Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Silencing the ACAT1 Gene in Human SH-SY5Y Neuroblastoma Cells Inhibits the Expression of Cyclo-Oxygenase 2 (COX2) and Reduces β-Amyloid-Induced Toxicity Due to Activation of Protein Kinase C (PKC) and ERK

doi: 10.12659/MSM.912862

Figure Lengend Snippet: Primary antibodies used in the present study.

Article Snippet: Rabbit anti-Cacna1b , 1: 1000 , Alomone Labs, Jerusalem, Israel.

Techniques:

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Silencing the ACAT1 Gene in Human SH-SY5Y Neuroblastoma Cells Inhibits the Expression of Cyclo-Oxygenase 2 (COX2) and Reduces β-Amyloid-Induced Toxicity Due to Activation of Protein Kinase C (PKC) and ERK

doi: 10.12659/MSM.912862

Figure Lengend Snippet: Effects of knockdown ACAT1 on CACNAs, PKC, and ERK phosphorylation and in Aβ-induced SH-SY5Y cells. ( A ) Western blot results showed that the relative expression of CACNA1b and CACNA2d1 was increased significantly, while the total PKC and the ratio of phospho-ERK (p-ERK)/ERK were decreased significantly after Aβ 25-35 treatment. ( B ) Knockdown of ACAT1 rescued the reduction of PKC and phosphorylation level of ERK, and also retarded the upregulation of CACNA1b and CACNA2d1 induced by Aβ. (** P <0.01 and * P <0.05).

Article Snippet: Rabbit anti-Cacna1b , 1: 1000 , Alomone Labs, Jerusalem, Israel.

Techniques: Western Blot, Expressing

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Upregulation of N-Type Voltage-Gated Calcium Channels Induces Neuropathic Pain in Experimental Autoimmune Neuritis

doi: 10.1155/2022/8547095

Figure Lengend Snippet: Primer sequences.

Article Snippet: On completion of a 1 h blocking step with 5% skimmed milk at ambient temperature, primary antibodies iNOS (ab49999, Abcam, UK), MCP-1 (ab7202, Abcam, UK), and Cav2.2 (ACC-002, AlomoneLabs, Israel) were served for overnight coincubation with the membrane at 4°C.

Techniques: Sequencing

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Upregulation of N-Type Voltage-Gated Calcium Channels Induces Neuropathic Pain in Experimental Autoimmune Neuritis

doi: 10.1155/2022/8547095

Figure Lengend Snippet: Effect of Cav2.2 on clinical score and sciatic nerve tissue of rats. (a) Clinical score record of rats during 18 days of immunization; (b) changes in body weight of rats during 18 days of immunization; (c) luxol fast blue (LFB) staining-based observation of the degree of cell infiltration and demyelination in sciatic nerve tissue of rats on the 18 th day of immunization. ∗∗ P < 0.01 vs. control group, ## P < 0.01 vs. EAN-shRNA group.

Article Snippet: On completion of a 1 h blocking step with 5% skimmed milk at ambient temperature, primary antibodies iNOS (ab49999, Abcam, UK), MCP-1 (ab7202, Abcam, UK), and Cav2.2 (ACC-002, AlomoneLabs, Israel) were served for overnight coincubation with the membrane at 4°C.

Techniques: Staining, shRNA

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Upregulation of N-Type Voltage-Gated Calcium Channels Induces Neuropathic Pain in Experimental Autoimmune Neuritis

doi: 10.1155/2022/8547095

Figure Lengend Snippet: Effect of Cav2.2 on allodynia in rats. (a–d) Measurement of withdrawal threshold to mechanical (a), thermal (b), and cold (c) stimulation, and mechanical hyperalgesia threshold (d) during 18 days of immunization. ∗∗ P < 0.01 vs. Control group, ## P < 0.01 vs. EAN-shRNA group.

Article Snippet: On completion of a 1 h blocking step with 5% skimmed milk at ambient temperature, primary antibodies iNOS (ab49999, Abcam, UK), MCP-1 (ab7202, Abcam, UK), and Cav2.2 (ACC-002, AlomoneLabs, Israel) were served for overnight coincubation with the membrane at 4°C.

