Journal: iScience

Article Title: Gαs slow conformational transition upon GTP binding and a novel Gαs regulator

doi: 10.1016/j.isci.2023.106603

Figure Lengend Snippet: Novel Gαs AHD-binding protein that facilitates AHD closing (A) Representative confocal images of HEK293T cells transiently expressing Gαs AHD-FLAG. HEK293T cells were stained with antibodies against FLAG (red) and MAGE D2 (green). Blue represents Hoechst 33342. Right: Representative co-localization tracer profile along the line indicated in the inset. Scale bar, 10 μm. (B) Immunoblot (IB) analysis of FLAG immunoprecipitates (IP line) and cell lysates (Input line) from HEK293T cells transiently co-expressing Gαs AHD-FLAG and MAGE D2-turboGFP. (C) Binding curve of the Gαs AHD with the MAGE D2 MHD analyzed by microscale thermophoresis (MST). A titration series of the Gαs AHD was performed, whereas the concentration of fluorescence-labeled MAGE D2 MHD was fixed (see for details). Error bars represent the standard error of the mean of more than three independent experiments. (D) Time-resolved tryptophan-induced bimane quenching analysis of the β 2 AR–Gs complex with or without the MAGE D2 MHD after GTPγS addition. The data are mean of the normalized fluorescence value of three independently labeled experiments and error bars represent the standard error of the mean. See also Figures S3 , , and .

Article Snippet: MAGE D2 Human Tagged ORF Clone , Origene , Cat# RG214066/A.

Techniques: Binding Assay, Expressing, Staining, Western Blot, Microscale Thermophoresis, Titration, Concentration Assay, Fluorescence, Labeling

Journal: iScience

Article Title: Gαs slow conformational transition upon GTP binding and a novel Gαs regulator

doi: 10.1016/j.isci.2023.106603

Figure Lengend Snippet: Summary cartoon illustrating the proposed sequence of events after GTP binding to a GPCR–Gs complex with or without MAGE D2 An agonist-activated receptor (R) induces GDP release from Gs, and GTP quickly binds to the empty nucleotide-binding pocket (step i). Upon binding of GTP, Gαs rapidly dissociates from the receptor and Gβγ (step ii). The AHD adopts a long-lived intermediate state until it is fully closed, a process that occurs slowly (step iii-a). Closing of the AHD is accelerated in the presence of MAGE D2 (step iii-b). RD indicates the Ras-like GTPase domain, and AHD indicates the α-helical domain.

Article Snippet: MAGE D2 Human Tagged ORF Clone , Origene , Cat# RG214066/A.

Techniques: Sequencing, Binding Assay

Journal: iScience

Article Title: Gαs slow conformational transition upon GTP binding and a novel Gαs regulator

doi: 10.1016/j.isci.2023.106603

Figure Lengend Snippet:

Article Snippet: MAGE D2 Human Tagged ORF Clone , Origene , Cat# RG214066/A.

Techniques: Recombinant, Magnetic Beads, Protease Inhibitor, Cotransfection, Expressing, Transfection, Labeling, Software