anti cav3 2  (Alomone Labs)


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    Structured Review

    Alomone Labs anti cav3 2
    Knockdown or overexpression of TRPC7 did not alter the expression of several important ion channels/pump in NRVMs. a – g Western blots showing the expression of a TRPC7, b HCN4, c Cav1.3, d IP3R1, e Cav3.1, f <t>Cav3.2,</t> g SERCA in NRVMs infected with different adenoviruses to knockdown or overexpress TRPC7. h – n Bar charts showing the quantification of each protein from a – g . To eliminate the loading bias, intensity of each target protein was normalized to that of its corresponding β-tubulin. TRPC7 was successfully knocked down or overexpressed in NRVMs but the change of TRPC7 expression did not alter the expression of HCN4, Cav1.3, IP3R1, Cav3.1, Cav3.2, and SERCA. Data were presented as mean ± SEM ( n = 4). * P
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    Images

    1) Product Images from "TRPC7 regulates the electrophysiological functions of embryonic stem cell-derived cardiomyocytes"

    Article Title: TRPC7 regulates the electrophysiological functions of embryonic stem cell-derived cardiomyocytes

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-021-02308-7

    Knockdown or overexpression of TRPC7 did not alter the expression of several important ion channels/pump in NRVMs. a – g Western blots showing the expression of a TRPC7, b HCN4, c Cav1.3, d IP3R1, e Cav3.1, f Cav3.2, g SERCA in NRVMs infected with different adenoviruses to knockdown or overexpress TRPC7. h – n Bar charts showing the quantification of each protein from a – g . To eliminate the loading bias, intensity of each target protein was normalized to that of its corresponding β-tubulin. TRPC7 was successfully knocked down or overexpressed in NRVMs but the change of TRPC7 expression did not alter the expression of HCN4, Cav1.3, IP3R1, Cav3.1, Cav3.2, and SERCA. Data were presented as mean ± SEM ( n = 4). * P
    Figure Legend Snippet: Knockdown or overexpression of TRPC7 did not alter the expression of several important ion channels/pump in NRVMs. a – g Western blots showing the expression of a TRPC7, b HCN4, c Cav1.3, d IP3R1, e Cav3.1, f Cav3.2, g SERCA in NRVMs infected with different adenoviruses to knockdown or overexpress TRPC7. h – n Bar charts showing the quantification of each protein from a – g . To eliminate the loading bias, intensity of each target protein was normalized to that of its corresponding β-tubulin. TRPC7 was successfully knocked down or overexpressed in NRVMs but the change of TRPC7 expression did not alter the expression of HCN4, Cav1.3, IP3R1, Cav3.1, Cav3.2, and SERCA. Data were presented as mean ± SEM ( n = 4). * P

    Techniques Used: Over Expression, Expressing, Western Blot, Infection

    2) Product Images from "Expression Pattern of T-Type Ca2+ Channels in Cerebellar Purkinje Cells after VEGF Treatment"

    Article Title: Expression Pattern of T-Type Ca2+ Channels in Cerebellar Purkinje Cells after VEGF Treatment

    Journal: Cells

    doi: 10.3390/cells10092277

    In vivo and in vitro expression of Cav3.2: Visualisation of the in vivo and in vitro distribution of Cav3.2 via anti-Cav3.2 antibodies. In dissociated PCs at 2div ( A – D ), Cav3.2 was prominent at the dendrites or axons, whereas in the cryosections of rat cerebella at p0 ( E – H ), the signal of Cav 3.2 was mostly at the soma of the PCs. In the age of p9 ( I – L ), there was a clear colocalization with the dendrites and the soma of the PCs. eGCL = external granular cell layer, ML = molecular layer, PCL = PC layer, iGCL = internal granular cell layer. Scale bar: 20 µm ( A , B , E , F , I , J ) and 2 µm ( C , D , G , H , K , L ).
    Figure Legend Snippet: In vivo and in vitro expression of Cav3.2: Visualisation of the in vivo and in vitro distribution of Cav3.2 via anti-Cav3.2 antibodies. In dissociated PCs at 2div ( A – D ), Cav3.2 was prominent at the dendrites or axons, whereas in the cryosections of rat cerebella at p0 ( E – H ), the signal of Cav 3.2 was mostly at the soma of the PCs. In the age of p9 ( I – L ), there was a clear colocalization with the dendrites and the soma of the PCs. eGCL = external granular cell layer, ML = molecular layer, PCL = PC layer, iGCL = internal granular cell layer. Scale bar: 20 µm ( A , B , E , F , I , J ) and 2 µm ( C , D , G , H , K , L ).

    Techniques Used: In Vivo, In Vitro, Expressing

    3) Product Images from "Down-regulation of T-type Cav3.2 channels by hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1): Evidence of a signaling complex"

    Article Title: Down-regulation of T-type Cav3.2 channels by hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1): Evidence of a signaling complex

    Journal: Channels

    doi: 10.1080/19336950.2017.1326233

    HCN1 channels alter Cav3.2 channel activity. Data shown are from whole-cell voltage clamp recordings from tsA-201 cells transiently transfected with Cav3.2 cDNA with or without HCN1 construct. (A) Representative Ba 2+ current traces recorded from Cav3.2-expressing cells with or without coexpression of HCN1 channels in response to 250 ms depolarizing steps varied from −80 to +40 mV from a holding potential of −100 mV. (B) Corresponding current-voltage relationship for Cav3.2-expressing cells with or without coexpression of HCN1 channels (without HCN1, n = 14; with HCN1, n = 21). (C) Corresponding mean activation time constants of Cav3.2 currents recorded at a command potential −20 mV (without HCN1, n = 14; with HCN1, n = 21). (D) Corresponding mean inactivation time constants of Cav3.2 currents recorded at −20 mV (without HCN1, n = 14; with HCN1, n = 21). (E) Normalized Cav3.2 channel activation curves from Cav3.2-expressing cells with coexpression of HCN1 channels showed a depolarizing shift in half activation voltage (without HCN1, n = 14; with HCN1, n = 21). (F, G) Summary of half activation voltage and maximal conductance (without HCN1, n = 14; with HCN1, n = 21). (H) Inactivation curves were determined in response to a pulse at −20 mV after 5 s-lasting depolarizing prepulses to different potentials from a holding potential of −110 mV (without HCN1, n = 16; with HCN1, n = 16). *, p
    Figure Legend Snippet: HCN1 channels alter Cav3.2 channel activity. Data shown are from whole-cell voltage clamp recordings from tsA-201 cells transiently transfected with Cav3.2 cDNA with or without HCN1 construct. (A) Representative Ba 2+ current traces recorded from Cav3.2-expressing cells with or without coexpression of HCN1 channels in response to 250 ms depolarizing steps varied from −80 to +40 mV from a holding potential of −100 mV. (B) Corresponding current-voltage relationship for Cav3.2-expressing cells with or without coexpression of HCN1 channels (without HCN1, n = 14; with HCN1, n = 21). (C) Corresponding mean activation time constants of Cav3.2 currents recorded at a command potential −20 mV (without HCN1, n = 14; with HCN1, n = 21). (D) Corresponding mean inactivation time constants of Cav3.2 currents recorded at −20 mV (without HCN1, n = 14; with HCN1, n = 21). (E) Normalized Cav3.2 channel activation curves from Cav3.2-expressing cells with coexpression of HCN1 channels showed a depolarizing shift in half activation voltage (without HCN1, n = 14; with HCN1, n = 21). (F, G) Summary of half activation voltage and maximal conductance (without HCN1, n = 14; with HCN1, n = 21). (H) Inactivation curves were determined in response to a pulse at −20 mV after 5 s-lasting depolarizing prepulses to different potentials from a holding potential of −110 mV (without HCN1, n = 16; with HCN1, n = 16). *, p

    Techniques Used: Activity Assay, Transfection, Construct, Expressing, Activation Assay

    HCN1 channels interact with Cav3.2 channels in mouse brain. (A) Co-immunoprecipitation of HCN1 and T-type Ca 2+ channel subunits using lysates from adult mouse brain showed that HCN1 channels associate with Cav3.2 T-type channels (lane 4). The samples were blotted with anti-HCN1 antibody. (B) western blot showing that HCN2 channels did not co-immunoprecipitate with any of the T-type channel subunits. HCN1 and HCN2 protein could both be detected in mouse brain homogenates (input). The Nuclear receptor related 1 protein (NURR1) here was used as an irrelevant antibody for the negative control. The samples were blotted with anti-HCN2 antibody. These experiments were repeated 4 times (with tissue obtained from 4 different mice) with identical results.
    Figure Legend Snippet: HCN1 channels interact with Cav3.2 channels in mouse brain. (A) Co-immunoprecipitation of HCN1 and T-type Ca 2+ channel subunits using lysates from adult mouse brain showed that HCN1 channels associate with Cav3.2 T-type channels (lane 4). The samples were blotted with anti-HCN1 antibody. (B) western blot showing that HCN2 channels did not co-immunoprecipitate with any of the T-type channel subunits. HCN1 and HCN2 protein could both be detected in mouse brain homogenates (input). The Nuclear receptor related 1 protein (NURR1) here was used as an irrelevant antibody for the negative control. The samples were blotted with anti-HCN2 antibody. These experiments were repeated 4 times (with tissue obtained from 4 different mice) with identical results.

