Journal: Endocrinology

Article Title: Role of regulator of G protein signaling in desensitization of the glucose-dependent insulinotropic peptide receptor.

doi: 10.1210/endo.139.11.6282

Figure Lengend Snippet: FIG. 7. Immunoblot detection of Gsa in RGS2-HA-transfected L293 cells in the presence (1) or the absence (2) of GIP stimulation. Cell lysates were immunoprecipitated with HA antiserum and resolved on SDS-PAGE gels. The detection of Gsa is shown at the top, and the bottom panel shows a representative of the SDS-PAGE gels stained with Coomassie brilliant blue.

Article Snippet: After electrophoresis, gels were either stained with Coomassie brilliant blue or transferred to a Hybond-C Extra filter (Amersham), and Gsa proteins were detected using Gsa antiserum (Santa Cruz Biotechnology) and the enhanced chemiluminescence system (Amersham) according to the manufacturer’s instruction.

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

Journal: Nature chemical biology

Article Title: Conformationally selective RNA aptamers allosterically modulate the β2-adrenoceptor.

doi: 10.1038/nchembio.2126

Figure Lengend Snippet: Figure 1 | Generation of conformation-specific RNA aptamers against the 2AR. (a) Schematic overview of the selection strategy, NGS, bioinformatics analysis, and characterization of candidate aptamers. Shown are ribbon diagram representations for selection against inactive β2AR (colored in blue; PDB 2RH1) and active β2AR bound to the high-affinity agonist BI167107 (colored in red; PDB 3SN6). MNG detergent micelles are represented in gray. Areas encircled in light pink are potential binding regions for aptamers to different β2AR conformations. (b) Scatter plot from NGS analysis, comparing fold enrichment (ratio of clones’ frequency in round 9 versus round 4) for the top 20 aptamer sequences after selection on unliganded β2AR (x axis) versus BI167107-bound β2AR (y axis). Each point in the plot represents a unique aptamer (the top seven candidate binders are color-coded in red, blue, or purple) according to their enrichment and selectivity to a selection target. (c) Bar graph showing top seven aptamers and their capacity to bind unliganded β2AR or BI167107-bound β2AR (relative to their ability to bind control aptamer CNT-Apt) as assessed by pulldown assay. Boxed bars denote the four aptamers that were selected for further characterization. Data shown represent the mean ± s.e.m. (*P < 0.05; **P < 0.01; ***P < 0.001) of three independent experiments, analyzed by one-way ANOVA followed by Fisher’s LSD (least significant difference) post hoc test.

Article Snippet: 12.5 μL of 4× SDS sample buffer was added to each eluted sample before performing a western blotting using anti-β2AR antibody (sc-569; Santa Cruz), and ethidium bromide (EtBr) staining for RNAs on a 10% TBE gel (Life Technologies).

Techniques: Selection, Binding Assay, Clone Assay, Control

Journal: Nature chemical biology

Article Title: Conformationally selective RNA aptamers allosterically modulate the β2-adrenoceptor.

doi: 10.1038/nchembio.2126

Figure Lengend Snippet: Figure 2 | Aptamers distinguish between inactive and active conformations of the 2AR. Binding kinetic profiles for the interactions of four biotinylated aptamers with BI167107-bound (active) β2AR or ICI-118,551-bound (inactive) β2AR as analyzed using biolayer interferometry (BLI). (a–d) Representative sensorgrams for the interactions of four biotinylated aptamers with BI167107-bound β2AR (left panels) or ICI-118,551-bound β2AR (right panels): A1 (a), A2 (b), A13 (c), and A16 (d). Data were globally fit to 1:1 binding model as described in the Online Methods. Kd (dissociation constant) is shown as the ratio of koff (dissociation) to kon (association) rate constants. Each Kd value represents the mean affinity values ± s.e.m. of three independent experiments. Blue, gray, green, and light blue curves represent the measured responses for each tested concentration of β2AR (bound to BI167107 or ICI-118,551); the overlay curves in red show the global fitting results of the binding data.

Article Snippet: 12.5 μL of 4× SDS sample buffer was added to each eluted sample before performing a western blotting using anti-β2AR antibody (sc-569; Santa Cruz), and ethidium bromide (EtBr) staining for RNAs on a 10% TBE gel (Life Technologies).

Techniques: Binding Assay, Concentration Assay

Journal: Nature chemical biology

Article Title: Conformationally selective RNA aptamers allosterically modulate the β2-adrenoceptor.

doi: 10.1038/nchembio.2126

Figure Lengend Snippet: Figure 4 | Influence of aptamers on conformational changes conferred by ligands. (a–d) Measurement of bimane fluorescence quenching shows that aptamers A1, A2, and A13 stabilize active forms of β2AR, while aptamer A16 stabilizes an inactive conformation. Bimane fluorescence quenching measurement detects conformational changes of the β2AR via movement of a bimane probe on TM6 (at C265) upon the binding of agonists (BI167107 (BI) or isoproterenol (ISO)) and/or aptamer A1 (a), A2 (b), A13 (c), or A16 (d). Fluorescence emission spectra showing ligand-induced conformational changes of bimane-labeled β2AR in the absence of agonists and aptamers (DMSO, black dashed line) or in the presence of full agonist (ISO, blue dashed line, or BI, red dashed line); inverse agonist ICI-118,551 (ICI, green dashed line); aptamer (A1, A2, A13, or A16, respectively; black solid line); or a combination of aptamer (A1, A2, A13, or A16) with ISO (blue solid line), BI (red solid line), or ICI (green solid line).

Article Snippet: 12.5 μL of 4× SDS sample buffer was added to each eluted sample before performing a western blotting using anti-β2AR antibody (sc-569; Santa Cruz), and ethidium bromide (EtBr) staining for RNAs on a 10% TBE gel (Life Technologies).

Techniques: Fluorescence, Binding Assay, Labeling

Journal: Nature chemical biology

Article Title: Conformationally selective RNA aptamers allosterically modulate the β2-adrenoceptor.

doi: 10.1038/nchembio.2126

Figure Lengend Snippet: Figure 6 | EM analysis and molecular architecture of 2AR–aptamer complexes. (a–d) EM characterization of purified β2AR–aptamer-Fab complexes. Shown at left in each panel is the 2D particle class average of anti-FLAG-Fab-labeled β2AR in complex with aptamer A1 (a), A2 (b), A13 (c), or A16 (d). Scale bars, 10 nm. Shown at right in each panel is a cartoon representations of the class average, with the various components in the 2D image maps (β2AR in red; detergent micelle labeled with “m” in light gray; anti-FLAG Fab antibody in maroon; the aptamers are in lime green (A1), medium purple (A2), pink (A13), and gray (A16)).

Article Snippet: 12.5 μL of 4× SDS sample buffer was added to each eluted sample before performing a western blotting using anti-β2AR antibody (sc-569; Santa Cruz), and ethidium bromide (EtBr) staining for RNAs on a 10% TBE gel (Life Technologies).

Techniques: Purification, Labeling