rabbit polyclonal igg anti ca v 1 2  (Alomone Labs)

Journal: Translational Psychiatry

Article Title: Identification of ultra-rare disruptive variants in voltage-gated calcium channel-encoding genes in Japanese samples of schizophrenia and autism spectrum disorder

doi: 10.1038/s41398-022-01851-y

Figure Lengend Snippet: a Three-dimensional (3D) model structure of the Ca v 1.2 channel with the folded N-terminal structure. b 3D model structure of the Ca v 1.2 channel with the CaM-binding structure, shown in complex with the N-lobe of CaM. c Enlarged view around the alanine 36 (A36) residue of the folded N-terminal structure. The A36 site is highlighted by green dotted circles. The N-terminal spatial Ca 2+ -transforming element (NSCaTE) region (47–68), Ca 2+ , and CaM are indicated in yellow, orange, and magenta, respectively. Molecular graphics were created using UCSF Chimera . d A schematic illustration of the hypothesis that the A36V mutation attenuates Ca 2+ -dependent inactivation (CDI) by conformational equilibrium shift favoring the folded structure.

Article Snippet: After pretreatment, cells were incubated overnight with rabbit polyclonal anti-Ca v 1.2 antibody (1:1000, Alomone Labs) in blocking solution, and then stained for 1 hour with an AlexaFluor 488-conjugated anti-rabbit IgG goat antibody (1:1000, Thermo Fisher Scientific) and Hoechst 33342 (Thermo Fisher Scientific) in PBS.

Techniques: Binding Assay, Mutagenesis

Journal: Translational Psychiatry

Article Title: Identification of ultra-rare disruptive variants in voltage-gated calcium channel-encoding genes in Japanese samples of schizophrenia and autism spectrum disorder

doi: 10.1038/s41398-022-01851-y

Figure Lengend Snippet: a Sanger sequencing results for the de novo variant p.A36V (left) and schematic illustration of the primary structure of the Ca v 1.2 channel (right). The red asterisk indicates the A36V mutation near the N-terminal spatial Ca 2+ -transforming element (NSCaTE). b The N-terminal amino acid sequences for Ca v 1.2 channels (short isoforms). The A36V mutation and the A39V Brugada mutation are indicated in red letters. c Expression of wild-type (WT) and A36V Ca v 1.2 channels in HEK293T cells as detected by anti-Ca v 1.2 antibody. d Membrane localization of WT and A36V Ca v 1.2 channels overexpressed in BHK cells. The plasma membrane was visualized by membrane-tethering red fluorescent protein (RFP-KRasCT). e The fluorescence intensity profiles of the line shown in Fig. 1d. f Plasma membrane to cytoplasm intensity ratio of Ca v 1.2. Statistical comparison was performed by two-tailed Welch’s t test (n.s., not significant). Data are presented as mean ± s.e.m.

Article Snippet: After pretreatment, cells were incubated overnight with rabbit polyclonal anti-Ca v 1.2 antibody (1:1000, Alomone Labs) in blocking solution, and then stained for 1 hour with an AlexaFluor 488-conjugated anti-rabbit IgG goat antibody (1:1000, Thermo Fisher Scientific) and Hoechst 33342 (Thermo Fisher Scientific) in PBS.

Techniques: Sequencing, Variant Assay, Mutagenesis, Expressing, Fluorescence, Two Tailed Test

Journal: Translational Psychiatry

Article Title: Identification of ultra-rare disruptive variants in voltage-gated calcium channel-encoding genes in Japanese samples of schizophrenia and autism spectrum disorder

doi: 10.1038/s41398-022-01851-y

Figure Lengend Snippet: a Families of Ba 2+ currents evoked by 30-ms depolarizing pulses from −30 to 60 mV with increments of 10 mV for wild-type (WT) and A36V neuronal Ca v 1.2 channels. b Current density–voltage ( I – V ) relationships. Data are expressed as mean ± s.e.m., WT: n = 18, A36V: n = 12. The values of G, Erev, V 0.5 , and k were −0.40, 63.0 mV, 7.6 mV, and 5.6 mV for WT channels, and −0.50, 61.3 mV, 6.7 mV, and 4.9 mV for A36V Ca v 1.2 channels. c Inactivation curves for WT (○, n = 9) and A36V (●, n = 4) neuronal Ca v 1.2 channels. Data are expressed as mean ± s.e.m. The values of V 0.5 , and k were (respectively) −37.6 mV and 11.5 mV for WT channels, and −41.6 mV and 12.1 mV for A36V Ca v 1.2 channels. d , g Ca 2+ -dependent inactivation (CDI) of neuronal ( d ) and cardiac ( g ) Ca v 1.2 channels. Ba 2+ (blue) and Ca 2+ (black) currents evoked by 350-ms step depolarization to 30 mV were normalized at their peak current amplitudes for WT and A36V Ca v 1.2 channels. e , f, h, i , Ratios of current amplitude to the peak amplitude were plotted against depolarizing time in the Ba 2+ ( e, h ) and the Ca 2+ ( f, i ) external solutions. The numbers of recorded cells were 10 and 15 for WT and A36V neuronal Ca v 1.2 channels ( e , f ), and 8 and 6 for WT and A36V cardiac Ca v 1.2 channels ( h – i ), respectively. Statistical comparison was performed by two-tailed non-paired Student’s t test (* p < 0.05). Data are presented as mean ± s.e.m.