    Techniques Used: Immunoprecipitation, Western Blot, Negative Control, Mouse Assay

    Cav3.2 co-expression does not alter current density or kinetics of HCN1 channels. (A) Sample I h currents recorded from tsA-201 cells without (black) or with co-expression of Cav3.2 channels (red) in response to a hyperpolarizing voltage step from −50 mV to −100 mV. The currents were leak subtracted using a P/4 protocol. To obtain stable recordings, the cells were held at −50 mV to minimize leakage currents from the opening of HCN1 channels at rest. To maximally recover Cav3.2 channels from inactivation and to measure I h , a 500 ms hyperpolarizing pulse to −100 mV was first applied. Then, a 200 ms pulse to −20 mV was applied to activate maximum calcium influx via Cav3.2. Finally, I h was activated by 1 s hyperpolarizing pulse to −100 mV (10 ms return to the holding at −50 mV before test pulse applied). The extracellular solution contained 2.5 mM Ca 2+ . (B) Effect of Cav3.2 coexpression on the mean peak current density of HCN1 channels (without Cav3.2, n = 9; with Cav3.2, n = 11). (C) Corresponding mean activation time constants of I h recorded at a command potential of −100 mV (before Cav3.2 activation, n = 9; after Cav3.2 activation, n = 11). (D) Normalized I h peak current recorded form cells co-expressing Cav3.2 and HCN1 (expressed as the current ratio obtained after and before application of a Cav3.2 activating pulse) before and after the bath application of dibutyryl-cAMP (n = 11).
    Figure Legend Snippet: Cav3.2 co-expression does not alter current density or kinetics of HCN1 channels. (A) Sample I h currents recorded from tsA-201 cells without (black) or with co-expression of Cav3.2 channels (red) in response to a hyperpolarizing voltage step from −50 mV to −100 mV. The currents were leak subtracted using a P/4 protocol. To obtain stable recordings, the cells were held at −50 mV to minimize leakage currents from the opening of HCN1 channels at rest. To maximally recover Cav3.2 channels from inactivation and to measure I h , a 500 ms hyperpolarizing pulse to −100 mV was first applied. Then, a 200 ms pulse to −20 mV was applied to activate maximum calcium influx via Cav3.2. Finally, I h was activated by 1 s hyperpolarizing pulse to −100 mV (10 ms return to the holding at −50 mV before test pulse applied). The extracellular solution contained 2.5 mM Ca 2+ . (B) Effect of Cav3.2 coexpression on the mean peak current density of HCN1 channels (without Cav3.2, n = 9; with Cav3.2, n = 11). (C) Corresponding mean activation time constants of I h recorded at a command potential of −100 mV (before Cav3.2 activation, n = 9; after Cav3.2 activation, n = 11). (D) Normalized I h peak current recorded form cells co-expressing Cav3.2 and HCN1 (expressed as the current ratio obtained after and before application of a Cav3.2 activating pulse) before and after the bath application of dibutyryl-cAMP (n = 11).

    Techniques Used: Expressing, Activation Assay

    HCN1 channels interact with the N terminus region of Cav3.2 channels. (A) Left column, western blot of transfected tsA-201 cell lysates showed the expression of HCN1 (input) in every condition. Right column, western blot showed co-immunoprecipitation between HCN1 and full length Cav3.2 (lane 2), but not between HCN1 and Cav3.2 I-II linker, II-III linker, and GFP-tag only (lanes 3, 4 and 5 respectively). Lane 1 showed mock transfection. Lane 6 showed IgG control. (B) Left column, western blot of transfected tsA-201 cell lysates showed the expression of HCN1 (input) in every condition. Right column, western blot showing co-immunoprecipitation between HCN1 and Cav3.2 full length (lane 2) and Cav3.2 N terminus (lane 4), but not between HCN1 and Cav3.2 III-IV linker, C terminus and GFP-tag only (lanes 3, 5 and 6 respectively). Lane 1 reflects mock transfection and lane 7 depicts IgG control. (C) Left column, western blot showing expression of HCN1 (input) in every condition. Right column, western blot showing co-immunoprecipitation between HCN1 and full length Cav3.2, N terminus and N3 terminus (amino acids 25–75) (lanes 2, 3 and 6). Lanes 4, 5 and 7 demonstrate that HCN1 did not co-immunoprecipitate with N1 (1–50) and N2 (51–100) terminus and with a GFP-tag alone. Lane 1 reflects mock transfection and lane 8 is an IgG control. (D) Left column, western blot showing Cav3.2 expression in every condition. Right column, western blot showing co-immunoprecipitation between full length Cav3.2 and HCN1 N terminus (lane 5), but not between Cav3.2 and HCN1 C terminus and mKate tag alone. Lane 1 reflects transfection and lane 3 indicates IgG control. For experiments represented in panels A, B and C, immunoprecipitations were done using an anti-GFP antibody and membranes were blotted with an anti-HCN1 antibody. For experiments represented in panel D, immunoprecipitation was performed with anti-tRFP (against mKate tag) and blots were done with anti-Cav3.2 antibody. These experiments were repeated 3 times with identical results.
    Figure Legend Snippet: HCN1 channels interact with the N terminus region of Cav3.2 channels. (A) Left column, western blot of transfected tsA-201 cell lysates showed the expression of HCN1 (input) in every condition. Right column, western blot showed co-immunoprecipitation between HCN1 and full length Cav3.2 (lane 2), but not between HCN1 and Cav3.2 I-II linker, II-III linker, and GFP-tag only (lanes 3, 4 and 5 respectively). Lane 1 showed mock transfection. Lane 6 showed IgG control. (B) Left column, western blot of transfected tsA-201 cell lysates showed the expression of HCN1 (input) in every condition. Right column, western blot showing co-immunoprecipitation between HCN1 and Cav3.2 full length (lane 2) and Cav3.2 N terminus (lane 4), but not between HCN1 and Cav3.2 III-IV linker, C terminus and GFP-tag only (lanes 3, 5 and 6 respectively). Lane 1 reflects mock transfection and lane 7 depicts IgG control. (C) Left column, western blot showing expression of HCN1 (input) in every condition. Right column, western blot showing co-immunoprecipitation between HCN1 and full length Cav3.2, N terminus and N3 terminus (amino acids 25–75) (lanes 2, 3 and 6). Lanes 4, 5 and 7 demonstrate that HCN1 did not co-immunoprecipitate with N1 (1–50) and N2 (51–100) terminus and with a GFP-tag alone. Lane 1 reflects mock transfection and lane 8 is an IgG control. (D) Left column, western blot showing Cav3.2 expression in every condition. Right column, western blot showing co-immunoprecipitation between full length Cav3.2 and HCN1 N terminus (lane 5), but not between Cav3.2 and HCN1 C terminus and mKate tag alone. Lane 1 reflects transfection and lane 3 indicates IgG control. For experiments represented in panels A, B and C, immunoprecipitations were done using an anti-GFP antibody and membranes were blotted with an anti-HCN1 antibody. For experiments represented in panel D, immunoprecipitation was performed with anti-tRFP (against mKate tag) and blots were done with anti-Cav3.2 antibody. These experiments were repeated 3 times with identical results.

    Techniques Used: Western Blot, Transfection, Expressing, Immunoprecipitation

    4) Product Images from "Caveolin-3 Regulates Protein Kinase A Modulation of the CaV3.2 (?1H) T-type Ca2+ Channels *"

    Article Title: Caveolin-3 Regulates Protein Kinase A Modulation of the CaV3.2 (?1H) T-type Ca2+ Channels *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.182550

    N terminus region of Cav-3 interacts with Ca v 3.2 channel protein and inhibits I Cav3.2 . A , diagram of the domain structure of Cav-3. GST was fused with full-length Cav-3 and the different Cav-3 domains and tested for an ability to interact with the Ca v 3.2 channel. B , representative Western blot analyses of the GST pulldown assay. Ca v 3.2 channels were expressed in HEK293 cells, and lysates were incubated with different GST-Cav-3 fusion proteins as described under “Experimental Procedures”: full-length ( GST-Cav-3FL ), Cav-3 N terminus ( GST-Cav-3NT ), Cav-3 scaffolding domain ( GST-Cav-3Scaf ), Cav-3 membrane domain ( GST-Cav-3Memb ), Cav-3 C terminus domain ( GST-Cav-3CT ), or GST alone. The pulldown samples were analyzed by probing with anti-GST and anti-Ca v 3.2 antibodies. C , HEK cells were transiently expressed with Ca v 3.2 alone or Ca v 3.2 with different Cav-3 domains ( Cav-3NT , Cav-3Scaf , Cav-3CT , and Cav-3Memb ). I Cav3.2 density was measured by the whole cell patch clamp technique and plotted against a change in test potentials. Data represent mean ± S.E. ( n = 7 each, *, p > 0.005 with respect to control) from three different transfections.
    Figure Legend Snippet: N terminus region of Cav-3 interacts with Ca v 3.2 channel protein and inhibits I Cav3.2 . A , diagram of the domain structure of Cav-3. GST was fused with full-length Cav-3 and the different Cav-3 domains and tested for an ability to interact with the Ca v 3.2 channel. B , representative Western blot analyses of the GST pulldown assay. Ca v 3.2 channels were expressed in HEK293 cells, and lysates were incubated with different GST-Cav-3 fusion proteins as described under “Experimental Procedures”: full-length ( GST-Cav-3FL ), Cav-3 N terminus ( GST-Cav-3NT ), Cav-3 scaffolding domain ( GST-Cav-3Scaf ), Cav-3 membrane domain ( GST-Cav-3Memb ), Cav-3 C terminus domain ( GST-Cav-3CT ), or GST alone. The pulldown samples were analyzed by probing with anti-GST and anti-Ca v 3.2 antibodies. C , HEK cells were transiently expressed with Ca v 3.2 alone or Ca v 3.2 with different Cav-3 domains ( Cav-3NT , Cav-3Scaf , Cav-3CT , and Cav-3Memb ). I Cav3.2 density was measured by the whole cell patch clamp technique and plotted against a change in test potentials. Data represent mean ± S.E. ( n = 7 each, *, p > 0.005 with respect to control) from three different transfections.

