Journal: Biomolecules

Article Title: Why Does Knocking Out NACHO, But Not RIC3, Completely Block Expression of α7 Nicotinic Receptors in Mouse Brain?

doi: 10.3390/biom10030470

Figure Lengend Snippet: RIC3 antibodies used in this paper.

Article Snippet: Alomone Laboratories ANC-020 anti-RIC3 antibody showed a similar band pattern and recognized both splice variants of human and mouse RIC3, and rat RIC3 ( C).

Techniques:

Journal: Biomolecules

Article Title: Why Does Knocking Out NACHO, But Not RIC3, Completely Block Expression of α7 Nicotinic Receptors in Mouse Brain?

doi: 10.3390/biom10030470

Figure Lengend Snippet: RIC3 sequences across species showing antibody antigens and mutations.

Article Snippet: Alomone Laboratories ANC-020 anti-RIC3 antibody showed a similar band pattern and recognized both splice variants of human and mouse RIC3, and rat RIC3 ( C).

Techniques:

Journal: Biomolecules

Article Title: Why Does Knocking Out NACHO, But Not RIC3, Completely Block Expression of α7 Nicotinic Receptors in Mouse Brain?

doi: 10.3390/biom10030470

Figure Lengend Snippet: Assessment of RIC3 antibodies by Western blot. ( A ) Anti-DDK shows RIC3 expression efficiency following cell transfections. DDK–tagged RIC3s showed as single bands around 40 kD with considerable differences among different species. Rat RIC3 was not tagged with DDK. ( B ) Thermo-Fisher anti-hRIC3 PA5-64196 (1:1000) recognizes both splice variants of human and mouse RIC3 (with multiple bands) and weakly stains rat RIC3, but not Xenopus. ( C ) Alomone Laboratories anti-RIC3 antibody (ANC-020, 1:1000) showed a similar but weaker pattern and recognized both splice variants of human and mouse RIC3 (with additional bands) and weakly rat RIC3. A high MW (~100 kD) band is non-specific due to its presence in the control. Numbers in parentheses refer to the amount of protein added to each well to account for differences in protein expression between transfections.

Article Snippet: Alomone Laboratories ANC-020 anti-RIC3 antibody showed a similar band pattern and recognized both splice variants of human and mouse RIC3, and rat RIC3 ( C).

Techniques: Western Blot, Expressing, Transfection

Journal: Biomolecules

Article Title: Why Does Knocking Out NACHO, But Not RIC3, Completely Block Expression of α7 Nicotinic Receptors in Mouse Brain?

doi: 10.3390/biom10030470

Figure Lengend Snippet: ( A ) Autoradiographic comparison of 125 I-αBGT binding between wild type and KO animal brain slices. Top row shows total binding for wild type (left), tmem35a KO (middle) and ric3 KO (right) brain sections. The bottom row shows corresponding non-specific binding. There was no specific binding in tmem35a KO, and significant loss of binding in specific brain structures in the ric3 KO brains (arrows). ( B ) Autoradiographic analysis of 125 I-αBGT binding using ImageJ. Significant loss of toxin binding was observed in the hippocampus and cortex of the ric3 KO compared to the corresponding structures in wild type (WT) animals (Specific binding is the difference between total binding and non-specific [NS] binding). The insets show typical sections and the areas used for analysis over two sections per condition (N = 8 areas per brain region, with a medial and lateral area for each brain side times two sections). This analysis was done on one experiment comparing one animal per condition since the two experiments performed so far were done using different batches of 125 I-αBGT with different specific activities and slightly different exposure times and are not easily comparable. Error bars represent standard deviations. *** p > 0.001, (** p < 0.01, * p < 0.05) by single factor ANOVA.

Article Snippet: Alomone Laboratories ANC-020 anti-RIC3 antibody showed a similar band pattern and recognized both splice variants of human and mouse RIC3, and rat RIC3 ( C).