Article Snippet: After pretreatment, cells were incubated overnight with rabbit polyclonal anti-Ca v 1.2 antibody (1:1000, Alomone Labs) in blocking solution, and then stained for 1 hour with an AlexaFluor 488-conjugated anti-rabbit IgG goat antibody (1:1000, Thermo Fisher Scientific) and Hoechst 33342 (Thermo Fisher Scientific) in PBS.

Techniques: Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: BMAL1 Disrupted Intrinsic Diurnal Oscillation in Rat Cerebrovascular Contractility of Simulated Microgravity Rats by Altering Circadian Regulation of miR-103/Ca V 1.2 Signal Pathway

doi: 10.3390/ijms20163947

Figure Lengend Snippet: Circadian rhythm of Ca V 1.2 activities in cerebrovascular SMCs isolated from CON and SUS rats at six different time points. ( A ) The representative families of inward currents recorded in cerebrovascular SMCs without Ca 2+ entry modulators, in the presence of agonist Bay K 8644 or antagonist nifedipine in the bath solution. ( B ) The peak current densities of Ca V 1.2 channel at +20 mV showed a circadian oscillation with the peak activities at ZT4 (the subjective light) and the trough activities at ZT16 (the subjective dark) in CON rats. ( C ) The peak current densities of Ca V 1.2 markedly increased at both ZT4 and ZT16 in SUS rats as compared with that in CON rats. ( D ) The diurnal variation in Ca V 1.2 activities (the difference value between ZT4 and ZT16 level) significantly decreased in SUS rats as compared with that in CON rats. Data are presented as box plots and 5th and 95th percentiles. n = 10, t -test or two-way ANOVA and Tukey’s multiple comparisons test, * p < 0.05, CON: 28-day simultaneous control rats; SUS: 28-day tail-suspended rats.

Article Snippet: The following antibodies were used: Rabbit anti-BMAL1 polyclonal antibody (1:1000, abcam, Eugene, USA), rabbit anti-Ca V 1.2 polyclonal antibody (1:200, Alomone Labs, Jerusalem, Israel), and mouse anti-β-actin monoclonal antibody (1:1000, Proteintech, Wuhan, China).

Techniques: Isolation

Journal: International Journal of Molecular Sciences

Article Title: BMAL1 Disrupted Intrinsic Diurnal Oscillation in Rat Cerebrovascular Contractility of Simulated Microgravity Rats by Altering Circadian Regulation of miR-103/Ca V 1.2 Signal Pathway

doi: 10.3390/ijms20163947

Figure Lengend Snippet: Circadian expression of Ca V 1.2 α1C-subunit in cerebral arteries isolated from CON and SUS rats at six different time points. ( A ) The representative protein expressions of Ca V 1.2 α1C-subunit are shown. ( B ) The mean data of protein expression display a circadian oscillation with the acrophase at subjective light-time and the trough at subjective dark-time. ( C ) The protein expression of Ca V 1.2 α1C-subunit markedly increased at both ZT4 and ZT16 in SUS rats as compared with that in CON rats. ( D ) The diurnal variation in protein expression of Ca V 1.2 α1C-subunit (the difference value between ZT4 and ZT16 level) significantly decreased in SUS rats as compared with that in CON rats. ( E ) both Ca V 1.2 mRNA expressions of cerebral arteries in SUS and CON rats remained constant throughout the course of a day. Data are presented as box plots and 5th and 95th percentiles. n = 6, t -test or two-way ANOVA and Tukey’s multiple comparisons test, * p < 0.05, ** p < 0.01, *** p < 0.001 as compared with CON. CON: 28-day simultaneous control rats; SUS: 28-day tail-suspended rats (the full-length Western blots for A are shown in the ).

Article Snippet: The following antibodies were used: Rabbit anti-BMAL1 polyclonal antibody (1:1000, abcam, Eugene, USA), rabbit anti-Ca V 1.2 polyclonal antibody (1:200, Alomone Labs, Jerusalem, Israel), and mouse anti-β-actin monoclonal antibody (1:1000, Proteintech, Wuhan, China).