    Techniques Used: Western Blot, GST Pulldown Assay, Incubation, Scaffolding, Patch Clamp, Transfection

    Cav-3 inhibits I Cav3.2 in mouse neonatal ventricular myocytes. A , average peak I Ca,T density in neonatal ventricular myocytes infected with AdGFP control or with either AdCa v 3.2 or AdCa v 3.2+Cav-3 as indicated in the bar plot . Cav-3 overexpression significantly inhibited the AdCa v 3.2-mediated increased I Cav3.2 . B , average densities of I Ca,L in neonatal ventricular myocytes infected with adenovirus as indicated. Adenovirus infection did not alter the average I Ca,L density in the cells. The data represent mean ± S.E. ( n = 3–5 cells, *, p > 0.005 with respect to AdCa v 3.2). Whole cell patch clamp analysis on the effect of siRNA-mediated knockdown of Cav-3 on I Ca,T in mouse neonatal ventricular myocytes. C , average peak I Ca,T density from nontransfected control ( NT ), GFP control, siRNA to Cav-3 or siRNA to GAPDH ( control )-transfected cells as indicated in the bar plot. Representative corresponding T-type current traces are shown above. D , average densities of I Ca,L in neonatal ventricular myocytes transfected as above and representative corresponding I Ca,L current traces are shown above the bar plot. The average peak I Ca,T or I Ca,L was not significantly different and the data represent mean ± S.E., number of cells used are indicated in parentheses.
    Figure Legend Snippet: Cav-3 inhibits I Cav3.2 in mouse neonatal ventricular myocytes. A , average peak I Ca,T density in neonatal ventricular myocytes infected with AdGFP control or with either AdCa v 3.2 or AdCa v 3.2+Cav-3 as indicated in the bar plot . Cav-3 overexpression significantly inhibited the AdCa v 3.2-mediated increased I Cav3.2 . B , average densities of I Ca,L in neonatal ventricular myocytes infected with adenovirus as indicated. Adenovirus infection did not alter the average I Ca,L density in the cells. The data represent mean ± S.E. ( n = 3–5 cells, *, p > 0.005 with respect to AdCa v 3.2). Whole cell patch clamp analysis on the effect of siRNA-mediated knockdown of Cav-3 on I Ca,T in mouse neonatal ventricular myocytes. C , average peak I Ca,T density from nontransfected control ( NT ), GFP control, siRNA to Cav-3 or siRNA to GAPDH ( control )-transfected cells as indicated in the bar plot. Representative corresponding T-type current traces are shown above. D , average densities of I Ca,L in neonatal ventricular myocytes transfected as above and representative corresponding I Ca,L current traces are shown above the bar plot. The average peak I Ca,T or I Ca,L was not significantly different and the data represent mean ± S.E., number of cells used are indicated in parentheses.

    Techniques Used: Infection, Over Expression, Patch Clamp, Transfection

    Co-expression of Cav-3 inhibits I Cav3.2 but not I Cav3.1 in HEK293 cells. Whole cell voltage clamp recordings of T-type Ca 2+ were performed in HEK293 cells expressing either Ca v 3.1 or Ca v 3.2 alone or co-expressing with Cav-3, by using a holding potential of −90 mV and step pulsed to 60 at 10 mV as shown in the inset. A , representative whole cell Ca 2+ current traces at −90, −30, and +20 mV from transiently transfected HEK293 cells with Ca v 3.1 alone and Ca v 3.1 with Cav-3. B , average current density plotted against the change in test potential for Ca v 3.1 (■) alone and with Ca v 3.1 + Cav3 (●). C , representative whole cell Ca 2+ current traces at −90, −30, and +20 mV from transiently transfected HEK293 cells with Ca v 3.2 alone and Ca v 3.2 with Cav-3. D , average current density plotted against the change in test potential for Ca v 3.2 (■) alone and with Ca v 3.2 + Cav-3 (●). Data represents mean ± S.E. ( n = 11 from 4 different transfections). Co-expression of Cav-3 does not alter plasma membrane expression of Ca v 3.2 channel protein. HEK293 cells were transfected with cDNAs of Ca v 3.2 + GFP or Ca v 3.2 + Cav-3 or GFP alone. E , cell lysates were precipitated with neutravidin beads and the sample was analyzed by Western blots by probing with anti-Ca v 3.2 or anti-β-actin antibodies as indicated in the representative immunoblot. Similar signal intensity for the biotinylated Ca v 3.2 protein was detected with Ca v 3.2 + GFP or Ca v 3.2 + Cav-3. F , a portion of the total lysate (50 μl) sample as input from 3 groups was also analyzed by probing with anti-Ca v 3.2 and anti-β-actin for loading control. Similar signal intensity for the Ca v 3.2 channel protein was detected with either Ca v 3.2 alone or with co-expression of Cav-3 and similar β-actin signal was detected between the three groups of cells demonstrating identical sample loading. Data are representative of three different experiments.
    Figure Legend Snippet: Co-expression of Cav-3 inhibits I Cav3.2 but not I Cav3.1 in HEK293 cells. Whole cell voltage clamp recordings of T-type Ca 2+ were performed in HEK293 cells expressing either Ca v 3.1 or Ca v 3.2 alone or co-expressing with Cav-3, by using a holding potential of −90 mV and step pulsed to 60 at 10 mV as shown in the inset. A , representative whole cell Ca 2+ current traces at −90, −30, and +20 mV from transiently transfected HEK293 cells with Ca v 3.1 alone and Ca v 3.1 with Cav-3. B , average current density plotted against the change in test potential for Ca v 3.1 (■) alone and with Ca v 3.1 + Cav3 (●). C , representative whole cell Ca 2+ current traces at −90, −30, and +20 mV from transiently transfected HEK293 cells with Ca v 3.2 alone and Ca v 3.2 with Cav-3. D , average current density plotted against the change in test potential for Ca v 3.2 (■) alone and with Ca v 3.2 + Cav-3 (●). Data represents mean ± S.E. ( n = 11 from 4 different transfections). Co-expression of Cav-3 does not alter plasma membrane expression of Ca v 3.2 channel protein. HEK293 cells were transfected with cDNAs of Ca v 3.2 + GFP or Ca v 3.2 + Cav-3 or GFP alone. E , cell lysates were precipitated with neutravidin beads and the sample was analyzed by Western blots by probing with anti-Ca v 3.2 or anti-β-actin antibodies as indicated in the representative immunoblot. Similar signal intensity for the biotinylated Ca v 3.2 protein was detected with Ca v 3.2 + GFP or Ca v 3.2 + Cav-3. F , a portion of the total lysate (50 μl) sample as input from 3 groups was also analyzed by probing with anti-Ca v 3.2 and anti-β-actin for loading control. Similar signal intensity for the Ca v 3.2 channel protein was detected with either Ca v 3.2 alone or with co-expression of Cav-3 and similar β-actin signal was detected between the three groups of cells demonstrating identical sample loading. Data are representative of three different experiments.

    Techniques Used: Expressing, Transfection, Western Blot

    5) Product Images from "Down-regulation of T-type Cav3.2 channels by hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1): Evidence of a signaling complex"

    Article Title: Down-regulation of T-type Cav3.2 channels by hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1): Evidence of a signaling complex

    Journal: Channels

    doi: 10.1080/19336950.2017.1326233

    HCN1 channels alter Cav3.2 channel activity. Data shown are from whole-cell voltage clamp recordings from tsA-201 cells transiently transfected with Cav3.2 cDNA with or without HCN1 construct. (A) Representative Ba 2+ current traces recorded from Cav3.2-expressing cells with or without coexpression of HCN1 channels in response to 250 ms depolarizing steps varied from −80 to +40 mV from a holding potential of −100 mV. (B) Corresponding current-voltage relationship for Cav3.2-expressing cells with or without coexpression of HCN1 channels (without HCN1, n = 14; with HCN1, n = 21). (C) Corresponding mean activation time constants of Cav3.2 currents recorded at a command potential −20 mV (without HCN1, n = 14; with HCN1, n = 21). (D) Corresponding mean inactivation time constants of Cav3.2 currents recorded at −20 mV (without HCN1, n = 14; with HCN1, n = 21). (E) Normalized Cav3.2 channel activation curves from Cav3.2-expressing cells with coexpression of HCN1 channels showed a depolarizing shift in half activation voltage (without HCN1, n = 14; with HCN1, n = 21). (F, G) Summary of half activation voltage and maximal conductance (without HCN1, n = 14; with HCN1, n = 21). (H) Inactivation curves were determined in response to a pulse at −20 mV after 5 s-lasting depolarizing prepulses to different potentials from a holding potential of −110 mV (without HCN1, n = 16; with HCN1, n = 16). *, p
    Figure Legend Snippet: HCN1 channels alter Cav3.2 channel activity. Data shown are from whole-cell voltage clamp recordings from tsA-201 cells transiently transfected with Cav3.2 cDNA with or without HCN1 construct. (A) Representative Ba 2+ current traces recorded from Cav3.2-expressing cells with or without coexpression of HCN1 channels in response to 250 ms depolarizing steps varied from −80 to +40 mV from a holding potential of −100 mV. (B) Corresponding current-voltage relationship for Cav3.2-expressing cells with or without coexpression of HCN1 channels (without HCN1, n = 14; with HCN1, n = 21). (C) Corresponding mean activation time constants of Cav3.2 currents recorded at a command potential −20 mV (without HCN1, n = 14; with HCN1, n = 21). (D) Corresponding mean inactivation time constants of Cav3.2 currents recorded at −20 mV (without HCN1, n = 14; with HCN1, n = 21). (E) Normalized Cav3.2 channel activation curves from Cav3.2-expressing cells with coexpression of HCN1 channels showed a depolarizing shift in half activation voltage (without HCN1, n = 14; with HCN1, n = 21). (F, G) Summary of half activation voltage and maximal conductance (without HCN1, n = 14; with HCN1, n = 21). (H) Inactivation curves were determined in response to a pulse at −20 mV after 5 s-lasting depolarizing prepulses to different potentials from a holding potential of −110 mV (without HCN1, n = 16; with HCN1, n = 16). *, p