Techniques: Binding Assay

Journal: Biomolecules

Article Title: Why Does Knocking Out NACHO, But Not RIC3, Completely Block Expression of α7 Nicotinic Receptors in Mouse Brain?

doi: 10.3390/biom10030470

Figure Lengend Snippet: The absence of NACHO in HEK cells has no effect on the ability of RIC3 to promote surface human α7nAChR expression, and the effects of the two chaperones are synergistic when expressed together. Binding assays in 24-well plates were performed as indicated in methods. Total cDNA in transfections was constant, with h chrna7 DNA (0.15 μg/well) that was equaled the sum of htmem35a and hric3 cDNA or RFP DNA (0.15 μg/well RFP DNA in transfection controls). The ratio of 3 parts h tmem35a cDNA to 1 part h ric3 cDNA (e.g., 0.11 µg h tmem35a and 0.04 µg h ric3 /well) produced the highest surface α7nAChR expression in HEK cells. In all 4 experiments, the combined effects were more than additive. In experiments where RIC3 or NACHO was the only chaperone, surface α7nAChR expression was comparable between these two chaperones as shown.

Article Snippet: Alomone Laboratories ANC-020 anti-RIC3 antibody showed a similar band pattern and recognized both splice variants of human and mouse RIC3, and rat RIC3 ( C).

Techniques: Expressing, Binding Assay, Transfection, Produced

Journal: The Journal of Neuroscience

Article Title: Influence of GABA A R Monoliganded States on GABAergic Responses

doi: 10.1523/JNEUROSCI.1453-10.2011

Figure Lengend Snippet: Characterization of α1α1F64Cβ1γ2 receptor gating properties. A, Summary of the dependence of current amplitude (left) and decay kinetics (right) upon the ratio between α1 and α1F64C overexpression. B, The 10–90% rise time of normalized α1α1F64Cβ1γ2 (thick line) and α1β1γ2 (thin line) currents. C, Comparison of the deactivation kinetics between α1α1F64Cβ1γ2 (left) and α1β1γ2 (right) normalized currents. D, Desensitization induced by 100 ms, 10 mm GABA pulse in α1α1F64Cβ1γ2 (left) and α1β1γ2 (right) receptors. E, Desensitization induced by 3000 ms, 10 mm GABA pulse in α1α1F64Cβ1γ2 (left) and α1β1γ2 (right). F, Currents elicited by two consecutive 10 ms pulses spaced by 100 ms from α1α1F64Cβ1γ2 (left) and α1β1γ2 (right) receptors. G, Typical response of α1α1F64Cβ1γ2 (left) and α1β1γ2 (right) currents to long (3000) ms conditioning pulse followed by a short (10 ms) test pulse. H, Summary of recovery from desensitization of paired pulse experiments shown in F. α1α1F64Cβ1γ2 currents (black circles), at the considered time points, showed immediate recovery, while the α1β1γ2 receptors (open circles) displayed biexponential recovery (τfast = 8 ± 0.2 and τslow = 321 ± 21 ms, Afast = 0.36 ± 0.03). I, Summary of recovery from desensitization of paired pulse experiments shown in G. Both α1α1F64Cβ1γ2 currents (black squares) α1β1γ2 currents (open squares) showed biexponential recovery (τw = 27.3 ± 2.7 and τw 8.4 ± 1.1 s, respectively). J, Summary of the dependence of α1β1γ2- and α1α1F64Cβ1γ2 currents deactivation kinetics on [GABA] (black, 1 ms pulse; gray, 20 ms pulse). K, Summary of the dependence of α1β1γ2- and α1α1F64Cβ1γ2 currents onset kinetics on [GABA] (3000 and 20 ms pulses for black and gray, respectively). Each data point results from at least 20 recordings.

