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

Journal: bioRxiv

Article Title: In vivo photopharmacology with light-activated opioid drugs

doi: 10.1101/2023.02.02.526901

Figure Lengend Snippet: (A) Reaction scheme depicting the one-step alkylation procedure used to synthesize DEAC-OXM from commercially available oxymorphone ( 2 ) and DEAC-Br, as well as its photochemical conversion to the proposed primary reaction product “rearranged DEAC-OXM” (RE-DEAC-OXM), which likely occurs via a 1,4-Photo-Claisen rearrangement. (B) High pressure liquid chromatography (HPLC) chromatograms measured at 220 nm indicating predominant photoconversion of DEAC-OXM to RE-DEAC-OXM, which has a similar retention time, along with a much smaller amount of OXM in PBS (pH 7.2). (C) (Top) LC-MS (mass spectrometry) chromatogram of the reaction mixture shown in B. (Bottom) MS traces revealing that RE-DEAC-OXM has the same molecular weight as DEAC-OXM (531 Da). (D) Agonist dose-response curves at the MOR for DEAC-OXM, RE-DEAC-OXM, and OXM using the GloSensor™ assay of cAMP signaling in HEK293T cells. The solid line depicts the best-fit sigmoidal function used to derive the indicated EC 50 value. OXM EC 50 = 1.3 nM, DEAC-OXM EC 50 = 380 nM, RE-DEAC-OXM EC 50 = 1.3 µM. Data were normalized to the response produced by DAMGO (1 µM) and are expressed as mean ± SEM (n=5-10 wells per concentration).

Article Snippet: Then the SSF-MOR plasmid, GloSensor (Promega) 22F cAMP dependent reporter plasmid (Promega), and Lipofectamine 2000 (Invitrogen) in Opti-MEM were added.

Techniques: High Performance Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Molecular Weight, Produced, Concentration Assay

Journal: bioRxiv

Article Title: In vivo photopharmacology with light-activated opioid drugs

doi: 10.1101/2023.02.02.526901

Figure Lengend Snippet: (A) Agonist dose-response curves at the mu opioid receptor (MOR) for PhOX and OXM using the GloSensor™ assay of cAMP signaling in HEK293T cells. The solid line depicts the best-fit sigmoidal function used to derive the indicated EC 50 value. OXM EC 50 = 1.4 nM. Data were normalized to the response produced by DAMGO (1 µM) and are expressed as mean ± SEM (n=5 wells per concentration). (B) Antagonist dose-response curves at the MOR for NLX and PhNX in the presence of DAMGO (100 nM) using the GloSensor™ assay. Data are presented as in A. NLX IC 50 = 86 nM. (C) Agonist dose-response curves at the MOR for DAMGO in the absence and presence of NLX (100 nM) or PhOX (300 nM) using the GloSensor™ assay. Data are presented as in A. DAMGO EC 50 = 0.7 nM, DAMGO + PhOX EC 50 = 0.9 nM, DAMGO + NLX EC 50 = 32 nM. (D) Agonist dose-response curves at the MOR for DAMGO and PhOX using a NanoBiT-based luminescence complementation assay of β-arrestin signaling in HEK293T cells (n=4 wells per concentration). DAMGO EC 50 = 11 nM. Data were normalized to the maximal response to DAMGO (10 µM) and are expressed as the mean ± SEM.

Article Snippet: Then the SSF-MOR plasmid, GloSensor (Promega) 22F cAMP dependent reporter plasmid (Promega), and Lipofectamine 2000 (Invitrogen) in Opti-MEM were added.

Techniques: Produced, Concentration Assay

Journal: bioRxiv

Article Title: Antagonistic interactions between odorants alter human odor perception

doi: 10.1101/2022.08.02.502184

Figure Lengend Snippet: A) Dose-response curves of OR2T11 against increasing concentrations of ionones and damascones, with the vapor concentration of CH 3 SH held constant at 7 ppm. IC50 value was calculated using Graph pad Prism software. B) Antagonistic effect of damascone & ionone analogs. 41 ppm H 2 S stimulation on human OR2T1, OR2T6 and OR2T11 were masked by 100 µM fragrance compounds. Multiple comparisons were performed using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test (*** p <0.001). C) Inhibitor odorants did not cause adverse effects on the assay system. OR2T11 and mouse Or2aj6 (Olfr171) (damascones responding receptor) expressing cells were stimulated by 100µM odorants and Glosensor buffer without any odorant as a negative control. Error bars indicate s.e.m (n=3) Multiple comparisons were performed using one-way ANOVA followed by Dunnett’s test (*** p <0.001).

Article Snippet: 18-24 hours after plating, cells were transfected with 80 ng/well of plasmids encoding ORs, 5 ng/well of RTP1S , and 10 ng/well of Glosensor plasmid (Promega).

Techniques: Concentration Assay, Software, Expressing, Negative Control