    Techniques Used: Activity Assay, Transfection, Construct, Expressing, Activation Assay

    HCN1 channels interact with Cav3.2 channels in mouse brain. (A) Co-immunoprecipitation of HCN1 and T-type Ca 2+ channel subunits using lysates from adult mouse brain showed that HCN1 channels associate with Cav3.2 T-type channels (lane 4). The samples were blotted with anti-HCN1 antibody. (B) western blot showing that HCN2 channels did not co-immunoprecipitate with any of the T-type channel subunits. HCN1 and HCN2 protein could both be detected in mouse brain homogenates (input). The Nuclear receptor related 1 protein (NURR1) here was used as an irrelevant antibody for the negative control. The samples were blotted with anti-HCN2 antibody. These experiments were repeated 4 times (with tissue obtained from 4 different mice) with identical results.
    Figure Legend Snippet: HCN1 channels interact with Cav3.2 channels in mouse brain. (A) Co-immunoprecipitation of HCN1 and T-type Ca 2+ channel subunits using lysates from adult mouse brain showed that HCN1 channels associate with Cav3.2 T-type channels (lane 4). The samples were blotted with anti-HCN1 antibody. (B) western blot showing that HCN2 channels did not co-immunoprecipitate with any of the T-type channel subunits. HCN1 and HCN2 protein could both be detected in mouse brain homogenates (input). The Nuclear receptor related 1 protein (NURR1) here was used as an irrelevant antibody for the negative control. The samples were blotted with anti-HCN2 antibody. These experiments were repeated 4 times (with tissue obtained from 4 different mice) with identical results.

    Techniques Used: Immunoprecipitation, Western Blot, Negative Control, Mouse Assay

    Cav3.2 co-expression does not alter current density or kinetics of HCN1 channels. (A) Sample I h currents recorded from tsA-201 cells without (black) or with co-expression of Cav3.2 channels (red) in response to a hyperpolarizing voltage step from −50 mV to −100 mV. The currents were leak subtracted using a P/4 protocol. To obtain stable recordings, the cells were held at −50 mV to minimize leakage currents from the opening of HCN1 channels at rest. To maximally recover Cav3.2 channels from inactivation and to measure I h , a 500 ms hyperpolarizing pulse to −100 mV was first applied. Then, a 200 ms pulse to −20 mV was applied to activate maximum calcium influx via Cav3.2. Finally, I h was activated by 1 s hyperpolarizing pulse to −100 mV (10 ms return to the holding at −50 mV before test pulse applied). The extracellular solution contained 2.5 mM Ca 2+ . (B) Effect of Cav3.2 coexpression on the mean peak current density of HCN1 channels (without Cav3.2, n = 9; with Cav3.2, n = 11). (C) Corresponding mean activation time constants of I h recorded at a command potential of −100 mV (before Cav3.2 activation, n = 9; after Cav3.2 activation, n = 11). (D) Normalized I h peak current recorded form cells co-expressing Cav3.2 and HCN1 (expressed as the current ratio obtained after and before application of a Cav3.2 activating pulse) before and after the bath application of dibutyryl-cAMP (n = 11).
    Figure Legend Snippet: Cav3.2 co-expression does not alter current density or kinetics of HCN1 channels. (A) Sample I h currents recorded from tsA-201 cells without (black) or with co-expression of Cav3.2 channels (red) in response to a hyperpolarizing voltage step from −50 mV to −100 mV. The currents were leak subtracted using a P/4 protocol. To obtain stable recordings, the cells were held at −50 mV to minimize leakage currents from the opening of HCN1 channels at rest. To maximally recover Cav3.2 channels from inactivation and to measure I h , a 500 ms hyperpolarizing pulse to −100 mV was first applied. Then, a 200 ms pulse to −20 mV was applied to activate maximum calcium influx via Cav3.2. Finally, I h was activated by 1 s hyperpolarizing pulse to −100 mV (10 ms return to the holding at −50 mV before test pulse applied). The extracellular solution contained 2.5 mM Ca 2+ . (B) Effect of Cav3.2 coexpression on the mean peak current density of HCN1 channels (without Cav3.2, n = 9; with Cav3.2, n = 11). (C) Corresponding mean activation time constants of I h recorded at a command potential of −100 mV (before Cav3.2 activation, n = 9; after Cav3.2 activation, n = 11). (D) Normalized I h peak current recorded form cells co-expressing Cav3.2 and HCN1 (expressed as the current ratio obtained after and before application of a Cav3.2 activating pulse) before and after the bath application of dibutyryl-cAMP (n = 11).

    Techniques Used: Expressing, Activation Assay

    HCN1 channels interact with the N terminus region of Cav3.2 channels. (A) Left column, western blot of transfected tsA-201 cell lysates showed the expression of HCN1 (input) in every condition. Right column, western blot showed co-immunoprecipitation between HCN1 and full length Cav3.2 (lane 2), but not between HCN1 and Cav3.2 I-II linker, II-III linker, and GFP-tag only (lanes 3, 4 and 5 respectively). Lane 1 showed mock transfection. Lane 6 showed IgG control. (B) Left column, western blot of transfected tsA-201 cell lysates showed the expression of HCN1 (input) in every condition. Right column, western blot showing co-immunoprecipitation between HCN1 and Cav3.2 full length (lane 2) and Cav3.2 N terminus (lane 4), but not between HCN1 and Cav3.2 III-IV linker, C terminus and GFP-tag only (lanes 3, 5 and 6 respectively). Lane 1 reflects mock transfection and lane 7 depicts IgG control. (C) Left column, western blot showing expression of HCN1 (input) in every condition. Right column, western blot showing co-immunoprecipitation between HCN1 and full length Cav3.2, N terminus and N3 terminus (amino acids 25–75) (lanes 2, 3 and 6). Lanes 4, 5 and 7 demonstrate that HCN1 did not co-immunoprecipitate with N1 (1–50) and N2 (51–100) terminus and with a GFP-tag alone. Lane 1 reflects mock transfection and lane 8 is an IgG control. (D) Left column, western blot showing Cav3.2 expression in every condition. Right column, western blot showing co-immunoprecipitation between full length Cav3.2 and HCN1 N terminus (lane 5), but not between Cav3.2 and HCN1 C terminus and mKate tag alone. Lane 1 reflects transfection and lane 3 indicates IgG control. For experiments represented in panels A, B and C, immunoprecipitations were done using an anti-GFP antibody and membranes were blotted with an anti-HCN1 antibody. For experiments represented in panel D, immunoprecipitation was performed with anti-tRFP (against mKate tag) and blots were done with anti-Cav3.2 antibody. These experiments were repeated 3 times with identical results.
    Figure Legend Snippet: HCN1 channels interact with the N terminus region of Cav3.2 channels. (A) Left column, western blot of transfected tsA-201 cell lysates showed the expression of HCN1 (input) in every condition. Right column, western blot showed co-immunoprecipitation between HCN1 and full length Cav3.2 (lane 2), but not between HCN1 and Cav3.2 I-II linker, II-III linker, and GFP-tag only (lanes 3, 4 and 5 respectively). Lane 1 showed mock transfection. Lane 6 showed IgG control. (B) Left column, western blot of transfected tsA-201 cell lysates showed the expression of HCN1 (input) in every condition. Right column, western blot showing co-immunoprecipitation between HCN1 and Cav3.2 full length (lane 2) and Cav3.2 N terminus (lane 4), but not between HCN1 and Cav3.2 III-IV linker, C terminus and GFP-tag only (lanes 3, 5 and 6 respectively). Lane 1 reflects mock transfection and lane 7 depicts IgG control. (C) Left column, western blot showing expression of HCN1 (input) in every condition. Right column, western blot showing co-immunoprecipitation between HCN1 and full length Cav3.2, N terminus and N3 terminus (amino acids 25–75) (lanes 2, 3 and 6). Lanes 4, 5 and 7 demonstrate that HCN1 did not co-immunoprecipitate with N1 (1–50) and N2 (51–100) terminus and with a GFP-tag alone. Lane 1 reflects mock transfection and lane 8 is an IgG control. (D) Left column, western blot showing Cav3.2 expression in every condition. Right column, western blot showing co-immunoprecipitation between full length Cav3.2 and HCN1 N terminus (lane 5), but not between Cav3.2 and HCN1 C terminus and mKate tag alone. Lane 1 reflects transfection and lane 3 indicates IgG control. For experiments represented in panels A, B and C, immunoprecipitations were done using an anti-GFP antibody and membranes were blotted with an anti-HCN1 antibody. For experiments represented in panel D, immunoprecipitation was performed with anti-tRFP (against mKate tag) and blots were done with anti-Cav3.2 antibody. These experiments were repeated 3 times with identical results.

