Journal: The Journal of Neuroscience

Article Title: Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Ca v 2.1 (P/Q-Type) Calcium Channels

doi: 10.1523/JNEUROSCI.3070-16.2017

Figure Lengend Snippet: RNF138 is a novel binding partner of CaV2.1 subunit. A, Bait construct for yeast two-hybrid screening comprises the pGilda plasmid containing the DNA-binding protein LexA fused in-frame with the distal C-terminal region (CaV2.1-C-ter) of human CaV2.1 long-isoform. Expression of the LexA-CaV2.1-C-ter fusion protein was only detected in EGY48 yeasts transformed with the bait-containing pGilda plasmid (LexA-CaV2.1-C-ter), but not with the pGilda vector per se (Vector). Protein samples (10 μl) were examined by immunoblotting with the anti-LexA (α-LexA) or anti-human CaV2.1 C-terminal (α-CaV2.1-CT) antibodies. Molecular weight marker (kDa) is labeled to the left. B, GST pull-down assay. Left, Association of Myc-tagged rat RNF138 (Myc-RNF138) with the GST fusion protein comprising the CaV2.1-C-ter fragment (GST-CaV2.1-C-ter), but not with the GST protein per se. Right, Additional assays with GST-CaV2.1 fusion proteins comprising CaV2.1 N-terminal region (CaV2.1-N-ter), domains I and II linker (CaV2.1-I-II), domains II and III linker (CaV2.1-II-III), domains III and IV linker (CaV2.1-III-IV), or proximal C-terminal region (CaV2.1-pC-ter). Lysates from HEK293T cells overexpressing Myc-RNF138 were used for GST pull-down assay, followed by immunoblotting with the anti-Myc antibody (α-Myc). GST and GST-CaV2.1 fusion proteins were detected with the anti-GST antibody (α-GST). Input represents ∼1% of total cell lysate volume. Arrowheads denote the location of GST or GST-CaV2.1 fusion protein bands. C, Coimmunoprecipitation of Myc-RNF138 with the human CaV2.1 long-isoform (CaV2.1-WT). Coexpression with the Myc vector was used as the control. HEK293T cell lysates were immunoprecipitated (IP) with α-Myc, followed by immunoblotting with α-Myc or the anti-human CaV2.1 C-terminal antibody (α-CaV2.1-CT). The apparent molecular weight of human CaV2.1 long-isoform is ∼280–290 kDa. Corresponding expression levels of human CaV2.1 and Myc-RNF138 in the lysates are shown in the “input” lane. Hereafter, input represents ∼10% of the total protein used for immunoprecipitation.

Article Snippet: Samples were then separated by 7.5–10% SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and detected using mouse anti-β-actin (1:5000; Millipore catalog #MAB1501, RRID:AB_2223041), rabbit anti-human Ca V 2.1-CT [1:2500; the C-terminal region (aa 2369–2473) of human Ca V 2.1 long-isoform], rabbit anti-human Ca V 2.1-(II-III) [1:2500; the linker region (aa 888–904) between transmembrane domains II and III of human Ca V 2.1], rabbit anti-rat Ca V 2.1 (1:200; Alomone Laboratories catalog #ACC-001, RRID:AB_2039764), rabbit anti-Flag (1:5000; Sigma-Aldrich catalog #F7425, RRID:AB_439687), rabbit anti-GAPDH (1:8000; GeneTex catalog #GTX100118, RRID:AB_1080976), rabbit anti-GFP (1:2500, Abcam catalog #ab290, RRID:AB_303395), mouse anti-GST tag (1:5000; Eno-Gene catalog #E12-007), rat anti-HA (1:5000; Roche catalog #11867423001, RRID:AB_10094468), rabbit anti-LexA (1:5000; Millipore catalog #06-719, RRID:AB_310223), mouse anti-Myc (1:5000; clone 9E10), mouse anti-postsynaptic density-95 (anti-PSD-95, 1:5000; UC Davis/NIH NeuroMab Facility catalog #75-028, RRID:AB_2307331), rabbit anti-RNF138 (1:200; Abcam catalog #ab92730, RRID:AB_2238719), mouse anti-synaptophysin (1:5000; clone 6D5AC4), rabbit anti-α-tubulin (1:5000; GeneTex catalog #GTX112141, RRID:AB_10722892), or mouse anti-ubiquitin (1:1000; Enzo Life Sciences catalog #BML-PW8810, RRID:AB_10541840) antibodies.