Techniques: Expressing, Isolation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: BMAL1 Disrupted Intrinsic Diurnal Oscillation in Rat Cerebrovascular Contractility of Simulated Microgravity Rats by Altering Circadian Regulation of miR-103/Ca V 1.2 Signal Pathway

doi: 10.3390/ijms20163947

Figure Lengend Snippet: Identification of Ca V 1.2 α1C-subunit as the down target of miR-103 in VSMCs. ( A ) Computational prediction of Ca 2+ signaling-related miRNAs by evaluating the published literatures and the prediction software programs of miRanDa, miRDB, and Targetscan. ( B ) qRT-PCR was performed to examine the mRNA levels of eight candidate miRNAs in cerebral VSMCs of SUS rats ( n = 3/group). ( C ) More samples were used to further confirm that miR-103 expression of cerebral arteries showed a circadian rhythm with a higher level during the subjective night than the subjective light in CON and SUS rats ( n = 6). ( D ) The expression of miR-103 markedly decreased at both ZT4 and ZT16 in SUS rats as compared with that in CON rats. ( E ) Schematic diagram of the presumptive binding sequences of miR-103 to CACNA1C 3′-UTR based on the TargetScan database prediction. Dual-luciferase activity assay was performed in A7r5 cells by co-transfecting miR-103 mimic with psiCHECK TM -2 vectors containing WT/MUT CACNA1C 3′-UTR. Renilla luciferase activity was normalized by Firefly luciferase activity (hRluc/hLuc). ( F , G ) Western blotting was used to show that overexpression or inhibition of miR-103 by mimic or inhibitor transfection significantly decreased or increased the protein expression of Ca V 1.2 channel in A7r5 cells, respectively. Data are presented as box plots and 5th and 95th percentiles. Each experiment in vitro was repeated three times, t -test or one(two)-way ANOVA and Tukey’s multiple comparisons test, * p < 0.05, ** p < 0.01, *** p < 0.001 as compared with CON (the full-length Western blots for F are shown in the ).

Article Snippet: The following antibodies were used: Rabbit anti-BMAL1 polyclonal antibody (1:1000, abcam, Eugene, USA), rabbit anti-Ca V 1.2 polyclonal antibody (1:200, Alomone Labs, Jerusalem, Israel), and mouse anti-β-actin monoclonal antibody (1:1000, Proteintech, Wuhan, China).

Techniques: Software, Quantitative RT-PCR, Expressing, Binding Assay, Luciferase, Activity Assay, Western Blot, Over Expression, Inhibition, Transfection, In Vitro

Journal: International Journal of Molecular Sciences

Article Title: BMAL1 Disrupted Intrinsic Diurnal Oscillation in Rat Cerebrovascular Contractility of Simulated Microgravity Rats by Altering Circadian Regulation of miR-103/Ca V 1.2 Signal Pathway

doi: 10.3390/ijms20163947

Figure Lengend Snippet: BMAL1 modulates the protein expression of Ca V 1.2 α1C-subunit by miRNA-103 signal pathway. ( A ) The mRNA levels of miR-103 in A7r5 cells transfected with BMAL1 overexpression plasmid. The representative protein expressions of Ca V 1.2 α1C-subunit and BMAL1 ( B ) and the mean data ( C , D ) with the treatment of miR-103 mimic and inhibitor in A7r5 cells transfected with BMAL1 overexpression plasmid, respectively. ( E ) the mRNA levels of miR-103 in A7r5 cells transfected with BMAL1 siRNA. The representative protein expressions of Ca V 1.2 α1C-subunit and BMAL1 ( F ) and the mean data ( G , H ) with the treatment of miR-103 mimic and inhibitor in A7r5 cells transfected with BMAL1 siRNA, respectively. Data are presented as box plots and 5th and 95th percentiles. Each experiment was repeated three times, one-way ANOVA and Tukey’s multiple comparisons test, ** p < 0.01, *** p < 0.001 as compared with the control (the full-length Western blots for B,F are shown in the ).

Article Snippet: The following antibodies were used: Rabbit anti-BMAL1 polyclonal antibody (1:1000, abcam, Eugene, USA), rabbit anti-Ca V 1.2 polyclonal antibody (1:200, Alomone Labs, Jerusalem, Israel), and mouse anti-β-actin monoclonal antibody (1:1000, Proteintech, Wuhan, China).

Techniques: Expressing, Transfection, Over Expression, Plasmid Preparation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: BMAL1 Disrupted Intrinsic Diurnal Oscillation in Rat Cerebrovascular Contractility of Simulated Microgravity Rats by Altering Circadian Regulation of miR-103/Ca V 1.2 Signal Pathway

doi: 10.3390/ijms20163947

Figure Lengend Snippet: A schematic model of the circadian regulation of BMAL1/miR-103/Ca V 1.2 signal pathway and the disruption of simulated microgravity.

Article Snippet: The following antibodies were used: Rabbit anti-BMAL1 polyclonal antibody (1:1000, abcam, Eugene, USA), rabbit anti-Ca V 1.2 polyclonal antibody (1:200, Alomone Labs, Jerusalem, Israel), and mouse anti-β-actin monoclonal antibody (1:1000, Proteintech, Wuhan, China).

Techniques: