Journal: Journal of Behavioral Addictions

Article Title: Differential transcriptome profile underlying risky choice in a rat gambling task

doi: 10.1556/2006.2022.00068

Figure Lengend Snippet: Validation for the down-regulation of genes in risk-seeking rats. The validation experiment detected significant reductions in the expression levels for a few genes by RT-qPCR (a) and further by western blot (b), similar to the findings in RNA-seq analysis. The values are mean +S.E.M. of relative expression of molecules in the risk-seeking group compared to the risk-averse group. * P < 0.05, ** P < 0.01, risk-seeking compared to risk-averse. The number of rats are 15 and 9 for the risk-averse and risk-seeking group, respectively

Article Snippet: Antibodies used to probe the blots were as following: CaV2.3 (1:20,000; Abclonal), teneurin4 (1:1,000; Abcam), CaV1.2 (1:30,000; Alomone Labs), CaV1.3 (1:1,000; Alomone Labs), CUB and sushi domain-containing protein (1:1,000; Abclonal), and GluN2B (1:2,000, Alomone Labs) diluted in PBS-T with 5% skim milk; PKCα (1:2,000, Cell Signaling Technology), adenyl cyclase type 1 (1:2,000; Abclonal), MeCP2 (1:10,000, Cell Signaling Technology), and β-actin (1:10,000; Abcam) diluted in PBS-T with 5% BSA.

Techniques: Biomarker Discovery, Expressing, Quantitative RT-PCR, Western Blot, RNA Sequencing, Analysis, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Transplantation of Neural Precursors Derived from Induced Pluripotent Cells Preserve Perineuronal Nets and Stimulate Neural Plasticity in ALS Rats

doi: 10.3390/ijms21249593

Figure Lengend Snippet: List of primary antibodies.

Article Snippet: Anti- l -type Ca 2+ CP α1C , L-type Ca 2+ CP α1C/ICC , Rabbit polyclonal , 1:200 , Alomone Labs, Jerusalem, Israel.

Techniques: Marker

Journal: Frontiers in Cell and Developmental Biology

Article Title: Embryo-derived trypsin-induced calcium entry is inhibited by endometrial infertility factor, LEFTY2

doi: 10.3389/fcell.2025.1499339

Figure Lengend Snippet: LEFTY2 decreases L-type ca 2+ Channel abundance. Cell cultures were treated with or without LEFTY2 (25 ng/mL) for 6 h either in the presence or absence of Trypsin (20 µg/mL, 24 h). (a) Uniform manifold approximation and projection (UMAP) clustering of tissues and cell types; Tissue compartments and cell types were annotated in the Single Cell Expression Atlas. (b) Expression of CACNA1C in single cells, presented as counts per million (CPM), overlaid on the UMAP map. (c) Arithmetic means ± SEM (n = 6) of transcript levels encoding the human CACNA1C transcript levels were determined by qRT-PCR, normalized to the levels of L19 mRNA and expressed in arbitrary units (a.u.). (d) Western blot analysis of LEFTY2 expression in Ishikawa cells (n = 4). GAPDH was used as a loading control. (e) IF microscopy of Ishikawa cells treated with or without LEFTY2 (25 ng/mL) for 6 h either in the presence or absence of Trypsin (20 µg/mL, 24 h) showing CACNA1C subcellular localization. CACNA1C fluorescence intensity quantification results were shown (right). CACNA1C: Alexa Fluor 568 (red); nucleus: DAPI (blue). Quantification performed from 3 experiments with >15 cells quantified for each condition. Scale bar = 25 µm. (f) Western blot analysis of COX2 expression in Ishikawa cells (n = 4). GAPDH was used as a loading control. (g) PGE 2 level determined by ELISA. The data are presented as mean ± SEM. One-way ANOVA was used to calculate statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Membranes were probed overnight at 4°C with antibodies: LEFTY2 (1:500, sc-365845, Santa Cruz, Germany), CACNA1C (1:200, ACC-013, Alomone Labs, Israel), COX2 (1:200, MA5-14568, ThermoFisher, Germany), GAPDH (1:1,000, #2118L, Cell Signaling, Germany) was used for loading control.