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

Techniques: Over Expression

Journal: The Journal of Neuroscience

Article Title: Influence of GABA A R Monoliganded States on GABAergic Responses

doi: 10.1523/JNEUROSCI.1453-10.2011

Figure Lengend Snippet: Model simulation of α1β1γ2 (doubly bound) and α1α1F64Cβ1γ2 (singly bound) gating properties. A, Gating scheme for GABAA receptor (Jones and Westbrook, 1995). In the present study, singly bound currents were simulated by using the R, AR, AR*, and AD states (in black). Doubly bound currents were simulated by adding the A2R, A2R* and A2D states (in gray). The rate constants used were: kon1 = 2.877 ± 0.177 ms−1 mm−1, koff = 0.267 ± 0.021 ms−1, β1 = 0.086 ± 0.007 ms−1, α1 = 0.208 ± 0.016 ms−1, d1 = 0.00023 ± 0.00002 ms−1, r1 = 0.00035 ± 0.00003 ms−1, kon2 = 18.29 ms−1 mm−1, β2 = 8.85 ms−1, α2 = 0.42 ms−1, d2 = 1.32 ms−1, r2 = 0.032 ms−1. B, Simulated (red) and experimental (black) onset of currents evoked by saturating and low GABA concentrations (as indicated) from singly bound receptors. C, Simulated (red) and experimental (black) currents showing the deactivation kinetics for singly and doubly bound receptor-mediated currents. D, Summary of simulated 10–90% rise-time dependence on the GABA concentration for singly and doubly bound receptor configurations. E, Simulated (red) desensitization for doubly and singly bound currents superimposed to experimental traces (black). F, Simulated and experimental currents (red and black, respectively), showing slow desensitization onset for singly bound currents. Doubly bound currents are not shown since more doubly bound desensitized state must be included in the model to adequately reproduce desensitization evoked by long GABA pulses. G, Recovery from desensitization using paired pulse protocol for simulated (red) and experimental currents (black). H, Recovery from desensitization of simulated (red) and experimental traces (black) after long conditioning pulses for singly bound currents. Note that in B–H, traces are normalized for better visualization of kinetic differences.

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

Techniques: Concentration Assay

Journal: The Journal of Neuroscience

Article Title: Influence of GABA A R Monoliganded States on GABAergic Responses

doi: 10.1523/JNEUROSCI.1453-10.2011

Figure Lengend Snippet: α1F64Cβ1γ2 receptors expressed at the cell surface are not functional. A, Single plane confocal images of EGFP fluorescence and surface α1 immunolabeling of HEK293 cells transfected with either α1β1γ2 (top) or α1F64Cβ1γ2 (bottom) subunits along with EGFP. Note that the anti-α1 antibody recognizes both α1 and α1F64C. Scale bars, 10 μm (n = 12). B, Line scans of fluorescence intensity of α1 immunolabeling in HEK293 cells transfected with α1β1γ2 (top) or α1F64Cβ1γ2 (bottom) along the white lines (20 μm) reported in A. C, Current traces elicited by applying GABA pulses (1 ms, 10 mm) to patches containing α1β1γ2 (top) and α1F64Cβ1γ2 (bottom) receptors (n = 20). D, Representative current traces elicited by applying pentobarbital (5 s, 500 μm) to patches containing α1β1γ2 (top) and α1F64Cβ1γ2 (bottom) receptors (n = 6). The presence of sizable currents from α1F64Cβ1γ2 indicates that mutant receptors, although not responsive to GABA, are expressed at the cell surface.

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

Techniques: Functional Assay, Fluorescence, Immunolabeling, Transfection, Mutagenesis

Journal: The Journal of Neuroscience

Article Title: Influence of GABA A R Monoliganded States on GABAergic Responses

doi: 10.1523/JNEUROSCI.1453-10.2011

Figure Lengend Snippet: α1HA-F64C coassembles with α1 subunit in the same GABAAR. A, Single plane confocal images of HEK293 cells overexpressing α1HA-F64Cβ1γ2 receptors and EGFP probed with anti-α1- and anti-HA antibodies as indicated. Scale bar, 10 μm (n = 15). B, Heteromerization of α1 and α1HA-F64C within the same receptor. Membrane fractions of HEK293 cells transfected with α1α1HA-F64Cβ1γ2 (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: Psychopharmacology

Article Title: Specificity of a rodent alpha(α)6 nicotinic acetylcholine receptor subunit antibody

doi: 10.1007/s00213-019-05413-x

Figure Lengend Snippet: Evaluation of an alpha(α)6 nicotinic acetylcholine receptor subunit antibody. A western blot of α6 nAChR subunit expression and antigen block in bilateral ventral tegmental tissue punches collected from male Sprague Dawley rats. n=2 animals total. GAPDH is used as a loading control.