Techniques: Binding Assay, Construct, Two Hybrid Screening, Plasmid Preparation, Expressing, Transformation Assay, Western Blot, Molecular Weight, Marker, Labeling, Pull Down Assay, Immunoprecipitation

Journal: The Journal of Neuroscience

Article Title: Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Ca v 2.1 (P/Q-Type) Calcium Channels

doi: 10.1523/JNEUROSCI.3070-16.2017

Figure Lengend Snippet: Colocalization of RNF138 and CaV2.1 in neurons. A, Interaction of rat CaV2.1 and RNF138 in the brain. Top, Verification of the specificity of the anti-rat CaV2.1 antibody (α-rCaV2.1). Rat CaV2.1 protein detection was prevented by preabsorbing the immunoblot with a control antigen peptide. Endogenous CaV2.1 in the rat brain comprises a major isoform with an apparent molecular weight of ∼190 kDa (labeled with black arrows), as well as a minor isoform of ∼220 kDa (Sakurai et al., 1995; Westenbroek et al., 1995). Bottom, Coimmunoprecipitation of endogenous RNF138 with CaV2.1. Rat forebrain lysates were immunoprecipitated with α-rCaV2.1 or the rabbit IgG. The protein band corresponding to rat RNF138 is highlighted with black arrowhead. B, Colocalization of rat CaV2.1 and RNF138 at presynaptic and postsynaptic compartments. Subcellular fractionation separated rat brains into homogenate (H), soluble (S1), crude membrane (P2), synaptosomal (SPM), and two postsynaptic density (PSD I, PSD II) fractions. Synaptophysin and PSD-95 serve as the presynaptic and the postsynaptic markers, respectively. The terms 40 μg and 20 μg refer to the amount of total protein loaded in each lane. C, Developmental protein expression patterns of rat CaV2.1 and RNF138 in cultured cortical neurons with different DIV. D, Suppression of endogenous CaV2.1 level in cultured cortical neurons infected with shRNA for CaV2.1 (shCaV2.1). shRNA for GFP (shGFP) was used as the infection control. For quantitative analyses of relative CaV2.1 protein level, CaV2.1 signals were standardized as the ratio to the β-actin loading control, which were then normalized to the shGFP infection control (n = 4). Asterisk denotes significant difference from the infection control (*p < 0.05). E, Representative confocal microscopic images of rat CaV2.1 (left) and RNF138 (right) immunofluorescence signals in DIV10 cortical neurons. First row, Distribution of CaV2.1 or RNF138 (green) along MAP2-positive (red) dendrites and somas, which is highlighted with white triangles and further demonstrated in the merge images. Second row, Distribution of CaV2.1 or RNF138 (green) along tau-positive (red) axons, which is highlighted with white arrows. Third and fourth rows, White arrowheads highlight the colocalization of CaV2.1 or RNF138 puncta (green) with synaptophysin or PSD95 puncta (red) along neurites, which is further indicated as yellow puncta in the merge images. Scale bar, 50 μm. F, Quantification of puncta density (puncta/100 μm) and puncta colocalization ratio (fraction of colocalization) in DIV10 cortical neurons. The analyses were based on 15–30 individual neurites associated with 3–6 different neurons. Fi, Estimated puncta densities per 100 μm neurite are ∼59.3 ± 1.8 (CaV2.1), 62.4 ± 1.6 (RNF138), 61.7 ± 2.0 (synaptophysin), and 49.2 ± 1.6 (PSD95). Fii, The estimated synaptophysin colocalization ratios are ∼65.4 ± 3.9% (CaV2.1) and 61.1 ± 2.4% (RNF138). Fiii, Estimated PSD95 colocalization ratios are ∼51.4 ± 2.1% (CaV2.1) and 52.5 ± 1.4% (RNF138). Fiv–Fv, Conversely, for synaptophysin, the estimated CaV2.1 and RNF138 colocalization ratios are ∼62.4 ± 2.0% and 62.2 ± 2.0%, respectively; for PSD95, the estimated CaV2.1 and RNF138 colocalization ratios are ∼67.0 ± 2.3% and 61.2 ± 1.8%, respectively.