Techniques: Tissue, Single Cell, Expressing, Quantitative RT-PCR, Western Blot, Analysis, Control, Microscopy, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Journal of Endocrinology

Article Title: CaV1.2 and CaV1.3 voltage-gated L-type Ca2+ channels in rat white fat adipocytes

doi: 10.1530/joe-19-0493

Figure Lengend Snippet: Figure 1 CaV1.1, 1.2 and 1.3 mRNA are expressed in rat adipocytes. (A) RT-CPR products (Rattus norvegicus) of voltage-dependent L-type calcium channels CaV1.1, CaV1.2, CaV1.3 and CaV1.4 alpha1 (Cacna1s, Cacna1c, Cacna1d and Cacna1f), beta2 (Cacnb1, Cacnb2, Cacnb3 and Cacnb4) and alpha2delta (Cacna2d1, Cacna2d2, Cacna2d3 and Cacna2d4) subunits for white fat adipocytes and for control: skeletal muscle for Cacna1s (CaV1.1) and whole brain for all other genes; L, DNA 50 bp ladder. PCR product sizes are in Table 1. (B) Relative expression of CaV1.1, CaV1.2 and CaV1.3 alpha-1 subunits as determined by qPCR. Data are normalized to mRNA expression of CaV1.2 in rat brain. Each point represents a different animal (n = 10), horizontal line is mean. Statistical significance is by one-way ANOVA, with Tukey’s multiple comparison test.

Article Snippet: Cells were permeablised in blocking buffer (PBS with 3% BSA and 0.5% Triton-100x) for 10 min then stained with primary anti-Calcium Channel L-type alpha 1C subunit (Cacna1C) antibody conjugated with Atto 594 (ACC-003-AG, Alomone) at 1:200 dilution for 16 h (Raifman et al. 2017).

Techniques: Control, Expressing, Comparison

Journal: Nature Communications

Article Title: Nanobody-thioesterase chimeras to specifically target protein palmitoylation

doi: 10.1038/s41467-025-56716-x

Figure Lengend Snippet: A Schematic. Nanobody binding to its palmitoylated target protein delivers a thioesterase which removes the 16 carbon fatty acid palmitate (depicted in blue) from the target (Created in BioRender. Fulton, H. (2025) https://BioRender.com/n03e444 ). B Impact of anti-GFP nanobody LaG16 fused to the amino or carboxyl terminus of thioesterases (APT1, APT2) on YFP-Ca(v)1.2 I-II-linker palmitoylation. UF: unfractionated cell lysate; P: purified palmitoylated proteins; Flot2: flotillin-2. Data are means ± SEM from n = 8 independent experiments. C Impact of LaG16 fused to catalytically inactive (‘dead’) APTs on YFP-Ca(v)1.2 I-II-linker palmitoylation. Data are means ± SEM from n = 4 independent experiments. D Structure (PDB: 6LR7) of eGFP (green) in complex with LaG16 (magenta) highlighting the positions of F30, T55 and G103 (cyan) in CDRs 1, 2 and 3. E Wild-type and mutated (‘3W’) LaG16 binding to YFP-Ca(v)1.2 I-II-linker assessed by immunoprecipitating the FLAG tagged nanobody. This experiment was repeated independently three times with similar results. F Impact of LaG16 and mutated LaG16 fused to APTs on YFP-Ca(v)1.2 I-II-linker palmitoylation. Data are means ± SEM from n = 4 independent experiments. Statistical comparisons: one-way ANOVA followed by Tukey’s multiple comparisons test. Source data are provided as a file.

Article Snippet: This investigation used an anti-Ca(v)1.2 α1C subunit antibody raised in guinea pig obtained from Alomone Labs, and antibodies against flotillin 2 from BD Biosciences, GAPDH, FLAG and HIS tags from Merck, and GFP from Protein Tech.

Techniques: Binding Assay, Purification, Co-Immunoprecipitation Assay

Journal: Nature Communications

Article Title: Nanobody-thioesterase chimeras to specifically target protein palmitoylation

doi: 10.1038/s41467-025-56716-x

Figure Lengend Snippet: A LAMA schematic (Created in BioRender. Fulton, H. (2025) https://BioRender.com/v66u239 ). B Impact of trimethoprim on LAMA binding to YFP-Ca(v)1.2 I-II-linker assessed by immunoprecipitating the FLAG tagged nanobody. This experiment was repeated independently three times with similar results. C Inserting dihydrofolate reductase (DHFR) at position G97 in CDR3 of the GFP enhancer nanobody generates a LAMA with potent depalmitoylating activity towards YFP-Ca(v)1.2 I-II-linker. Treatment with 10 µM trimethoprim restores target protein palmitoylation. Data are means ± SEM from n = 5 independent experiments. D Inserting DHFR at position F98 in CDR3 of the GFP enhancer nanobody generates a LAMA with potent, trimethoprim-insensitive depalmitoylating activity towards YFP-tagged Ca(v)1.2 I-II-linker. Data are means ± SEM from n = 5 independent experiments. Statistical comparisons: one-way ANOVA followed by Tukey’s multiple comparisons test. Source data are provided as a file.

Article Snippet: This investigation used an anti-Ca(v)1.2 α1C subunit antibody raised in guinea pig obtained from Alomone Labs, and antibodies against flotillin 2 from BD Biosciences, GAPDH, FLAG and HIS tags from Merck, and GFP from Protein Tech.