    Techniques Used: Western Blot, Transfection, Expressing, Immunoprecipitation

    6) Product Images from "Id2 Represses Aldosterone-Stimulated Cardiac T-Type Calcium Channels Expression"

    Article Title: Id2 Represses Aldosterone-Stimulated Cardiac T-Type Calcium Channels Expression

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms22073561

    Manipulation of Id2′s expression altered of voltage-gated calcium channel expression in neonatal rat ventricular cardiomyocytes and prevented aldosterone-stimulated increased. ( A ) 2, CaV3.1, and CaV3.2 were measured by RT-qPCR in neonatal rat ventricular cardiomyocytes treated and non-treated with 1 µmol/L Aldo for 24 h upon Id2 overexpression. Bar graphs shows the mean expression of CaV1.2 mRNA (left), CaV3.1 mRNA (middle), and CaV3.2 (right) ( n = 5). ( B ) The mRNA expression of CaV1.2, CaV3.1, and CaV3.2 were measured by real-time qPCR in neonatal rat ventricular cardiomyocytes treated with Id2 siRNA-2, siRNA-5 or Luciferase1 siRNA for 48 h. Bar graphs shows the mean expression of CaV1.2 mRNA (left), CaV3.1 mRNA (middle), and CaV3.2 (right) ( n = 3). The data were evaluated by one-way analysis of variance (ANOVA) followed by Tukey–Kramer’s post hoc test. Data are the mean ± s.e.m, * p
    Figure Legend Snippet: Manipulation of Id2′s expression altered of voltage-gated calcium channel expression in neonatal rat ventricular cardiomyocytes and prevented aldosterone-stimulated increased. ( A ) 2, CaV3.1, and CaV3.2 were measured by RT-qPCR in neonatal rat ventricular cardiomyocytes treated and non-treated with 1 µmol/L Aldo for 24 h upon Id2 overexpression. Bar graphs shows the mean expression of CaV1.2 mRNA (left), CaV3.1 mRNA (middle), and CaV3.2 (right) ( n = 5). ( B ) The mRNA expression of CaV1.2, CaV3.1, and CaV3.2 were measured by real-time qPCR in neonatal rat ventricular cardiomyocytes treated with Id2 siRNA-2, siRNA-5 or Luciferase1 siRNA for 48 h. Bar graphs shows the mean expression of CaV1.2 mRNA (left), CaV3.1 mRNA (middle), and CaV3.2 (right) ( n = 3). The data were evaluated by one-way analysis of variance (ANOVA) followed by Tukey–Kramer’s post hoc test. Data are the mean ± s.e.m, * p

    Techniques Used: Expressing, Quantitative RT-PCR, Over Expression, Real-time Polymerase Chain Reaction

    7) Product Images from "Transmitter and ion channel profiles of neurons in the primate abducens and trochlear nuclei"

    Article Title: Transmitter and ion channel profiles of neurons in the primate abducens and trochlear nuclei

    Journal: Brain Structure & Function

    doi: 10.1007/s00429-021-02315-7

    Low-voltage activated calcium channel family (Cav3) subunits in the abducens and trochlear nucleus. a Consecutive coronal paraffin sections through the abducens nucleus (nVI) ( a ) depicting the immunolabeling for Cav3.1 (first panel), Cav3.2 (third panel) and Cav3.3 (fourth panel) subunits with combined immunostaining for ChAT (brown) and ACAN (black) (second panel) as reference. b, c Close-up of Cav subunit expression in nVI MIF (red arrowheads) and SIF motoneurons (MNs) (green arrows) and INTs (blue arrow). d Consecutive coronal paraffin sections through the trochlear nucleus (nVI) depicting the immunolabeling for Cav3.1 (first panel), Cav3.2 (second panel) and Cav3.3 (fourth panel) subunits with combined immunostaining for ChAT (brown) and ACAN (black) (third panel) as reference. Thin dashed lines in d indicate the border of nIV and thick dashed lines indicate the boundary between the MIF and SIF MNs. e Close-up of Cav subunit expression in MIF (red arrowheads) and SIF MNs (green arrow) on different sections as those illustrated in d . Note the weak Cav3.1 expression along the membrane of some SIF MNs (left column, black star). Red dashed lines indicate the tentative position of the border delineating the dorsal cap of nIV. f Cerebellar Purkinje cells located on the same consecutive sections as nVI as controls for immunopositivity of the different Cav subunits. Scale bar indicates 100 μm in a, d, f and 50 μm in b, c, e
    Figure Legend Snippet: Low-voltage activated calcium channel family (Cav3) subunits in the abducens and trochlear nucleus. a Consecutive coronal paraffin sections through the abducens nucleus (nVI) ( a ) depicting the immunolabeling for Cav3.1 (first panel), Cav3.2 (third panel) and Cav3.3 (fourth panel) subunits with combined immunostaining for ChAT (brown) and ACAN (black) (second panel) as reference. b, c Close-up of Cav subunit expression in nVI MIF (red arrowheads) and SIF motoneurons (MNs) (green arrows) and INTs (blue arrow). d Consecutive coronal paraffin sections through the trochlear nucleus (nVI) depicting the immunolabeling for Cav3.1 (first panel), Cav3.2 (second panel) and Cav3.3 (fourth panel) subunits with combined immunostaining for ChAT (brown) and ACAN (black) (third panel) as reference. Thin dashed lines in d indicate the border of nIV and thick dashed lines indicate the boundary between the MIF and SIF MNs. e Close-up of Cav subunit expression in MIF (red arrowheads) and SIF MNs (green arrow) on different sections as those illustrated in d . Note the weak Cav3.1 expression along the membrane of some SIF MNs (left column, black star). Red dashed lines indicate the tentative position of the border delineating the dorsal cap of nIV. f Cerebellar Purkinje cells located on the same consecutive sections as nVI as controls for immunopositivity of the different Cav subunits. Scale bar indicates 100 μm in a, d, f and 50 μm in b, c, e

    Techniques Used: Immunolabeling, Immunostaining, Expressing

    8) Product Images from "Developmentally Regulated Rebound Depolarization Enhances Spike Timing Precision in Auditory Midbrain Neurons"

    Article Title: Developmentally Regulated Rebound Depolarization Enhances Spike Timing Precision in Auditory Midbrain Neurons

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2020.00236

    Developmental regulation of rebound depolarization in inferior colliculus (IC) neurons. (A) Distribution of rebound (red circle) and non-rebound (blue triangle) neurons in the IC. The IC was divided into the ICX (lateral region), ICC (middle region), and ICD (mediodorsal region). (B) Responses of a rebound neuron (top traces) and a non-rebound neuron (bottom traces) to a positive (60 pA) and a negative (−100 pA) current pulse. Arrowhead points to the rebound. (C) The rebound and anode break spikes (arrow) were blocked by 5 μm mibefradil. (D) Proportions of rebound neurons before (P9–11), during (P12–13), and after (P14–21) the onset of hearing. (E–G) Double-labeling immunocytochemistry with T-type calcium channel isoforms CaV3.1 ( E , green), CaV3.2 ( F , green), and CaV3.3 ( G , green) and the microtubule-associated protein 2 (MAP2; E – G , red) in the IC from P10 and P21 rats demonstrates a developmental increase in the expression of CaV3.1, CaV3.2, and CaV3.3, with the highest changes in the CaV3.2 expression in IC neurons. Scale bars 20 μm.
    Figure Legend Snippet: Developmental regulation of rebound depolarization in inferior colliculus (IC) neurons. (A) Distribution of rebound (red circle) and non-rebound (blue triangle) neurons in the IC. The IC was divided into the ICX (lateral region), ICC (middle region), and ICD (mediodorsal region). (B) Responses of a rebound neuron (top traces) and a non-rebound neuron (bottom traces) to a positive (60 pA) and a negative (−100 pA) current pulse. Arrowhead points to the rebound. (C) The rebound and anode break spikes (arrow) were blocked by 5 μm mibefradil. (D) Proportions of rebound neurons before (P9–11), during (P12–13), and after (P14–21) the onset of hearing. (E–G) Double-labeling immunocytochemistry with T-type calcium channel isoforms CaV3.1 ( E , green), CaV3.2 ( F , green), and CaV3.3 ( G , green) and the microtubule-associated protein 2 (MAP2; E – G , red) in the IC from P10 and P21 rats demonstrates a developmental increase in the expression of CaV3.1, CaV3.2, and CaV3.3, with the highest changes in the CaV3.2 expression in IC neurons. Scale bars 20 μm.

    Techniques Used: Immunocytochemistry, Labeling, Expressing

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    Alomone Labs anti cav3 2
    Knockdown or overexpression of TRPC7 did not alter the expression of several important ion channels/pump in NRVMs. a – g Western blots showing the expression of a TRPC7, b HCN4, c Cav1.3, d IP3R1, e Cav3.1, f <t>Cav3.2,</t> g SERCA in NRVMs infected with different adenoviruses to knockdown or overexpress TRPC7. h – n Bar charts showing the quantification of each protein from a – g . To eliminate the loading bias, intensity of each target protein was normalized to that of its corresponding β-tubulin. TRPC7 was successfully knocked down or overexpressed in NRVMs but the change of TRPC7 expression did not alter the expression of HCN4, Cav1.3, IP3R1, Cav3.1, Cav3.2, and SERCA. Data were presented as mean ± SEM ( n = 4). * P
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    Knockdown or overexpression of TRPC7 did not alter the expression of several important ion channels/pump in NRVMs. a – g Western blots showing the expression of a TRPC7, b HCN4, c Cav1.3, d IP3R1, e Cav3.1, f Cav3.2, g SERCA in NRVMs infected with different adenoviruses to knockdown or overexpress TRPC7. h – n Bar charts showing the quantification of each protein from a – g . To eliminate the loading bias, intensity of each target protein was normalized to that of its corresponding β-tubulin. TRPC7 was successfully knocked down or overexpressed in NRVMs but the change of TRPC7 expression did not alter the expression of HCN4, Cav1.3, IP3R1, Cav3.1, Cav3.2, and SERCA. Data were presented as mean ± SEM ( n = 4). * P

