talk-1 antibody  (Novus Biologicals)

Journal: Molecular Pharmacology

Article Title: Identification of potent and selective small molecule TALK-1 inhibitors that promote β -cell function

doi: 10.1016/j.molpha.2026.100130

Figure Lengend Snippet: High-throughput screening approach to identify TALK-1 inhibitors. (A) Diagram of the HTS pipeline to identify TALK-1 inhibitors. (B) Western blot showing tetracycline (Tet.) induction of TALK-1 ion channel expression in the T-Rex-TALK-1 cell line. (C) Schematic of Tl + flux assay. Green circles represent thallos dye. (D) Representative Tl + flux trace of Tet. induced and uninduced T-REx-TALK-1 cells before and after the indicated concentrations of Tl + addition ( n = 12 well/condition). (E) TALK-1 inhibitors identified in the pilot screens. (F) TALK-1 niclosamide CRC (IC 50 = 292 nM; 95% confidence interval [CI], 252–337 nM; n = 6 wells/concentration; error bars represent standard deviation). (G) Scatter plot of induced T-REx-TALK-1 cells treated with vehicle (DMSO) or niclosamide (10 μ M) in a checkerboard pattern. Z′ calculation for this 384-well plate was 0.664.

Article Snippet: The membrane was then blotted with primary antibodies: anti-TALK-1 antibody (APC-170, Alomone Labs) or anti-TALK-2 antibody (NBP1-92041, Novus Biologicals, LLC), followed by the secondary antibody: anti-Rabbit IgG (H+L), HRP conjugate.

Techniques: High Throughput Screening Assay, Western Blot, Expressing, Flux Assay, Concentration Assay, Standard Deviation

Journal: Molecular Pharmacology

Article Title: Identification of potent and selective small molecule TALK-1 inhibitors that promote β -cell function

doi: 10.1016/j.molpha.2026.100130

Figure Lengend Snippet: Primary TALK-1 HTS identified potent TALK-1 inhibitors. (A) Cartoon schematic of the workflow used for the full T-REx-TALK-1 HTS. (B) Scatter plot of a 384-well plate showing the percent inhibition of tetracycline (Tet)-induced T-REx-TALK-1 Tl + flux with TALK-1 inhibitors identified during the primary HTS. (C) Representative Tl + traces for Tet-induced T-REx-TALK-1 cells treated with either VHL, niclosamide, or VU0544194. (D) Confirmatory screen of the remaining TALK-1 inhibitors identified in the primary HTS: percent inhibition of Tl + flux slope. (E) Percent inhibition of Tl + flux AUC in uninduced T-REx-TALK-1 cells. (F) Percent inhibition of Tl + flux slope in T-REx-Kir4.2 cells. (G) Representative Tl + flux traces with either VHL, VU0646169, or niclosamide in Tet-induced T-REx-TALK-1 cells. (H) Representative Tl + flux traces comparing Tl + flux inhibition of VU0646169 in the uninduced T-REx-TALK-1 cells. (I) Representative Tl + flux traces comparing Tl + flux inhibition of VU0646169 in the T-REx-Kir4.2 cells.

Article Snippet: The membrane was then blotted with primary antibodies: anti-TALK-1 antibody (APC-170, Alomone Labs) or anti-TALK-2 antibody (NBP1-92041, Novus Biologicals, LLC), followed by the secondary antibody: anti-Rabbit IgG (H+L), HRP conjugate.

Techniques: Inhibition

Journal: Molecular Pharmacology

Article Title: Identification of potent and selective small molecule TALK-1 inhibitors that promote β -cell function

doi: 10.1016/j.molpha.2026.100130

Figure Lengend Snippet: Potencies of TALK-1 inhibitors were measured using 7-point CRC assays. (A–F) Concentration response curves (CRCs) and structures of 6 molecules tested in triplicate using a 7-point, 3-fold serial dilution series from 50 μ M to 400 nM. Percent inhibition of AUC of Tl + flux by the small molecule compounds was calculated using the formula: ([Compound AUC – VHL AUC]/[Niclosamide AUC – VHL AUC]) × 100, and IC 50 values were calculated using a 4-parameter logistic fit (Hill equation). VU0630026: 2.61 μ M (95% CI, 0.29–4.171 μ M); VU0646163: 5.15 μ M (95% CI, 3.62–7.64 μ M); VU0621279: 2.29 μ M (95% CI, 1.44–3.13 μ M); VU0544194: 5.20 μ M (95% CI, 3.96–6.92 μ M); VU0532253: 5.52 μ M (90% CI, 3.5–13.6 μ M); VU0520878: 1.27 μ M (90% CI, 0.0165–2.01 μ M) ( n = 3 replicates/concentration; error bars represent standard deviation).

Article Snippet: The membrane was then blotted with primary antibodies: anti-TALK-1 antibody (APC-170, Alomone Labs) or anti-TALK-2 antibody (NBP1-92041, Novus Biologicals, LLC), followed by the secondary antibody: anti-Rabbit IgG (H+L), HRP conjugate.

Techniques: Concentration Assay, Serial Dilution, Inhibition, Standard Deviation

Journal: Molecular Pharmacology

Article Title: Identification of potent and selective small molecule TALK-1 inhibitors that promote β -cell function

doi: 10.1016/j.molpha.2026.100130

Figure Lengend Snippet: Secondary screens for TALK-1 channel inhibitor selectivity. (A) Western blot showing tetracycline-induced TALK-2 expression in the T-REx-TALK-2 cell line. (B) Optimization of Tl + concentration was performed by testing 0.5, 1, 2, and 3 mM Tl + in tetracycline-induced or uninduced T-REx-TALK-2 cells ( n = 12 wells/condition). (C) AUC of Tl + flux (1 mM) in tetracycline-induced (+) or uninduced (-) T-REx-TALK-1 and T-REx-TALK-2 cells (AUC: T-REx-TALK-1 induced 339.5 ± 10.96 vs uninduced 211.1 ± 9.09; T-REx-TALK-2 induced 307.7 ± 0.420 vs uninduced 182.1 ± 1.554; n = 3; statistical analysis was performed using Student’s t test; ∗∗∗ P < .001, ∗∗∗∗ P < .0001). (D) Scatter plot showing Tl + AUC of tetracycline-induced T-REx-TALK-2 cells treated with TALK-1 inhibitor compounds ( n = 3 replicates/compound; error bars represent SEM). (E) Representative Tl + flux traces for either VHL, VU0547061, or uninduced T-REx-TALK-2. (F) The number of TALK-1 nonselective inhibitors identified in secondary screens against TALK-2, TASK-3, K ATP , and hERG channels.

Article Snippet: The membrane was then blotted with primary antibodies: anti-TALK-1 antibody (APC-170, Alomone Labs) or anti-TALK-2 antibody (NBP1-92041, Novus Biologicals, LLC), followed by the secondary antibody: anti-Rabbit IgG (H+L), HRP conjugate.

Techniques: Western Blot, Expressing, Concentration Assay

Journal: Molecular Pharmacology

Article Title: Identification of potent and selective small molecule TALK-1 inhibitors that promote β -cell function

doi: 10.1016/j.molpha.2026.100130

Figure Lengend Snippet: Testing the impact of VU0544194 (K2P16-i7) on Ca 2+ handling. (A) Schematic of SERCA inhibition with CPA to evaluate [Ca 2+ ] ER storage. (B) Average [Ca 2+ ] C traces for CPA-induced [Ca 2+ ] ER release measured in T-REx-TALK-1 cells with or without tetracycline induction, pretreated (1.5 hours) with VHL or K2P16-i7 (10 μ M). (C) Quantification of AUC for the respective CPA-induced [Ca 2+ ] C responses from (B) (VHL uninduced 225.5 ± 2.2; VHL induced 186.9 ± 1.99; K2P16-i7 uninduced 240.5 ± 6.46; K2P16-i7 induced 211.4 ± 7.27; n = 4 days (avg of 12 wells/d); statistical analysis performed using Tukey’s multiple comparisons test; ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001). (D) Structure of K2P16-i7, a TALK-1-selective lead compound. (E) Physicochemical properties of K2P16-i7 indicate alignment with Lipinski’s rule of five for drug-likeness of molecules.

Article Snippet: The membrane was then blotted with primary antibodies: anti-TALK-1 antibody (APC-170, Alomone Labs) or anti-TALK-2 antibody (NBP1-92041, Novus Biologicals, LLC), followed by the secondary antibody: anti-Rabbit IgG (H+L), HRP conjugate.

