Journal: Journal of the Endocrine Society

Article Title: Real-Time Signaling Assays Demonstrate Somatostatin Agonist Bias for Ion Channel Regulation in Somatotroph Tumor Cells

doi: 10.1210/js.2018-00115

Figure Lengend Snippet: Effect of anti-G βγ ‒binding peptide on SS analogue signaling. Cells were incubated with (A) membrane potential dye, (B) cAMP GloSensor luciferin, or (C–E) intracellular Ca 2+ dye in the presence or absence of the G protein βγ subunit peptide blocker MPS-phosducin-like protein C-terminus (MPS-Phos, 1 µM). Cells were pretreated with MPS-Phos for 15 minutes at 28°C for cAMP GloSensor experiments or for 15 minutes at 37°C for the other experiments. (A) Hyperpolarization responses after simulation with SS14 (100 nM) or SS14 (100 nM) + MPS-Phos (1 µM). Treatment groups were not significantly different ( P = 0.5; two-tailed t test). (B) Inhibition of NKH477 (10 µM) stimulated cAMP production by SS14 (100 nM) or SOM230 (1 µM) in the absence or presence of MPS-Phos (1 µM). Response of MPS-Phos‒treated groups was not significantly different from that of nontreated groups (two-tailed t test). (C) HEK293-Sstr2A cells were incubated with the FLUO-8 am dye for 45 minutes, washed, and then incubated with the anti- βγ peptide MPS-Phos (2 µM) for 15 minutes. Cells were then treated with saturating concentrations of SS14 (100 nM). The effect of MPS-Phos was significant ( P < 0.0001; two-tailed t test). Data are mean ± SEM from two different experiments, three replicates per group per experiment. (D) Intracellular Ca 2+ levels after stimulation with SS14 (100 nM) or SS14 (100 nM) + MPS-Phos (1 µM). The effect of MPS-Phos was significant ( P < 0.0001; two-tailed t test). (E) Intracellular Ca 2+ levels after stimulation with SOM230 (1 µM) or SOM230 (1 µM) + MPS-Phos (1 µM). The effect of MPS-Phos was significant ( P < 0.0001; two-tailed t test). Data are expressed as a ratio of fluorescence intensity (F 1 /F 0 ). (A, B, D, and E) Data show the mean ± SEM from three independent experiments, three replicates per group per experiment. Black arrows indicate the addition of agonists 30 seconds after the start of the experiment. -, no ligand; SOM, SOM230; SS, somatostatin 14.

Article Snippet: Stable GH12C1 cell lines expressing a cAMP biosensor were created by transfecting the 22F GloSensor cAMP biosensor (Promega) into GH12C1-HA3-rSstr2A clone #35 cells using FuGENE (Promega) and selecting with 200 μg/mL of hygromycin B. Clonal cell lines were isolated by limiting dilution and were screened for biosensor activity.

Techniques: Incubation, Membrane, Two Tailed Test, Inhibition, Fluorescence

Journal: Chemical Senses

Article Title: The stability of tastant detection by mouse lingual chemosensory tissue requires Regulator of G protein Signaling-21 (RGS21)

doi: 10.1093/chemse/bjab048

Figure Lengend Snippet: RGS21 binds gustducin-α and reduces bitterant signaling in a bitterant-responsive cultured cell line. (A) COS7 cell monolayers were transiently transfected with plasmid DNA encoding gustducin-α tagged with an HA epitope (“HA-Gα gust”). Forty-eight hours post transfection, cell monolayers were lysed in buffer containing GDP, Mg2+ and AlF4– (“AMF”) or GDP alone, as indicated. Clarified cell lysates were incubated with 10 μg of purified His6-RGS21 protein at 4°C overnight with NTA-agarose. Samples were centrifuged, agarose beads washed four times in lysis buffer, and precipitated proteins (“P”:) eluted by boiling for 5 minutes in loading buffer prior to being resolved by SDS–PAGE electrophoresis on the basis of molecular weight (“kDa” = kiloDalton), transferred to a nitrocellulose membrane, and detected using anti-HA antibody immunoblotting (“IB”:) and chemiluminescence. (B) Overexpression of wild-type RGS21, but not a loss-of-function, point mutant RGS21, leads to inhibition of bitterant signaling-induced reduction of cAMP levels. Cultures of the 16HBE cell line were transiently transfected with GloSensor cAMP biosensor cDNA and expression plasmids containing open reading frames for wild-type (“wt”) RGS21, or Arg-126-to-Glu point mutated (“R>E”) RGS21, or empty pcDNA3.1 vector, as indicated. Inhibition of forskolin-stimulated cAMP production (“100% maximum”; dotted line) by treatment with indicated concentration of the bitterant denatonium benzoate (i.e. EC50 previously established as per Supplementary Fig. S1) was determined 24 hr post-transfection by detection of GloSensor-dependent luminescence. Asterisks denote statistically significant differences as determined by one-way ANOVA with Bonferroni’s post-test: **, P < 0.01; ns, not significant (P >> 0.05).

Article Snippet: Briefly, monolayer cultures of the 16HBE cell line were transiently cotransfected using FuGENE6 (Promega) with the GloSensor cAMP-biosensor cDNA (Promega) and pcDNA3.1-based expression plasmids encoding either wild-type RGS21 open-reading frame, or a GAP-dead, loss-of-function point-mutant version (R126E a.k.a.

Techniques: Cell Culture, Transfection, Plasmid Preparation, Incubation, Purification, Lysis, SDS Page, Electrophoresis, Molecular Weight, Western Blot, Over Expression, Mutagenesis, Inhibition, Expressing, Concentration Assay

Journal: Cell

Article Title: Human Gain-of-Function MC4R Variants Show Signaling Bias and Protect against Obesity

doi: 10.1016/j.cell.2019.03.044

Figure Lengend Snippet:

Article Snippet: GloSensor cAMP biosensor - pGloSensor 20F plasmid , Promega , Cat#E1171.

Techniques: Virus, Recombinant, Plasmid Preparation, Software