Article Snippet: Thus, the purpose of this study is to validate the specificity of the commercially available polyclonal α6 nAChR subunit antibody from Alomone Labs (cat. #: ANC-006, Jerusalem, Israel).

Techniques: Western Blot, Expressing, Blocking Assay

Journal: Psychopharmacology

Article Title: Specificity of a rodent alpha(α)6 nicotinic acetylcholine receptor subunit antibody

doi: 10.1007/s00213-019-05413-x

Figure Lengend Snippet: Genetic validation of an alpha(α)6 nicotinic acetylcholine receptor subunit antibody. A.) Western blot and B.) relative band intensity quantification of α6 nAChR subunit expression and antigen block from whole brain tissue collected from male and female WT and α6 KO C57BL/6J mice, n=4 animals/genotype; **p< 0.01 and ***p< 0.001 α6 antibody vs. α6 antibody + antigen normalized signal. GAPDH is used as a loading control.

Article Snippet: Thus, the purpose of this study is to validate the specificity of the commercially available polyclonal α6 nAChR subunit antibody from Alomone Labs (cat. #: ANC-006, Jerusalem, Israel).

Techniques: Western Blot, Expressing, Blocking Assay

Journal: International Journal of Molecular Sciences

Article Title: α3β4 Acetylcholine Nicotinic Receptors Are Components of the Secretory Machinery Clusters in Chromaffin Cells

doi: 10.3390/ijms23169101

Figure Lengend Snippet: Membrane colocalization and overlapping between α3β4 nAChR (red) and DBH (green) after secretion stimulus: in each image we individually selected several regions of interest (ROIs) ( a , b ) and proceeded to analyze them using ImageJ JACoP complement (See ), obtaining the values of the Pearson’s and Manders coefficients, whose averages are shown in , as well as their corresponding scatterplot graphs, whose examples are shown ( d ). The averaged Pearson’s coeficients for 50% threshold images show a significant colocalization between endogenous α3β4 nAChRs structures and the DBH sites in both stimulation conditions to those obtained for randomly simulated α3β4 nAChRs structures ( ( c )). Furthermore, the averaged coefficients obtained for the stimulus with ACh are significantly higher than those obtained when stimulating with a high K + ( p value *** < 0.0001; ). ( d ) A scatterplot or fluorogram for pixel colocalization of the red channel (AChR structures) and green channel (DBH) comparing three examples of the images obtained in each of the three analyzed conditions. Bars represent 1 µm.

Article Snippet: We revealed the immunological location of the endogenous β4 subunits using a rabbit polyclonal antibody anti-Nicotinic Acetylcholine Receptor β4 (extracellular)-Atto-594 (alomone Labs, Cat #: ANC-014-AR; Lot: ANC014ARAN0150, Jerusalem, Israel).

Techniques: Membrane

Journal: International Journal of Molecular Sciences

Article Title: α3β4 Acetylcholine Nicotinic Receptors Are Components of the Secretory Machinery Clusters in Chromaffin Cells

doi: 10.3390/ijms23169101

Figure Lengend Snippet: Parameters used in the simulation of buffered calcium diffusion in a conical domain.

Article Snippet: We revealed the immunological location of the endogenous β4 subunits using a rabbit polyclonal antibody anti-Nicotinic Acetylcholine Receptor β4 (extracellular)-Atto-594 (alomone Labs, Cat #: ANC-014-AR; Lot: ANC014ARAN0150, Jerusalem, Israel).

Techniques: Diffusion-based Assay, Concentration Assay, Binding Assay