multiple-comparisons test in F and unpaired t -test in C, E, G. **** P < 0.0001, *** P < 0.001, ns = not significant.

Article Snippet: MC2R-Tango (a gift from Bryan Roth; Addgene plasmid #66428 ( Kroeze et al. 2015 )) and MRAP1_pcDNA6.2/EmGFP-Bsd (a gift from Roger Reeves; Addgene plasmid #176937 ( Moyer et al. 2023 )) plasmids were purchased from Addgene and used as templates to generate pEGFP-C1-MC2R and pmCherry-C1-MRAP1 expression constructs by standard cloning procedures and oligonucleotides obtained from Merck (sequences listed in Supplementary Table 1).

Techniques: Variant Assay, Activity Assay, Expressing, Concentration Assay, Transfection, Luciferase

The Lys58Gln Gα s variant enhances cell growth and apoptosis. (A) Cell viability in Gα s wild-type and Gα s -Lys58Gln variant cell lines transiently expressing MC2R and MRAP1 measured over 96 h. Data were expressed corrected to the total protein, then normalized to hour 0. (B) Apoptosis measured over 96 h was expressed corrected to the total protein, then normalised to hour 0. Statistical analyses were performed by unpaired t -test in n = 5 replicates. ** P < 0.01, * P < 0.05.

Journal: Endocrine Oncology

Article Title: Characterisation of a GNAS variant linked to cortisol-producing adrenocortical adenoma

doi: 10.1530/EO-25-0009

Figure Lengend Snippet: The Lys58Gln Gα s variant enhances cell growth and apoptosis. (A) Cell viability in Gα s wild-type and Gα s -Lys58Gln variant cell lines transiently expressing MC2R and MRAP1 measured over 96 h. Data were expressed corrected to the total protein, then normalized to hour 0. (B) Apoptosis measured over 96 h was expressed corrected to the total protein, then normalised to hour 0. Statistical analyses were performed by unpaired t -test in n = 5 replicates. ** P < 0.01, * P < 0.05.

Article Snippet: MC2R-Tango (a gift from Bryan Roth; Addgene plasmid #66428 ( Kroeze et al. 2015 )) and MRAP1_pcDNA6.2/EmGFP-Bsd (a gift from Roger Reeves; Addgene plasmid #176937 ( Moyer et al. 2023 )) plasmids were purchased from Addgene and used as templates to generate pEGFP-C1-MC2R and pmCherry-C1-MRAP1 expression constructs by standard cloning procedures and oligonucleotides obtained from Merck (sequences listed in Supplementary Table 1).

Techniques: Variant Assay, Expressing