    Journal: Stem Cell Research & Therapy

    Article Title: TRPC7 regulates the electrophysiological functions of embryonic stem cell-derived cardiomyocytes

    doi: 10.1186/s13287-021-02308-7

    Figure Lengend Snippet: Knockdown or overexpression of TRPC7 did not alter the expression of several important ion channels/pump in NRVMs. a – g Western blots showing the expression of a TRPC7, b HCN4, c Cav1.3, d IP3R1, e Cav3.1, f Cav3.2, g SERCA in NRVMs infected with different adenoviruses to knockdown or overexpress TRPC7. h – n Bar charts showing the quantification of each protein from a – g . To eliminate the loading bias, intensity of each target protein was normalized to that of its corresponding β-tubulin. TRPC7 was successfully knocked down or overexpressed in NRVMs but the change of TRPC7 expression did not alter the expression of HCN4, Cav1.3, IP3R1, Cav3.1, Cav3.2, and SERCA. Data were presented as mean ± SEM ( n = 4). * P

    Article Snippet: Antibodies used were anti-TRPC7 1:500 (HPA031126, Sigma), anti-β-tubulin 1:1000 (15,115, Cell Signaling, Danvers, Massachusetts, USA), anti-HCN4 1:200 (APC-052, Alomone), anti-Cav1.3 1:100 (ACC-005, Alomone), anti-Cav3.1 1:200 (ACC-021, Alomone), anti-Cav3.2 1:200 (ACC-025, Alomone), anti-RyR2 1:1000 (MA3-916, Invitrogen), anti-SERCA 1:200 (sc-30110, Santa Cruz), anti-IP3R 1:500 (ACC-019, Alomone), anti-p(S2814)RyR2 1:5000 (A010-31, Badrilla), anti-phospholamban (PLN) 1:1000 (A010-14, Badrilla), anti-p(T17) PLN 1:5000 (A010-13, Badrilla), HRP-conjugated goat anti-rabbit secondary antibody 1:5000 (Dako, Zug, Switzerland), and HRP-conjugated goat anti-mouse secondary antibody 1:5000 (Dako).

    Techniques: Over Expression, Expressing, Western Blot, Infection

    In vivo and in vitro expression of Cav3.2: Visualisation of the in vivo and in vitro distribution of Cav3.2 via anti-Cav3.2 antibodies. In dissociated PCs at 2div ( A – D ), Cav3.2 was prominent at the dendrites or axons, whereas in the cryosections of rat cerebella at p0 ( E – H ), the signal of Cav 3.2 was mostly at the soma of the PCs. In the age of p9 ( I – L ), there was a clear colocalization with the dendrites and the soma of the PCs. eGCL = external granular cell layer, ML = molecular layer, PCL = PC layer, iGCL = internal granular cell layer. Scale bar: 20 µm ( A , B , E , F , I , J ) and 2 µm ( C , D , G , H , K , L ).

    Journal: Cells

    Article Title: Expression Pattern of T-Type Ca2+ Channels in Cerebellar Purkinje Cells after VEGF Treatment

    doi: 10.3390/cells10092277

    Figure Lengend Snippet: In vivo and in vitro expression of Cav3.2: Visualisation of the in vivo and in vitro distribution of Cav3.2 via anti-Cav3.2 antibodies. In dissociated PCs at 2div ( A – D ), Cav3.2 was prominent at the dendrites or axons, whereas in the cryosections of rat cerebella at p0 ( E – H ), the signal of Cav 3.2 was mostly at the soma of the PCs. In the age of p9 ( I – L ), there was a clear colocalization with the dendrites and the soma of the PCs. eGCL = external granular cell layer, ML = molecular layer, PCL = PC layer, iGCL = internal granular cell layer. Scale bar: 20 µm ( A , B , E , F , I , J ) and 2 µm ( C , D , G , H , K , L ).

    Article Snippet: On the next day, the samples were washed with PBS three times for 10 min and incubated with secondary anti-mouse TRITC antibody (goat, 1:1000 T5393, Sigma-Aldrich) at room temperature for 2 h, respectively, 2.5 h. After additional washing, the second primary antibody anti-Cav3.1 (rabbit, 1:1500 ACC21, Alomone Labs), anti-Cav3.2 (Rabbit, 1:1500 ACC25, Alomone Labs) or anti-Cav3.3 (rabbit, 1:1000 ACC-009, Alomone Labs) was applied at 4 °C overnight.

    Techniques: In Vivo, In Vitro, Expressing

    HCN1 channels alter Cav3.2 channel activity. Data shown are from whole-cell voltage clamp recordings from tsA-201 cells transiently transfected with Cav3.2 cDNA with or without HCN1 construct. (A) Representative Ba 2+ current traces recorded from Cav3.2-expressing cells with or without coexpression of HCN1 channels in response to 250 ms depolarizing steps varied from −80 to +40 mV from a holding potential of −100 mV. (B) Corresponding current-voltage relationship for Cav3.2-expressing cells with or without coexpression of HCN1 channels (without HCN1, n = 14; with HCN1, n = 21). (C) Corresponding mean activation time constants of Cav3.2 currents recorded at a command potential −20 mV (without HCN1, n = 14; with HCN1, n = 21). (D) Corresponding mean inactivation time constants of Cav3.2 currents recorded at −20 mV (without HCN1, n = 14; with HCN1, n = 21). (E) Normalized Cav3.2 channel activation curves from Cav3.2-expressing cells with coexpression of HCN1 channels showed a depolarizing shift in half activation voltage (without HCN1, n = 14; with HCN1, n = 21). (F, G) Summary of half activation voltage and maximal conductance (without HCN1, n = 14; with HCN1, n = 21). (H) Inactivation curves were determined in response to a pulse at −20 mV after 5 s-lasting depolarizing prepulses to different potentials from a holding potential of −110 mV (without HCN1, n = 16; with HCN1, n = 16). *, p

    Journal: Channels

    Article Title: Down-regulation of T-type Cav3.2 channels by hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1): Evidence of a signaling complex

    doi: 10.1080/19336950.2017.1326233

    Figure Lengend Snippet: HCN1 channels alter Cav3.2 channel activity. Data shown are from whole-cell voltage clamp recordings from tsA-201 cells transiently transfected with Cav3.2 cDNA with or without HCN1 construct. (A) Representative Ba 2+ current traces recorded from Cav3.2-expressing cells with or without coexpression of HCN1 channels in response to 250 ms depolarizing steps varied from −80 to +40 mV from a holding potential of −100 mV. (B) Corresponding current-voltage relationship for Cav3.2-expressing cells with or without coexpression of HCN1 channels (without HCN1, n = 14; with HCN1, n = 21). (C) Corresponding mean activation time constants of Cav3.2 currents recorded at a command potential −20 mV (without HCN1, n = 14; with HCN1, n = 21). (D) Corresponding mean inactivation time constants of Cav3.2 currents recorded at −20 mV (without HCN1, n = 14; with HCN1, n = 21). (E) Normalized Cav3.2 channel activation curves from Cav3.2-expressing cells with coexpression of HCN1 channels showed a depolarizing shift in half activation voltage (without HCN1, n = 14; with HCN1, n = 21). (F, G) Summary of half activation voltage and maximal conductance (without HCN1, n = 14; with HCN1, n = 21). (H) Inactivation curves were determined in response to a pulse at −20 mV after 5 s-lasting depolarizing prepulses to different potentials from a holding potential of −110 mV (without HCN1, n = 16; with HCN1, n = 16). *, p

    Article Snippet: Samples were transferred to a 0.2 μm nitrocellulose membrane (Bio-Rad) and western blot analysis was performed using one of the following antibodies: anti-HCN1 (1:300, Neuromab, 75–110), anti-HCN2 (1:300, Neuromab, 71–37), anti-Cav3.1 (1:200, Alomone, ACC-021), anti-Cav3.2 (1:200, Santa Cruz, sc-25691), and anti-Cav3.3 (1:200, Alomone, ACC-0009).

    Techniques: Activity Assay, Transfection, Construct, Expressing, Activation Assay

    HCN1 channels interact with Cav3.2 channels in mouse brain. (A) Co-immunoprecipitation of HCN1 and T-type Ca 2+ channel subunits using lysates from adult mouse brain showed that HCN1 channels associate with Cav3.2 T-type channels (lane 4). The samples were blotted with anti-HCN1 antibody. (B) western blot showing that HCN2 channels did not co-immunoprecipitate with any of the T-type channel subunits. HCN1 and HCN2 protein could both be detected in mouse brain homogenates (input). The Nuclear receptor related 1 protein (NURR1) here was used as an irrelevant antibody for the negative control. The samples were blotted with anti-HCN2 antibody. These experiments were repeated 4 times (with tissue obtained from 4 different mice) with identical results.

    Journal: Channels

    Article Title: Down-regulation of T-type Cav3.2 channels by hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1): Evidence of a signaling complex

    doi: 10.1080/19336950.2017.1326233

    Figure Lengend Snippet: HCN1 channels interact with Cav3.2 channels in mouse brain. (A) Co-immunoprecipitation of HCN1 and T-type Ca 2+ channel subunits using lysates from adult mouse brain showed that HCN1 channels associate with Cav3.2 T-type channels (lane 4). The samples were blotted with anti-HCN1 antibody. (B) western blot showing that HCN2 channels did not co-immunoprecipitate with any of the T-type channel subunits. HCN1 and HCN2 protein could both be detected in mouse brain homogenates (input). The Nuclear receptor related 1 protein (NURR1) here was used as an irrelevant antibody for the negative control. The samples were blotted with anti-HCN2 antibody. These experiments were repeated 4 times (with tissue obtained from 4 different mice) with identical results.

    Article Snippet: Samples were transferred to a 0.2 μm nitrocellulose membrane (Bio-Rad) and western blot analysis was performed using one of the following antibodies: anti-HCN1 (1:300, Neuromab, 75–110), anti-HCN2 (1:300, Neuromab, 71–37), anti-Cav3.1 (1:200, Alomone, ACC-021), anti-Cav3.2 (1:200, Santa Cruz, sc-25691), and anti-Cav3.3 (1:200, Alomone, ACC-0009).