Article Snippet: Samples were then separated by 7.5–10% SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and detected using mouse anti-β-actin (1:5000; Millipore catalog #MAB1501, RRID:AB_2223041), rabbit anti-human Ca V 2.1-CT [1:2500; the C-terminal region (aa 2369–2473) of human Ca V 2.1 long-isoform], rabbit anti-human Ca V 2.1-(II-III) [1:2500; the linker region (aa 888–904) between transmembrane domains II and III of human Ca V 2.1], rabbit anti-rat Ca V 2.1 (1:200; Alomone Laboratories catalog #ACC-001, RRID:AB_2039764), rabbit anti-Flag (1:5000; Sigma-Aldrich catalog #F7425, RRID:AB_439687), rabbit anti-GAPDH (1:8000; GeneTex catalog #GTX100118, RRID:AB_1080976), rabbit anti-GFP (1:2500, Abcam catalog #ab290, RRID:AB_303395), mouse anti-GST tag (1:5000; Eno-Gene catalog #E12-007), rat anti-HA (1:5000; Roche catalog #11867423001, RRID:AB_10094468), rabbit anti-LexA (1:5000; Millipore catalog #06-719, RRID:AB_310223), mouse anti-Myc (1:5000; clone 9E10), mouse anti-postsynaptic density-95 (anti-PSD-95, 1:5000; UC Davis/NIH NeuroMab Facility catalog #75-028, RRID:AB_2307331), rabbit anti-RNF138 (1:200; Abcam catalog #ab92730, RRID:AB_2238719), mouse anti-synaptophysin (1:5000; clone 6D5AC4), rabbit anti-α-tubulin (1:5000; GeneTex catalog #GTX112141, RRID:AB_10722892), or mouse anti-ubiquitin (1:1000; Enzo Life Sciences catalog #BML-PW8810, RRID:AB_10541840) antibodies.

Techniques: Western Blot, Molecular Weight, Labeling, Immunoprecipitation, Fractionation, Expressing, Cell Culture, Infection, shRNA, Immunofluorescence

Journal: The Journal of Neuroscience

Article Title: Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Ca v 2.1 (P/Q-Type) Calcium Channels

doi: 10.1523/JNEUROSCI.3070-16.2017

Figure Lengend Snippet: Regulation of CaV2.1 protein expression by RNF138. A, Human CaV2.1 long-isoform (CaV2.1-WT) was coexpressed with the Myc vector (−), Myc-tagged rat RNF138 (Myc-RNF138), or Myc-tagged human RNF128 (Myc-RNF128) in the molar ratio of 1:3 in HEK293T cells. Top, Representative CaV2.1 protein level in response to different coexpression conditions. Bottom, Quantification of relative CaV2.1 protein level. CaV2.1 signals were standardized as the ratio to the cognate loading control, followed by normalization to the corresponding vector control (n = 6–12). B, Coexpression of rat RNF138 (Flag-RNF138 or Myc-RNF138) with Myc-tagged human CaV2.1 short-isoform [CaV2.1(Short)-6myc; ∼260–270 kDa], HA-tagged rat CaV1.2 (CaV1.2-HA; ∼260 kDa), or Flag-tagged bovine CaV2.2 (CaV2.2-Flag; ∼255–280 kDa) in HEK293T cells. For quantification, data in each coexpression condition were normalized to the vector control (n = 3–4). C, Treatment with 10 μm MG132 for 24 h reverses RNF138-mediated suppression of CaV2.1 protein level in HEK293T cells. Data were normalized to the DMSO treatment (−) control (n = 6–9). D, Effect of Myc-tagged rat RNF138-H36E (Myc-RNF138-H36E) on CaV2.1 protein level in HEK293T cells (n = 12). E, Effect of Myc-tagged human RNF138 (Myc-hRNF138) wild-type (WT) and N-terminal (residues 1–64) deletion mutant (Myc-hRNF138-ΔN) on CaV2.1 protein level in HEK293T cells (n = 5). F, Suppression of endogenous RNF138 expression with shRNA for RNF138 (shRNF138-1, shRNF138-2) enhances CaV2.1 protein level in HEK293T (left) and human neuroblastoma NT2 (right) cells. Data were normalized to the shRNA for GFP (shGFP) infection control (n = 5–6). G, shRNA-mediated silencing of endogenous rat RNF138 expression results in the upregulation of endogenous rat CaV2.1 protein level in cultured cortical neurons (n = 5). The arrow refers to the major, 190 kDa rat CaV2.1 isoform. Tubulin or actin signals were used as loading control and for quantification standardization. Asterisks denote significant difference from the control (*p < 0.05).

