Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Association of the α 2 δ 1 Subunit with Ca v 3.2 Enhances Membrane Expression and Regulates Mechanically Induced ATP Release in MLO-Y4 Osteocytes

doi: 10.1002/jbmr.437

Figure Lengend Snippet: RT-PCR primers used for detection of VSCC subunits and osteocyte markers

Article Snippet: The rabbit anti-β 3 antibody was purchased from Alomone Research Laboratories (Jerusalem, Israel).

Techniques: Sequencing

Journal: Neurobiology of disease

Article Title: Inhibitory and excitatory synaptic neuroadaptations in the diazepam tolerant brain

doi: 10.1016/j.nbd.2023.106248

Figure Lengend Snippet: Synaptic and extrasynaptic GABA A receptor and gephyrin redistribution with seven-day DZP treatment in vivo. (A-F) Mice were treated i.p. once daily for seven days with Veh or DZP. Western blot analysis of collected cortical tissue was performed to assess protein levels of GABA A R subunits and gephyrin from total (A, B), synaptic (C, D), and extrasynaptic (E, F) fractions. Representative blots show five mice from each treatment group. Proteins quantified by western blot were normalized to GAPDH or KIR3.2 loading control. (B) Measurements of total protein levels revealed a significant increase in α1 ( p = 0.0018), β3 ( p = 0.0005), and γ2 ( p = 0.0002) subunits. The amount of synaptic α1 ( p = 0.0019), α4 ( p = 0.0028), and γ2 ( p = 0.0007) subunits also increased (D), while extrasynaptic α1 ( p = 0.0034) and α4 ( p = 0.0010) subunits were decreased and gephyrin ( p = 0.0040) was increased (F) (* p ≤ 0.05, ** p < 0.01, *** p < 0.001, Student’s t -test; n = 5–10 mice per treatment; error bars ± S.E.M.).

Article Snippet: Primary antibodies: GAPDH (RRID: AB_561053, #2118, Cell Signaling); NMDAR Receptor 1 (GluN1) (RRID: AB_1904067, #5704, Cell Signaling); NMDA NR2B subunit (GluN2B) (RRID: AB_397797, #610417, BD Biosciences); NMDA NR2A subunit (GluN2A) (RRID: AB_2492170, #1500-NR2A, PhosphoSolutions); GABA A Receptor α1 (RRID: AB_310272, #06–868, Millipore); GABA A Receptor α4 (RRID: AB_2492103, #845-GA4C, PhosphoSolutions); GABA A Receptor α5 (RRID: AB_2619944, #224503, Synaptic Systems); GABA A Receptor β3 (RRID: AB_2492110, #863-GB3C, PhosphoSolutions); GABA A Receptor γ2 (RRID: AB_2263066, #224003, Synaptic Systems; RRID: AB_10594245, #224004, Synaptic Systems); gephyrin (RRID: AB_640963, #sc-14003, Santa Cruz Biotechnology); Kir3.2 (RRID: AB_2040115, #APC-006, Alomone Labs).

Techniques: In Vivo, Western Blot

Journal: Neurobiology of disease

Article Title: Inhibitory and excitatory synaptic neuroadaptations in the diazepam tolerant brain

doi: 10.1016/j.nbd.2023.106248

Figure Lengend Snippet: γ2 containing GABA A receptor composition is unchanged and tonic inhibition is reduced in DZP mice. (A) Immunoprecipitation of γ2-GABA A R from seven-day Veh- or DZP-treated mouse cortex was analyzed by DIA mass spectrometry to assess changes in receptor subunit composition ( n = 4 mice per treatment group). The intensity of α1–5 subunit-specific peptides are shown. Inset: Relative abundance (%) of α and β subunits associated with γ2 after seven-day DZP treatment. (B,C) (B) Left: representative traces with mIPSCs from seven-day DZP-treated animals before (dark red) and after (gray) 300 nM Ro 15–4513 application. Right: averaged mIPSCs before and after Ro 15–4513. (C) Quantification shows inverse agonist activity of Ro 15–4513, consistent with predominant receptors composed of γ2 with α1, α2, α3, α5-GABA A R subunits ( n = 5 cells; amplitude, p = 0.0191; frequency, p = 0.0179; tau, p = 0.0026). (D) GABA A R-mediated tonic current was measured in acute cortical slices from mice treated i.p. once daily for seven days with Veh or DZP. Picrotoxin-sensitive changes in holding current (V hold = −70 mV) were used to measure tonic inhibition in cortical slices from seven-day Veh- or DZP-treated mice. (E) Quantification revealed that GABA A R-mediated tonic current was significantly reduced (p = 0.0084) in DZP-treated mice ( n = 8) relative to Veh-treated mice ( n = 6). (E: ** p ≤ 0.01, Student’s t -test; C: * p ≤ 0.05, **p ≤ 0.01, paired t-test; error bars ± S.E.M.).

