anti α1 antibody  (Alomone Labs)

Journal: The Journal of Neuroscience

Article Title: Influence of GABAAR Monoliganded States on GABAergic Responses

doi: 10.1523/JNEUROSCI.1453-10.2011

Figure Lengend Snippet: α1 HA-F64C coassembles with α1 subunit in the same GABA A R. A , Single plane confocal images of HEK293 cells overexpressing α1 HA-F64C β1γ2 receptors and EGFP probed with anti-α1- and anti-HA antibodies as indicated. Scale bar, 10 μm ( n = 15). B , Heteromerization of α1 and α1 HA-F64C within the same receptor. Membrane fractions of HEK293 cells transfected with α1α1 HA-F64C β1γ2 (INPUT) were immunoprecipitated with the anti-HA antibody (IP anti-HA) and immunoblotted with the anti-α1 (top) or anti-HA antibody (bottom) ( n = 3).

Article Snippet: Anti-α1 antibody (Alomone Labs) was directed against the N-terminal region of the α1subunit.

Techniques: Transfection, Immunoprecipitation

Journal: bioRxiv

Article Title: Pannexin-1 mediated ATP release in adipocytes is sensitive to glucose and insulin and modulates lipolysis and macrophage migration

doi: 10.1101/380469

Figure Lengend Snippet: Cell model of adrenergically induced signaling resulting in ATP release and autocrine and paracrine effects in white adipose tissue. (A) Adrenergic stimulation of adipocytes induces cAMP increase, PKA activation, boost in cell metabolism and opening of Panx1 mediating ATP release. Insulin tightly regulates this pathway via activation of PDE3, which hydrolyses cAMP and downregulates the activation of downstream events, i.e. reduces ATP release. (B) In pathological conditions, such as insulin deficiency and/or insulin resistance, insulin regulation of cAMP levels would be missing, and thus PKA and Panx1 activation, and finally ATP release, would be unhindered. Extracellular ATP acts as an autocrine signal, stimulating intracellular Ca 2+ signaling and lipolysis via P2 receptors activation. Extracellular ATP functions also as a paracrine signal, for example, as a chemoattractant for adipose tissue macrophages, stimulating their migration by activating of P2X7 receptors. The model predicts that elevated extracellular ATP (as in b) would lead to stronger and perhaps detrimental auto-/paracrine signaling in white adipose tissue.

Article Snippet: Membranes were blocked with 5% skim milk solution in TBS-Tween (0.1 %) buffer for 1 h at room temperature and incubated overnight at 4°C with primary antibody against P2X7R (1:500 rabbit polyclonal, Alomone APR-004), pannexin-1 (1:1000 rabbit polyclonal, Alomone ACC-234) and vinculin (1:1000 mouse monoclonal, Sigma-Aldrich V9131).

Techniques: Activation Assay, Migration

Journal: bioRxiv

Article Title: Pannexin-1 mediated ATP release in adipocytes is sensitive to glucose and insulin and modulates lipolysis and macrophage migration

doi: 10.1101/380469

Figure Lengend Snippet: Adipocytes express Panx1 that is involved in the adrenergically stimulated pore formation. (A) Representative gel of Panx1 mRNA expression and (B) representative western blot of Panx1 protein expression in CH3H10T1/2 pre-adipocytes and mature adipocytes. Vinculin was used as a loading control. (C) Western blot quantification of Panx1A and Panx1C isoforms expression. (D) Representative recordings of EtBr (0.5 μM) uptake in single mature adipocytes (n=30) stimulated with 5 μM phenylephrine (PE) and fluorescence was recorded every 2 min for 75 min. (E) Representative images of brightfield (grey) and EtBr fluorescence (red) after 70 min of stimulation with control buffer (Ctrl) or phenylephrine (PE) in presence or absence of 100 μM 10 Panx (10Panx) or 30 μM Carbenoxolone (CBX). (F) The time-course of EtBr uptake after stimulation with control buffer (Ctrl) or phenylephrine (PE) alone, and in combination with 100 μM 10 Panx or 30 μM CBX. The graph shows the mean ± s.e.m. responses of 90 cells per condition per independent experiment. (g) Quantification of EtBr uptake as difference between the basal fluorescence (before stimulation) and the peak uptake (70 min) with the indicated treatment. All data are shown as mean values ± s.e.m. and significance related to the control condition is indicated p

Article Snippet: Membranes were blocked with 5% skim milk solution in TBS-Tween (0.1 %) buffer for 1 h at room temperature and incubated overnight at 4°C with primary antibody against P2X7R (1:500 rabbit polyclonal, Alomone APR-004), pannexin-1 (1:1000 rabbit polyclonal, Alomone ACC-234) and vinculin (1:1000 mouse monoclonal, Sigma-Aldrich V9131).

Techniques: Expressing, Western Blot, Fluorescence