Techniques: shRNA

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Upregulation of N-Type Voltage-Gated Calcium Channels Induces Neuropathic Pain in Experimental Autoimmune Neuritis

doi: 10.1155/2022/8547095

Figure Lengend Snippet: Effect of Cav2.2 on rat inflammatory response. (a) ELISA-based measurement of IL-6 and TNF- α expression in rat serum; (b) qRT-PCR-based measurement of mRNA expression of iNOS, MCP-1, and Cav2.2 in rat sciatic nerve; (c) western blot-based measurement of protein expression of iNOS, MCP-1, and Cav2.2 in rat sciatic nerve. ∗ P < 0.05 and ∗∗ P < 0.01 vs. control group, # P < 0.05 and ## P < 0.01 vs. EAN-shRNA group.

Article Snippet: On completion of a 1 h blocking step with 5% skimmed milk at ambient temperature, primary antibodies iNOS (ab49999, Abcam, UK), MCP-1 (ab7202, Abcam, UK), and Cav2.2 (ACC-002, AlomoneLabs, Israel) were served for overnight coincubation with the membrane at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Western Blot, shRNA

Journal: PLoS ONE

Article Title: Differential Calcium Signaling Mediated by Voltage-Gated Calcium Channels in Rat Retinal Ganglion Cells and Their Unmyelinated Axons

doi: 10.1371/journal.pone.0084507

Figure Lengend Snippet: Ca channel α 1 subunits co-express with the ganglion cell antibody RBPMS in vertical sections showing the inner plexiform layer (IPL), the ganglion cell layer (GCL) and the nerve fiber layer (NFL). ( Top row ) Double immunostaining with α1C subunit and RBPMS antibodies shows colocalization in the RGC somata (filled arrow). RGC axons (arrowhead) as well as putative Müller and neuronal cell processes in the IPL showed immunostaining. Control preadsorption of α1C antibody with the immunization peptide showed no staining. ( Second row ) Double immunostaining with α1D subunit and RBPMS antibodies shows colocalization in the RGC somata (filled arrow). Numerous processes in the IPL as well as the RGC axons throughout the NFL (arrowhead) were also labelled. Control preadsorption of α1D antibody with the immunization peptide showed no staining. ( Third row ) Double immunostaining with α1B subunit and RBPMS antibodies shows colocalization in RGC somata (filled arrow). Processes in the IPL, putative displaced amacrine cells in the GCL (open arrows) and RGC axons throughout the NFL (arrowheads) showed immunostaining. Control preadsorption of α1B antibody with the immunization peptide showed no staining. ( Bottom row ) Double immunostaining with α1A subunit and RBPMS antibodies shows colocalization in the RGC somata (filled arrow). Labelling in the IPL and in putative displaced amacrine cells in the GCL (open arrows) was also seen. Control preadsorption of α1A antibody with the immunization peptide showed no staining. Scale bar is 20 µm.

Article Snippet: The following polyclonal antibodies were used to obtain staining of the α1 subunits: α1C (Cav1.2, Alomone, ACC-003, 1∶1000, Jerusalem, Israel), α1D (Cav1.3, Sigma, C1728, St. Louis, MO, 1∶100), α1A (Cav2.1, Synaptic Systems, 152–103, 1∶7500, Göttingen, Germany), α1B (Cav2.2, Alomone, ACC-002, 1∶1000, Jerusalem, Israel).

Techniques: Double Immunostaining, Immunostaining, Staining

Journal: PLoS ONE

Article Title: Differential Calcium Signaling Mediated by Voltage-Gated Calcium Channels in Rat Retinal Ganglion Cells and Their Unmyelinated Axons

doi: 10.1371/journal.pone.0084507

Figure Lengend Snippet: R at wholemount retina labelled with RBPMS (red), α1B VGCC subunit (green), and NF-M (white) antibodies. α1B colocalized with RBPMS in RGC somata (filled arrow), NF-M in RGC axons (arrowhead) and putative displaced amacrine cells (open arrow). Stack of six optical sections each of 0.3 µm thickness. Scale bar is 20 µm.

Article Snippet: The following polyclonal antibodies were used to obtain staining of the α1 subunits: α1C (Cav1.2, Alomone, ACC-003, 1∶1000, Jerusalem, Israel), α1D (Cav1.3, Sigma, C1728, St. Louis, MO, 1∶100), α1A (Cav2.1, Synaptic Systems, 152–103, 1∶7500, Göttingen, Germany), α1B (Cav2.2, Alomone, ACC-002, 1∶1000, Jerusalem, Israel).