Article Snippet: Primary antibodies: GAPDH (RRID: AB_561053, #2118, Cell Signaling); NMDAR Receptor 1 (GluN1) (RRID: AB_1904067, #5704, Cell Signaling); NMDA NR2B subunit (GluN2B) (RRID: AB_397797, #610417, BD Biosciences); NMDA NR2A subunit (GluN2A) (RRID: AB_2492170, #1500-NR2A, PhosphoSolutions); GABA A Receptor α1 (RRID: AB_310272, #06–868, Millipore); GABA A Receptor α4 (RRID: AB_2492103, #845-GA4C, PhosphoSolutions); GABA A Receptor α5 (RRID: AB_2619944, #224503, Synaptic Systems); GABA A Receptor β3 (RRID: AB_2492110, #863-GB3C, PhosphoSolutions); GABA A Receptor γ2 (RRID: AB_2263066, #224003, Synaptic Systems; RRID: AB_10594245, #224004, Synaptic Systems); gephyrin (RRID: AB_640963, #sc-14003, Santa Cruz Biotechnology); Kir3.2 (RRID: AB_2040115, #APC-006, Alomone Labs).

Techniques: Inhibition, Immunoprecipitation, Mass Spectrometry, Activity Assay

Journal: eLife

Article Title: A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain

doi: 10.7554/eLife.43322

Figure Lengend Snippet: Non-R-mAb antibodies used in this study. Table lists Abs used in this study outside of the R-mAbs whose generation is described here. For each Ab the name, immunogen used in Ab generation, source and RRID number in the Antibody Registry, form and concentration/dilution used, and specific use in this paper is detailed.

Article Snippet: N87/25 , Fusion protein aa 370–433 of mouse GABA-A-receptor β3 subunit , Mouse IgG1 mAb, NeuroMab RRID: AB_10673389 , Tissue culture supernatant, 1:5 , 4.

Techniques: Concentration Assay, Affinity Purification, Recombinant

Journal: British Journal of Pharmacology

Article Title: 2?-Methoxy-6-methylflavone: a novel anxiolytic and sedative with subtype selective activating and modulating actions at GABA A receptors

doi: 10.1111/j.1476-5381.2011.01604.x

Figure Lengend Snippet: 2′MeO6MF is a positive modulator of α1-containing GABAA receptors expressed in Xenopus oocytes. (A) Representative current trace showing the potentiation of GABA (EC10: 10 µM) by various concentrations of 2′MeO6MF (1, 10, 30, 100 and 300 µM) at human recombinant α1β2γ2L GABAA receptors. Horizontal bars show the duration of drug application. In the absence of GABA, 2′MeO6MF (300 µM) produced ≍2% of the current produced by the maximum GABA concentration. Note the rebound current following removal of 2′MeO6MF (indicated by the arrows) (B) 2′MeO6MF potentiates the response to GABA (EC10) at recombinant α1β2, α1β1γ2, α1β2γ2L and α1β3γ2L receptors expressed in oocytes. Control GABA concentration at each receptor subtype was 10 µM. Data are mean ± SEM (n = 3–6 oocytes). (C) Representative current trace from an oocyte showing potentiation of GABA (10 µM) by 2′MeO6MF (100 µM) at recombinant α1β2γ2L GABAA receptors. As shown, the enhancement of GABA (10 µM) by 2′MeO6MF (100 µM) was not inhibited by flumazenil (1 and 10 µM) whereas potentiation of GABA (10 µM) by diazepam (1 µM) was inhibited by flumazenil (1 and 10 µM). Horizontal bars show the duration of drug application.

Article Snippet: GABA receptor subunit constructs Human α1, α2, β2 and γ2L DNA in pcDM8 were provided by Dr Paul Whiting (Merck, Sharpe and Dohme Research Labs, Harlow, UK), α3 and β3 in pGEMHE, α5 in pcDNA3 and β1 in PCDM8 were a gift from Dr Bjarke Ebert (H. Lundbeck A/S, Valby, Denmark).

Techniques: Recombinant, Produced, Concentration Assay, Control

Journal: British Journal of Pharmacology

Article Title: 2?-Methoxy-6-methylflavone: a novel anxiolytic and sedative with subtype selective activating and modulating actions at GABA A receptors

doi: 10.1111/j.1476-5381.2011.01604.x

Figure Lengend Snippet: The effects of 2′MeO6MF at the various recombinant ionotropic GABA receptor subtypes expressed in Xenopus oocytes

Article Snippet: GABA receptor subunit constructs Human α1, α2, β2 and γ2L DNA in pcDM8 were provided by Dr Paul Whiting (Merck, Sharpe and Dohme Research Labs, Harlow, UK), α3 and β3 in pGEMHE, α5 in pcDNA3 and β1 in PCDM8 were a gift from Dr Bjarke Ebert (H. Lundbeck A/S, Valby, Denmark).