    Techniques: Immunoprecipitation, Western Blot, Negative Control, Mouse Assay

    Cav3.2 co-expression does not alter current density or kinetics of HCN1 channels. (A) Sample I h currents recorded from tsA-201 cells without (black) or with co-expression of Cav3.2 channels (red) in response to a hyperpolarizing voltage step from −50 mV to −100 mV. The currents were leak subtracted using a P/4 protocol. To obtain stable recordings, the cells were held at −50 mV to minimize leakage currents from the opening of HCN1 channels at rest. To maximally recover Cav3.2 channels from inactivation and to measure I h , a 500 ms hyperpolarizing pulse to −100 mV was first applied. Then, a 200 ms pulse to −20 mV was applied to activate maximum calcium influx via Cav3.2. Finally, I h was activated by 1 s hyperpolarizing pulse to −100 mV (10 ms return to the holding at −50 mV before test pulse applied). The extracellular solution contained 2.5 mM Ca 2+ . (B) Effect of Cav3.2 coexpression on the mean peak current density of HCN1 channels (without Cav3.2, n = 9; with Cav3.2, n = 11). (C) Corresponding mean activation time constants of I h recorded at a command potential of −100 mV (before Cav3.2 activation, n = 9; after Cav3.2 activation, n = 11). (D) Normalized I h peak current recorded form cells co-expressing Cav3.2 and HCN1 (expressed as the current ratio obtained after and before application of a Cav3.2 activating pulse) before and after the bath application of dibutyryl-cAMP (n = 11).

    Journal: Channels

    Article Title: Down-regulation of T-type Cav3.2 channels by hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1): Evidence of a signaling complex

    doi: 10.1080/19336950.2017.1326233

    Figure Lengend Snippet: Cav3.2 co-expression does not alter current density or kinetics of HCN1 channels. (A) Sample I h currents recorded from tsA-201 cells without (black) or with co-expression of Cav3.2 channels (red) in response to a hyperpolarizing voltage step from −50 mV to −100 mV. The currents were leak subtracted using a P/4 protocol. To obtain stable recordings, the cells were held at −50 mV to minimize leakage currents from the opening of HCN1 channels at rest. To maximally recover Cav3.2 channels from inactivation and to measure I h , a 500 ms hyperpolarizing pulse to −100 mV was first applied. Then, a 200 ms pulse to −20 mV was applied to activate maximum calcium influx via Cav3.2. Finally, I h was activated by 1 s hyperpolarizing pulse to −100 mV (10 ms return to the holding at −50 mV before test pulse applied). The extracellular solution contained 2.5 mM Ca 2+ . (B) Effect of Cav3.2 coexpression on the mean peak current density of HCN1 channels (without Cav3.2, n = 9; with Cav3.2, n = 11). (C) Corresponding mean activation time constants of I h recorded at a command potential of −100 mV (before Cav3.2 activation, n = 9; after Cav3.2 activation, n = 11). (D) Normalized I h peak current recorded form cells co-expressing Cav3.2 and HCN1 (expressed as the current ratio obtained after and before application of a Cav3.2 activating pulse) before and after the bath application of dibutyryl-cAMP (n = 11).

    Article Snippet: Samples were transferred to a 0.2 μm nitrocellulose membrane (Bio-Rad) and western blot analysis was performed using one of the following antibodies: anti-HCN1 (1:300, Neuromab, 75–110), anti-HCN2 (1:300, Neuromab, 71–37), anti-Cav3.1 (1:200, Alomone, ACC-021), anti-Cav3.2 (1:200, Santa Cruz, sc-25691), and anti-Cav3.3 (1:200, Alomone, ACC-0009).

    Techniques: Expressing, Activation Assay

    HCN1 channels interact with the N terminus region of Cav3.2 channels. (A) Left column, western blot of transfected tsA-201 cell lysates showed the expression of HCN1 (input) in every condition. Right column, western blot showed co-immunoprecipitation between HCN1 and full length Cav3.2 (lane 2), but not between HCN1 and Cav3.2 I-II linker, II-III linker, and GFP-tag only (lanes 3, 4 and 5 respectively). Lane 1 showed mock transfection. Lane 6 showed IgG control. (B) Left column, western blot of transfected tsA-201 cell lysates showed the expression of HCN1 (input) in every condition. Right column, western blot showing co-immunoprecipitation between HCN1 and Cav3.2 full length (lane 2) and Cav3.2 N terminus (lane 4), but not between HCN1 and Cav3.2 III-IV linker, C terminus and GFP-tag only (lanes 3, 5 and 6 respectively). Lane 1 reflects mock transfection and lane 7 depicts IgG control. (C) Left column, western blot showing expression of HCN1 (input) in every condition. Right column, western blot showing co-immunoprecipitation between HCN1 and full length Cav3.2, N terminus and N3 terminus (amino acids 25–75) (lanes 2, 3 and 6). Lanes 4, 5 and 7 demonstrate that HCN1 did not co-immunoprecipitate with N1 (1–50) and N2 (51–100) terminus and with a GFP-tag alone. Lane 1 reflects mock transfection and lane 8 is an IgG control. (D) Left column, western blot showing Cav3.2 expression in every condition. Right column, western blot showing co-immunoprecipitation between full length Cav3.2 and HCN1 N terminus (lane 5), but not between Cav3.2 and HCN1 C terminus and mKate tag alone. Lane 1 reflects transfection and lane 3 indicates IgG control. For experiments represented in panels A, B and C, immunoprecipitations were done using an anti-GFP antibody and membranes were blotted with an anti-HCN1 antibody. For experiments represented in panel D, immunoprecipitation was performed with anti-tRFP (against mKate tag) and blots were done with anti-Cav3.2 antibody. These experiments were repeated 3 times with identical results.

    Journal: Channels

    Article Title: Down-regulation of T-type Cav3.2 channels by hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1): Evidence of a signaling complex

    doi: 10.1080/19336950.2017.1326233

    Figure Lengend Snippet: HCN1 channels interact with the N terminus region of Cav3.2 channels. (A) Left column, western blot of transfected tsA-201 cell lysates showed the expression of HCN1 (input) in every condition. Right column, western blot showed co-immunoprecipitation between HCN1 and full length Cav3.2 (lane 2), but not between HCN1 and Cav3.2 I-II linker, II-III linker, and GFP-tag only (lanes 3, 4 and 5 respectively). Lane 1 showed mock transfection. Lane 6 showed IgG control. (B) Left column, western blot of transfected tsA-201 cell lysates showed the expression of HCN1 (input) in every condition. Right column, western blot showing co-immunoprecipitation between HCN1 and Cav3.2 full length (lane 2) and Cav3.2 N terminus (lane 4), but not between HCN1 and Cav3.2 III-IV linker, C terminus and GFP-tag only (lanes 3, 5 and 6 respectively). Lane 1 reflects mock transfection and lane 7 depicts IgG control. (C) Left column, western blot showing expression of HCN1 (input) in every condition. Right column, western blot showing co-immunoprecipitation between HCN1 and full length Cav3.2, N terminus and N3 terminus (amino acids 25–75) (lanes 2, 3 and 6). Lanes 4, 5 and 7 demonstrate that HCN1 did not co-immunoprecipitate with N1 (1–50) and N2 (51–100) terminus and with a GFP-tag alone. Lane 1 reflects mock transfection and lane 8 is an IgG control. (D) Left column, western blot showing Cav3.2 expression in every condition. Right column, western blot showing co-immunoprecipitation between full length Cav3.2 and HCN1 N terminus (lane 5), but not between Cav3.2 and HCN1 C terminus and mKate tag alone. Lane 1 reflects transfection and lane 3 indicates IgG control. For experiments represented in panels A, B and C, immunoprecipitations were done using an anti-GFP antibody and membranes were blotted with an anti-HCN1 antibody. For experiments represented in panel D, immunoprecipitation was performed with anti-tRFP (against mKate tag) and blots were done with anti-Cav3.2 antibody. These experiments were repeated 3 times with identical results.

    Article Snippet: Samples were transferred to a 0.2 μm nitrocellulose membrane (Bio-Rad) and western blot analysis was performed using one of the following antibodies: anti-HCN1 (1:300, Neuromab, 75–110), anti-HCN2 (1:300, Neuromab, 71–37), anti-Cav3.1 (1:200, Alomone, ACC-021), anti-Cav3.2 (1:200, Santa Cruz, sc-25691), and anti-Cav3.3 (1:200, Alomone, ACC-0009).

    Techniques: Western Blot, Transfection, Expressing, Immunoprecipitation

    N terminus region of Cav-3 interacts with Ca v 3.2 channel protein and inhibits I Cav3.2 . A , diagram of the domain structure of Cav-3. GST was fused with full-length Cav-3 and the different Cav-3 domains and tested for an ability to interact with the Ca v 3.2 channel. B , representative Western blot analyses of the GST pulldown assay. Ca v 3.2 channels were expressed in HEK293 cells, and lysates were incubated with different GST-Cav-3 fusion proteins as described under “Experimental Procedures”: full-length ( GST-Cav-3FL ), Cav-3 N terminus ( GST-Cav-3NT ), Cav-3 scaffolding domain ( GST-Cav-3Scaf ), Cav-3 membrane domain ( GST-Cav-3Memb ), Cav-3 C terminus domain ( GST-Cav-3CT ), or GST alone. The pulldown samples were analyzed by probing with anti-GST and anti-Ca v 3.2 antibodies. C , HEK cells were transiently expressed with Ca v 3.2 alone or Ca v 3.2 with different Cav-3 domains ( Cav-3NT , Cav-3Scaf , Cav-3CT , and Cav-3Memb ). I Cav3.2 density was measured by the whole cell patch clamp technique and plotted against a change in test potentials. Data represent mean ± S.E. ( n = 7 each, *, p > 0.005 with respect to control) from three different transfections.