Techniques:

Journal: Molecular Brain

Article Title: Differential regulation of Cav2.2 channel exon 37 variants by alternatively spliced μ-opioid receptors

doi: 10.1186/s13041-019-0524-6

Figure Lengend Snippet: Schematic representation of Cav2.2 channels and mMORs. a The Cav2.2 proximal C-terminus is in part encoded by the mutually exclusive exons 37a and 37b. The illustration shows the amino acids encoded by each exon. Shown in red is the tyrosine that was mutated for the experiments in Fig. . b MOR full-length distal C-terminal variants are produced by alternative splicing and amino acid sequences encoded by exon 4, 7 and 7–8-9 are depicted

Article Snippet: Biotinylated proteins and lysates were resolved by SDS-PAGE and analyzed by western blot using the antibodies anti-Cav2.2 (1:500, Alomone ACC-002) and anti-Na/K ATPase (1:5000, Abcam AB 7671).

Techniques: Produced

Journal: Molecular Brain

Article Title: Differential regulation of Cav2.2 channel exon 37 variants by alternatively spliced μ-opioid receptors

doi: 10.1186/s13041-019-0524-6

Figure Lengend Snippet: Inhibition of Src and Cav2.2-37a Y1747F abolish the effect of mMOR1C on Cav2.2-37a peak current density. a Peak current density of Cav2.2-37a channels treated overnight with vehicle (0.1% water or 500 ng/ml of PTX overnight. b Peak current density of Cav2.2-37a channels treated for 4 h with vehicle (0.004% DMSO) or 2 μM of the Src inhibitor PP1. c Peak current density of the Cav2.2-37a Y1747F mutant in the absence and the presence of mMOR1C. The number of cells recorded is indicated in parentheses, asterisks denote significance at the *0.05 and **0.01 levels ( a and b – ANOVA, c - Mann-Whitney test)

Article Snippet: Biotinylated proteins and lysates were resolved by SDS-PAGE and analyzed by western blot using the antibodies anti-Cav2.2 (1:500, Alomone ACC-002) and anti-Na/K ATPase (1:5000, Abcam AB 7671).

Techniques: Inhibition, Mutagenesis, MANN-WHITNEY

Journal: Molecular Brain

Article Title: Differential regulation of Cav2.2 channel exon 37 variants by alternatively spliced μ-opioid receptors

doi: 10.1186/s13041-019-0524-6

Figure Lengend Snippet: Peak current densities (I peak) of Cav2.2e37a and Cav2.2e37b channels coexpressed with mMOR1, mMOR1C or mMOR1O. a Representative whole cell current traces recorded in response to depolarizing steps from − 60 mV to + 40 mV from a holding potential of − 80 mV from tsA-201 cells expressing Cav2.2-37a/Cavβ1/Cavα2δ-1 or Cav2.2-37b /Cavβ1/Cavα2δ-1 channels plus/minus mMOR1C. b Average current density-voltage relationships for cells expressing Cav2.2-37a channels with or without mMOR1C. Inset: Corresponding mean half activation potentials. c Average current density-voltage relationships for cells expressing Cav2.2-37b channels with or without mMOR1C. Inset: Corresponding mean half activation potentials. d Average peak current density for whole cell calcium currents recorded from cells expressing Cav2.2e37a/Cavβ1/Cavα2δ-1 in the presence of mMOR1, mMOR1C and mMOR1O. e Average peak current density recorded from tsA-201 cells expressing Cav2.2e37b/Cavβ1/Cavα2δ-1 in the presence of mMOR1, mMOR1C or mMOR1O. The numbers in parentheses represent the number of cells recorded. n.s. – not significant, asterisks denote significance at the *0.05 and ***0.001 levels ( d – ANOVA; e - Kruskal-Wallis test)

Article Snippet: Biotinylated proteins and lysates were resolved by SDS-PAGE and analyzed by western blot using the antibodies anti-Cav2.2 (1:500, Alomone ACC-002) and anti-Na/K ATPase (1:5000, Abcam AB 7671).