Techniques: Recombinant

Journal: British Journal of Pharmacology

Article Title: 2?-Methoxy-6-methylflavone: a novel anxiolytic and sedative with subtype selective activating and modulating actions at GABA A receptors

doi: 10.1111/j.1476-5381.2011.01604.x

Figure Lengend Snippet: 2′MeO6MF directly activates human recombinant α2β2/3γ2L and α2β2/3 GABAA receptors expressed in oocytes. (A) Current trace showing the effects of increasing concentrations of 2′MeO6MF on recombinant α2β2γ2L GABAA receptors expressed in Xenopus oocytes against the maximum effect of GABA (3000 µM). Horizontal bars indicate duration of drug application. (B) Representative trace showing the potentiation of GABA (EC10: 5 µM) in the presence of diazepam (1 µM) and this effect was attenuated by flumazenil (10 µM) at α2β2γ2L GABAA receptors. In contrast, the direct activation of 2′MeO6MF (30 µM) on the same receptor was not attenuated by flumazenil (10 µM). (C) Representative trace showing that a 3 min pre-incubation with bicuculline (10 µM) attenuates the response produced by 2′MeO6MF (100 µM) by 56 ± 4% at α2β2γ2L GABAA receptors. Gabazine (10 µM and 100 µM) also attenuated the response produced by 2′MeO6MF on α2β2γ2L GABAA receptors by 45 ± 4% and 100% respectively. (D) Concentration–response curves for 2′MeO6MF alone and 2′MeO6MF in the presence of bicuculline (10 µM) and gabazine (1 µM) at α2β2γ2L GABAA receptors expressed in Xenopus oocytes. Data are mean ± SEM (n = 4–6 oocytes). (E) Concentration–response curves for 2′MeO6MF on α2β3γ2L, α2β2γ2L, α2β3 and α2β2 recombinant GABAA receptors expressed in oocytes. Currents are expressed as a percentage of the peak current elicited by 3 mM GABA. Data are mean ± SEM (n = 4–6 oocytes).

Article Snippet: GABA receptor subunit constructs Human α1, α2, β2 and γ2L DNA in pcDM8 were provided by Dr Paul Whiting (Merck, Sharpe and Dohme Research Labs, Harlow, UK), α3 and β3 in pGEMHE, α5 in pcDNA3 and β1 in PCDM8 were a gift from Dr Bjarke Ebert (H. Lundbeck A/S, Valby, Denmark).

Techniques: Recombinant, Activation Assay, Incubation, Produced, Concentration Assay

Journal: British Journal of Pharmacology

Article Title: 2?-Methoxy-6-methylflavone: a novel anxiolytic and sedative with subtype selective activating and modulating actions at GABA A receptors

doi: 10.1111/j.1476-5381.2011.01604.x

Figure Lengend Snippet: (A) Representative trace showing the activation of 2′MeO6MF (300 µM) is significantly reduced at the mutant α2β2N265Sγ2L GABAA receptor. In contrast, 2′MeO6MF (300 µM) was able to positively modulate GABA (10 µM) by 454 ± 16%. Horizontal bars show duration of drug application. (B) Concentration–response curves for 2′MeO6MF in the presence of GABA (EC10) at α2β1, α2β1γ2L and α2β2N265Sγ2L. Control GABA dose was 10 µM. Data are mean ± SEM (n = 4 oocytes). (C) Representative trace showing 2′MeO6MF (100 µM) directly activating recombinant α2β1S265Nγ2L mutant GABAA receptors. The current generated by 2′MeO6MF (100 µM) in the presence of GABA (10 µM) is the sum of the individual currents produced by GABA (10 µM) and 2′MeO6MF (100 µM) alone. Horizontal bars show duration of drug application. (D) Concentration–response curve for 2′MeO6MF expressed relative the maximal GABA concentration (3 mM) at α2β1S265Nγ2L GABAA receptors showing that the converse mutation converted 2′MeO6MF from a potentiator to a direct activator. Data are mean ± SEM for three to four oocytes.

Article Snippet: GABA receptor subunit constructs Human α1, α2, β2 and γ2L DNA in pcDM8 were provided by Dr Paul Whiting (Merck, Sharpe and Dohme Research Labs, Harlow, UK), α3 and β3 in pGEMHE, α5 in pcDNA3 and β1 in PCDM8 were a gift from Dr Bjarke Ebert (H. Lundbeck A/S, Valby, Denmark).

Techniques: Activation Assay, Mutagenesis, Concentration Assay, Control, Recombinant, Generated, Produced