Techniques: Binding Assay, Activity Assay, Immunoprecipitation

Journal: Nature Communications

Article Title: Nanobody-thioesterase chimeras to specifically target protein palmitoylation

doi: 10.1038/s41467-025-56716-x

Figure Lengend Snippet: A APT2-Nanobody-F3 fusion protein recruitment to the Ca(v)1.2 complex assessed by immunoprecipitating the FLAG tagged nanobody. Experiment was repeated independently three times with similar results. B APT2 fused to Ca(v)1.2 beta subunit nanobody F3 depalmitoylates Ca(v)1.2 α1C subunit only when co-expressed with HIS-tagged beta2b subunit to facilitate nanobody recruitment to Ca(v)1.2. Data are means ± SEM from n = 5 independent experiments. Statistical comparison: unpaired two-tailed t-test. C 293 T-REx cells stably expressing α1C and transfected with β2b and α2δ1 subunits, plus either APT2-F3 or catalytically inactive (S122A) APT2-F3, were voltage-clamped at −60 mV. 500 ms voltage steps from −60 mV to +60 mV at 10 mV increments were applied at 1 Hz. Representative examples of currents from cells expressing inactive (black, left) or active (red, right) APT2 fused to nanobody F3 with barium as charge carrier are shown. D Current voltage relationship for Ca(v)1.2 mediated barium currents in the presence of active APT2-F3 (red, data are means ± SEM from n = 9 independent experiments) or catalytically inactive APT2-F3 (black, data are means ± SEM from n = 8 independent experiments). E A Boltzmann function was fitted to the I–V relationships to yield V50 and Gmax parameters for catalytically inactive APT2-F3 (black, data are means ± SEM from n = 8 independent experiments) or APT2-F3 (red, data are means ± SEM from n = 7 independent experiments). Statistical comparison: unpaired two-tailed t-test. F G–V curves were drawn by calculating G/Gmax using parameters obtained from the Boltzmann fits of I–V curves from individual cells. Data are means ± SEM from n = 8 (catalytically inactive APT2-F3, black) and n = 7 (APT2-F3, red) independent experiments. Statistical comparisons: unpaired t-test. Source data are provided as a file.

Article Snippet: This investigation used an anti-Ca(v)1.2 α1C subunit antibody raised in guinea pig obtained from Alomone Labs, and antibodies against flotillin 2 from BD Biosciences, GAPDH, FLAG and HIS tags from Merck, and GFP from Protein Tech.

Techniques: Comparison, Two Tailed Test, Stable Transfection, Expressing, Transfection, Co-Immunoprecipitation Assay, Patch Clamp

Journal: Journal of Biological Chemistry

Article Title: Regulation of Gene Transcription by Voltage-gated L-type Calcium Channel, Cav1.3

doi: 10.1074/jbc.m114.586883

Figure Lengend Snippet: FIGURE 3. The C terminus of the Cav1.3 channel is localized in the nucleus of cardiac myocytes from wild-type mice. a, immunofluorescence confocal microscopic images of neonatal mouse cardiomyocytes stained using an antibody recognizing an epitope in the II-III cytoplasmic loop of the Cav1.3 channel (amino acids 859–875, loop antibody, upper panels). No detectable nuclear fluorescence is observed. In contrast, a high nuclear fluorescence signal is detected when an antibody that recognizes the C terminus of Cav1.3 (amino acids 1661–1990) is used (lower panels). b, isolated nuclei from adult atrial myocytes. The C-terminal antibody of the Cav1.3 channel detects a high nuclear fluorescence in contrast to the antibody directed against the II-III cytoplasmic loop. Nuclei were stained using DAPI (blue). c, adult atrial myo- cytes co-stained with anti-Cav1.3 (loop antibody, red) and -actinin2 (green) antibodies. Scale bars are 10 m. Right panel shows the merged image at higher magnification. d, isolated nuclei from adult atrial myocytes. Two dif- ferent monoclonal antibodies for the Cav1.3 Ca2 channel were used includ- ing: 1) anti-Cav1.3 Ca2 channel (NeuroMab clone L48A/9) directed against amino acids 859–875 in the N terminus of rat Cav1.3, and 2) anti-Cav1.3 Ca2 channel (NeuroMab clone N38/8) directed against amino acids 2025–2161 in

Article Snippet: 9) Monoclonal anti-Cav1.3 Ca2 channel (NeuroMab clone L48A/9) directed against amino acids 859 – 875 in the N terminus of rat Cav1.3, and 10) monoclonal anti-Cav1.3 Ca2 channel (NeuroMab clone N38/8) directed against amino acids 2025– 2161 in the C terminus of rat Cav1.3.

Techniques: Immunofluorescence, Staining, Fluorescence, Isolation, Bioprocessing