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