Techniques: Expressing, Activation Assay

Journal: Molecular Brain

Article Title: Differential regulation of Cav2.2 channel exon 37 variants by alternatively spliced μ-opioid receptors

doi: 10.1186/s13041-019-0524-6

Figure Lengend Snippet: Biotinylation of Cav2.2e37a/Cavβ1/Cavα2δ-1 in the presence of mMOR1, mMOR1C and mMOR1O.Biotinylated cell surface proteins were isolated and normalized to Na/K-ATPase levels. a Representative blot of Cav2.2-37a surface and total expression (top blots) and Na/K-ATPase surface and total expression (bottom blots). b Quantification of plasma membrane Cav2.2-37a/ Cavβ1/Cavα2δ-1 channel expression in the absence and the presence of mMOR1, mMOR1C or mMOR1O (normalized by Na/K-ATPase cell surface expression). c Quantification of total Cav2.2-37a/Cavβ1/Cavα2δ-1 expression in the absence or the presence of mMOR1, mMOR1C or mMOR1O (normalized by total Na/K-ATPase expression). d Normalized surface to total expression of Cav2.2-37a channels. Data are from 4 independent experiments. n.s. – not significant (Kruskal-Wallis test)

Article Snippet: Biotinylated proteins and lysates were resolved by SDS-PAGE and analyzed by western blot using the antibodies anti-Cav2.2 (1:500, Alomone ACC-002) and anti-Na/K ATPase (1:5000, Abcam AB 7671).

Techniques: Isolation, Expressing

Journal: Molecular Brain

Article Title: Differential regulation of Cav2.2 channel exon 37 variants by alternatively spliced μ-opioid receptors

doi: 10.1186/s13041-019-0524-6

Figure Lengend Snippet: G protein modulation of Cav2.2-37a and Cav2.2-37b channels following activation of mMOR1, mMOR1C and mMOR1O. a Representative set of Cav2.2-37a currents in the presence of mMOR1C, recorded before or after the application of 10 μM DAMGO. As outlined in the Results section, P1 represents the first current in each trace evoked by a test depolarization to + 10 mV, P2 is the second inward current in a given trace evoked by a 10 mV test depolarization (P2) preceded by a strong depolarizing prepulse (PP, note that the pre-pulse-evoked outward current is not shown in the figure). Relief of Gβγ modulation by the pre-pulse is observed by the increase in current amplitude seen during P2 in the presence of DAMGO. b Percentage of peak current inhibition (during P1) of Cav2.2e-37a currents after application of 10 μM DAMGO. c Percentage of peak current inhibition (during P1) of Cav2.2e-37b currents after application of 10 μM DAMGO. d Voltage dependent pre-pulse facilitation measured in the presence of DAMGO in tsA-201 cells expressing Cav2.2-37a channels with mMOR1, mMOR1C or mMOR1O. The data points reflect the current evoked by test pulse P2 normalized to the current evoked by test pulse P1. e Voltage dependent pre-pulse facilitation measured in the presence of DAMGO in tsA-201 cells expressing Cav2.2-37b channels with mMOR1, mMOR1C or mMOR1O. The data points reflect the current evoked by the test pulse P2 normalized to the current evoked by test pulse P1. The number of cells recorded are indicated in parentheses, asterisks denote significance at the *0.05, **0.01, and ***0.001 levels (unpaired Wilcoxon test)

Article Snippet: Biotinylated proteins and lysates were resolved by SDS-PAGE and analyzed by western blot using the antibodies anti-Cav2.2 (1:500, Alomone ACC-002) and anti-Na/K ATPase (1:5000, Abcam AB 7671).

Techniques: Activation Assay, Inhibition, Expressing

Journal: Molecular Brain

Article Title: Differential regulation of Cav2.2 channel exon 37 variants by alternatively spliced μ-opioid receptors

doi: 10.1186/s13041-019-0524-6

Figure Lengend Snippet: Voltage dependent and voltage independent components of DAMGO-induced modulation of Cav2.2 variants by the various MORs. a Voltage-dependent and independent inhibition of Cav2.2-37a channels coexpressed with mMOR1, mMOR1C and mMOR1O. b Voltage-dependent and independent inhibition of Cav2.2-37b channels coexpressed with mMOR1, mMOR1C and mMOR1O. The number of cells recorded are indicated in parentheses, asterisks denote significance at the *0.05 and ***0.001 levels (t-test) between voltage-dependent and voltage-independent modulation for each receptor-channel combination

Article Snippet: Biotinylated proteins and lysates were resolved by SDS-PAGE and analyzed by western blot using the antibodies anti-Cav2.2 (1:500, Alomone ACC-002) and anti-Na/K ATPase (1:5000, Abcam AB 7671).

Techniques: Inhibition