Techniques: Inhibition

Journal: Molecular Pharmacology

Article Title: Identification of potent and selective small molecule TALK-1 inhibitors that promote β -cell function

doi: 10.1016/j.molpha.2026.100130

Figure Lengend Snippet: K2P16-i7 increases β -cell Ca 2+ in a TALK-1–dependent manner. (A) Representative Ca 2+ oscillations from control islet clusters with glucose stimulation (16.7 mM) and K2P16-i7 addition. (B) Quantification of control islet cluster Ca 2+ plateau fraction before and after addition of K2P16-i7 (16.7 mM glucose 46.53% ± 1.97%; K2P16-i7 64.67% ± 3.86%; n = 4 mice; paired t test; ∗∗ P < .01). (C) Representative Ca 2+ oscillations from TALK-1 KO mouse islet clusters at 16.7 mM glucose and K2P16-i7 addition. (D) Quantification of TALK-1 KO islet Ca 2+ plateau fraction before and after addition of K2P16-i7 (16.7 mM glucose 48.16% ± 1.51%; K2P16-i7 55.46% ± 4.07%; n = 3 mice; paired t test; P = .113; 95% CI, –4.28 to 18.88).

Article Snippet: The membrane was then blotted with primary antibodies: anti-TALK-1 antibody (APC-170, Alomone Labs) or anti-TALK-2 antibody (NBP1-92041, Novus Biologicals, LLC), followed by the secondary antibody: anti-Rabbit IgG (H+L), HRP conjugate.

Techniques: Control

Journal: Molecular Pharmacology

Article Title: Identification of potent and selective small molecule TALK-1 inhibitors that promote β -cell function

doi: 10.1016/j.molpha.2026.100130

Figure Lengend Snippet: Identification of similar molecular scaffolds with improved potency to inhibit TALK-1. (A) Molecular structures of K2P16-i7-4 and K2P16-i7-19 with highlighted regions that are different from K2P16-i7. (B) 11-point CRCs of K2P16-i7 (IC 50 = 6.512 μ M; 95% CI, 2.96–10 μ M), K2P16-i7-19 (IC 50 = 4.054 μ M: the model failed to generate a reliable confidence interval due to insufficient constraint of the fit), and K2P16-i7-4 (IC 50 = 1.609 μ M: the model failed to generate a reliable confidence interval due to insufficient constraint of the fit) ( n = 6 wells/compound). (C) Average CPA-induced [Ca 2+ ] C traces measured in T-REx-TALK-1 cells with or without tetracycline induction, pretreated with VHL, K2P16-i7-4, or K2P16-i7-19 at 10 μ M concentration. (D) Quantification of the AUC for CPA responses from T-REx-TALK-1 cells treated as indicated. Statistical analysis performed using one-way ANOVA (with a Tukey’s multiple comparisons follow-up test) (VHL uninduced 127.5 ± 2.93; VHL induced 36.53 ± 2.99; K2P16-i7-4 83.00 ± 3.32; K2P16-i7-19 73.37 ± 4.44; mean ± SD; n = 11 technical replicates). (E) Representative CPA-induced [Ca 2+ ] C release from the ER measured in dispersed islet clusters from C57Bl/6J mice pretreated with VHL or 10 μ M K2P16-i7-4. (F) Quantification of the CPA-induced area under the Ca 2+ curve in islet cells pretreated with K2P16-i7-4 or VHL (VHL 10.30 ± 0.329; K2P16-i7-4 11.72 ± 0.511; islet clusters from 2 independent islet preps: n = 50 VHL, n = 54 K2P16-i7-4). Statistical analysis was performed using an unpaired t test.

Article Snippet: The membrane was then blotted with primary antibodies: anti-TALK-1 antibody (APC-170, Alomone Labs) or anti-TALK-2 antibody (NBP1-92041, Novus Biologicals, LLC), followed by the secondary antibody: anti-Rabbit IgG (H+L), HRP conjugate.

Techniques: Concentration Assay

Journal:

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Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rehydrated 5-μm sections were stained with primary antibodies against insulin (dilution 1:500; Dako, Santa Clara, CA, USA), somatostatin (dilution 1:250; sc-55565, Santa Cruz Biotechnology Inc., Dallas, TX, USA), and TALK-1 (dilution 1:175; APC-170, Alomone Labs, Jerusalem, Israel; or dilution 1:175; Prestige Antibodies Novus Biologicals #NBP1–83071) followed by secondary antibodies (dilution 1:500; anti-guinea pig Alexa Fluor 647, dilution 1:500; anti-mouse Alexa Fluor 488, and dilution 1:500; anti-rabbit Alexa Fluor 546). β-cell proliferation in response to HFD feeding was quantified by immunostaining for Ki-67 (dilution 1:300; ab15580, Abcam) and utilizing CytoNuclear FL v1.2 algorithm macros generated in HALO software (Indica Labs) to count insulin and Ki-67 double-positive cells.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Software