    Journal: The Journal of Biological Chemistry

    Article Title: Caveolin-3 Regulates Protein Kinase A Modulation of the CaV3.2 (?1H) T-type Ca2+ Channels *

    doi: 10.1074/jbc.M110.182550

    Figure Lengend Snippet: N terminus region of Cav-3 interacts with Ca v 3.2 channel protein and inhibits I Cav3.2 . A , diagram of the domain structure of Cav-3. GST was fused with full-length Cav-3 and the different Cav-3 domains and tested for an ability to interact with the Ca v 3.2 channel. B , representative Western blot analyses of the GST pulldown assay. Ca v 3.2 channels were expressed in HEK293 cells, and lysates were incubated with different GST-Cav-3 fusion proteins as described under “Experimental Procedures”: full-length ( GST-Cav-3FL ), Cav-3 N terminus ( GST-Cav-3NT ), Cav-3 scaffolding domain ( GST-Cav-3Scaf ), Cav-3 membrane domain ( GST-Cav-3Memb ), Cav-3 C terminus domain ( GST-Cav-3CT ), or GST alone. The pulldown samples were analyzed by probing with anti-GST and anti-Ca v 3.2 antibodies. C , HEK cells were transiently expressed with Ca v 3.2 alone or Ca v 3.2 with different Cav-3 domains ( Cav-3NT , Cav-3Scaf , Cav-3CT , and Cav-3Memb ). I Cav3.2 density was measured by the whole cell patch clamp technique and plotted against a change in test potentials. Data represent mean ± S.E. ( n = 7 each, *, p > 0.005 with respect to control) from three different transfections.

    Article Snippet: The eluted sample was then separated and analyzed by SDS-PAGE and Western blot, respectively, by probing with anti-Cav3.2 (1:100, rabbit polyclonal, Alomone Labs) and anti-GST antibody (1:500, mouse monoclonal; BD Biosciences).

    Techniques: Western Blot, GST Pulldown Assay, Incubation, Scaffolding, Patch Clamp, Transfection

    Cav-3 inhibits I Cav3.2 in mouse neonatal ventricular myocytes. A , average peak I Ca,T density in neonatal ventricular myocytes infected with AdGFP control or with either AdCa v 3.2 or AdCa v 3.2+Cav-3 as indicated in the bar plot . Cav-3 overexpression significantly inhibited the AdCa v 3.2-mediated increased I Cav3.2 . B , average densities of I Ca,L in neonatal ventricular myocytes infected with adenovirus as indicated. Adenovirus infection did not alter the average I Ca,L density in the cells. The data represent mean ± S.E. ( n = 3–5 cells, *, p > 0.005 with respect to AdCa v 3.2). Whole cell patch clamp analysis on the effect of siRNA-mediated knockdown of Cav-3 on I Ca,T in mouse neonatal ventricular myocytes. C , average peak I Ca,T density from nontransfected control ( NT ), GFP control, siRNA to Cav-3 or siRNA to GAPDH ( control )-transfected cells as indicated in the bar plot. Representative corresponding T-type current traces are shown above. D , average densities of I Ca,L in neonatal ventricular myocytes transfected as above and representative corresponding I Ca,L current traces are shown above the bar plot. The average peak I Ca,T or I Ca,L was not significantly different and the data represent mean ± S.E., number of cells used are indicated in parentheses.

    Journal: The Journal of Biological Chemistry

    Article Title: Caveolin-3 Regulates Protein Kinase A Modulation of the CaV3.2 (?1H) T-type Ca2+ Channels *

    doi: 10.1074/jbc.M110.182550

    Figure Lengend Snippet: Cav-3 inhibits I Cav3.2 in mouse neonatal ventricular myocytes. A , average peak I Ca,T density in neonatal ventricular myocytes infected with AdGFP control or with either AdCa v 3.2 or AdCa v 3.2+Cav-3 as indicated in the bar plot . Cav-3 overexpression significantly inhibited the AdCa v 3.2-mediated increased I Cav3.2 . B , average densities of I Ca,L in neonatal ventricular myocytes infected with adenovirus as indicated. Adenovirus infection did not alter the average I Ca,L density in the cells. The data represent mean ± S.E. ( n = 3–5 cells, *, p > 0.005 with respect to AdCa v 3.2). Whole cell patch clamp analysis on the effect of siRNA-mediated knockdown of Cav-3 on I Ca,T in mouse neonatal ventricular myocytes. C , average peak I Ca,T density from nontransfected control ( NT ), GFP control, siRNA to Cav-3 or siRNA to GAPDH ( control )-transfected cells as indicated in the bar plot. Representative corresponding T-type current traces are shown above. D , average densities of I Ca,L in neonatal ventricular myocytes transfected as above and representative corresponding I Ca,L current traces are shown above the bar plot. The average peak I Ca,T or I Ca,L was not significantly different and the data represent mean ± S.E., number of cells used are indicated in parentheses.

    Article Snippet: The eluted sample was then separated and analyzed by SDS-PAGE and Western blot, respectively, by probing with anti-Cav3.2 (1:100, rabbit polyclonal, Alomone Labs) and anti-GST antibody (1:500, mouse monoclonal; BD Biosciences).

    Techniques: Infection, Over Expression, Patch Clamp, Transfection

    Co-expression of Cav-3 inhibits I Cav3.2 but not I Cav3.1 in HEK293 cells. Whole cell voltage clamp recordings of T-type Ca 2+ were performed in HEK293 cells expressing either Ca v 3.1 or Ca v 3.2 alone or co-expressing with Cav-3, by using a holding potential of −90 mV and step pulsed to 60 at 10 mV as shown in the inset. A , representative whole cell Ca 2+ current traces at −90, −30, and +20 mV from transiently transfected HEK293 cells with Ca v 3.1 alone and Ca v 3.1 with Cav-3. B , average current density plotted against the change in test potential for Ca v 3.1 (■) alone and with Ca v 3.1 + Cav3 (●). C , representative whole cell Ca 2+ current traces at −90, −30, and +20 mV from transiently transfected HEK293 cells with Ca v 3.2 alone and Ca v 3.2 with Cav-3. D , average current density plotted against the change in test potential for Ca v 3.2 (■) alone and with Ca v 3.2 + Cav-3 (●). Data represents mean ± S.E. ( n = 11 from 4 different transfections). Co-expression of Cav-3 does not alter plasma membrane expression of Ca v 3.2 channel protein. HEK293 cells were transfected with cDNAs of Ca v 3.2 + GFP or Ca v 3.2 + Cav-3 or GFP alone. E , cell lysates were precipitated with neutravidin beads and the sample was analyzed by Western blots by probing with anti-Ca v 3.2 or anti-β-actin antibodies as indicated in the representative immunoblot. Similar signal intensity for the biotinylated Ca v 3.2 protein was detected with Ca v 3.2 + GFP or Ca v 3.2 + Cav-3. F , a portion of the total lysate (50 μl) sample as input from 3 groups was also analyzed by probing with anti-Ca v 3.2 and anti-β-actin for loading control. Similar signal intensity for the Ca v 3.2 channel protein was detected with either Ca v 3.2 alone or with co-expression of Cav-3 and similar β-actin signal was detected between the three groups of cells demonstrating identical sample loading. Data are representative of three different experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: Caveolin-3 Regulates Protein Kinase A Modulation of the CaV3.2 (?1H) T-type Ca2+ Channels *

    doi: 10.1074/jbc.M110.182550

    Figure Lengend Snippet: Co-expression of Cav-3 inhibits I Cav3.2 but not I Cav3.1 in HEK293 cells. Whole cell voltage clamp recordings of T-type Ca 2+ were performed in HEK293 cells expressing either Ca v 3.1 or Ca v 3.2 alone or co-expressing with Cav-3, by using a holding potential of −90 mV and step pulsed to 60 at 10 mV as shown in the inset. A , representative whole cell Ca 2+ current traces at −90, −30, and +20 mV from transiently transfected HEK293 cells with Ca v 3.1 alone and Ca v 3.1 with Cav-3. B , average current density plotted against the change in test potential for Ca v 3.1 (■) alone and with Ca v 3.1 + Cav3 (●). C , representative whole cell Ca 2+ current traces at −90, −30, and +20 mV from transiently transfected HEK293 cells with Ca v 3.2 alone and Ca v 3.2 with Cav-3. D , average current density plotted against the change in test potential for Ca v 3.2 (■) alone and with Ca v 3.2 + Cav-3 (●). Data represents mean ± S.E. ( n = 11 from 4 different transfections). Co-expression of Cav-3 does not alter plasma membrane expression of Ca v 3.2 channel protein. HEK293 cells were transfected with cDNAs of Ca v 3.2 + GFP or Ca v 3.2 + Cav-3 or GFP alone. E , cell lysates were precipitated with neutravidin beads and the sample was analyzed by Western blots by probing with anti-Ca v 3.2 or anti-β-actin antibodies as indicated in the representative immunoblot. Similar signal intensity for the biotinylated Ca v 3.2 protein was detected with Ca v 3.2 + GFP or Ca v 3.2 + Cav-3. F , a portion of the total lysate (50 μl) sample as input from 3 groups was also analyzed by probing with anti-Ca v 3.2 and anti-β-actin for loading control. Similar signal intensity for the Ca v 3.2 channel protein was detected with either Ca v 3.2 alone or with co-expression of Cav-3 and similar β-actin signal was detected between the three groups of cells demonstrating identical sample loading. Data are representative of three different experiments.

    Article Snippet: The eluted sample was then separated and analyzed by SDS-PAGE and Western blot, respectively, by probing with anti-Cav3.2 (1:100, rabbit polyclonal, Alomone Labs) and anti-GST antibody (1:500, mouse monoclonal; BD Biosciences).

    Techniques: Expressing, Transfection, Western Blot