Article Snippet: Samples were then separated by 7.5–10% SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and detected using mouse anti-β-actin (1:5000; Millipore catalog #MAB1501, RRID:AB_2223041), rabbit anti-human Ca V 2.1-CT [1:2500; the C-terminal region (aa 2369–2473) of human Ca V 2.1 long-isoform], rabbit anti-human Ca V 2.1-(II-III) [1:2500; the linker region (aa 888–904) between transmembrane domains II and III of human Ca V 2.1], rabbit anti-rat Ca V 2.1 (1:200; Alomone Laboratories catalog #ACC-001, RRID:AB_2039764), rabbit anti-Flag (1:5000; Sigma-Aldrich catalog #F7425, RRID:AB_439687), rabbit anti-GAPDH (1:8000; GeneTex catalog #GTX100118, RRID:AB_1080976), rabbit anti-GFP (1:2500, Abcam catalog #ab290, RRID:AB_303395), mouse anti-GST tag (1:5000; Eno-Gene catalog #E12-007), rat anti-HA (1:5000; Roche catalog #11867423001, RRID:AB_10094468), rabbit anti-LexA (1:5000; Millipore catalog #06-719, RRID:AB_310223), mouse anti-Myc (1:5000; clone 9E10), mouse anti-postsynaptic density-95 (anti-PSD-95, 1:5000; UC Davis/NIH NeuroMab Facility catalog #75-028, RRID:AB_2307331), rabbit anti-RNF138 (1:200; Abcam catalog #ab92730, RRID:AB_2238719), mouse anti-synaptophysin (1:5000; clone 6D5AC4), rabbit anti-α-tubulin (1:5000; GeneTex catalog #GTX112141, RRID:AB_10722892), or mouse anti-ubiquitin (1:1000; Enzo Life Sciences catalog #BML-PW8810, RRID:AB_10541840) antibodies.

Techniques: Expressing, Plasmid Preparation, Mutagenesis, shRNA, Infection, Cell Culture

Journal: The Journal of Neuroscience

Article Title: Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Ca v 2.1 (P/Q-Type) Calcium Channels

doi: 10.1523/JNEUROSCI.3070-16.2017

Figure Lengend Snippet: RNF138 reduces CaV2.1 protein stability. A, Lack of effect of RNF138/RNF138-H36E overexpression on human CaV2.1 mRNA level in HEK293T cells subject to the indicated transfection condition (p > 0.05; n = 3). To rule out the potential contamination arising from human CaV2.1 plasmid in RNA prepared from transfected cells, RT-PCR was performed in the absence (left) or presence (right) of DNase I treatment before reverse transcription reaction. Also shown is the blank control that involves identical PCR in the absence of cDNA template (vertical arrows). The signals of CaV2.1 were standardized as the ratio to those of cognate GAPDH, followed by normalization to the corresponding Myc vector control. B, RNF138 knock-down does not significantly change rat CaV2.1 mRNA level in neurons (p > 0.05; n = 3). RT-PCR analyses were based on RNA extracted from cultured cortical neurons subject to the indicated shRNA infection. Standardized CaV2.1 signals were normalized to the shGFP infection control. C, Representative immunoblots showing the effect of RNF128, RNF138, or RNF138-H36E coexpression on protein stability of human CaV2.1 subunit. CaV2.1 protein turnover kinetics in HEK293T cells was analyzed by applying cycloheximide (CHX) with the indicated treatment durations (h). Coexpression with the Myc vector was used as the control experiment. D, Quantification of CaV2.1 protein half-life in the presence of Myc vector (black), RNF128 (green), RNF138 (blue), or RNF138-H36E (red). Left, Normalized CaV2.1 protein densities with respect to cycloheximide treatment durations. Data points represent the average of 7–8 independent experiments. Center, Same data points were transformed into a semilogarithmic plot, which is subject to single linear-regression analyses (solid lines; top) or double linear-regression analyses (solid lines; bottom with RNF138 only). Right, Comparison of CaV2.1 protein half-life values derived from linear-regression analyses. The estimated CaV2.1 protein half-life values based on single linear-regression analyses (top right) are ∼8.1 ± 0.3 (with vector; n = 8), 8.7 ± 1.3 (with RNF128; n = 7), 3.1 ± 0.4 (with RNF138; n = 8), and 10.9 ± 0.7 (with RNF138-H36E; n = 8) h. Based on double linear-regression analyses (bottom right), the estimated CaV2.1 protein half-life values in the presence of RNF138 are ∼1.3 ± 0.3 h (fast component) and 5.5 ± 0.6 h (slow component). E, Representative immunoblots showing the effect of shRNA knock-down of endogenous RNF138 on CaV2.1 protein turnover kinetics in HEK293T cells. shGFP infection was used as the control experiment. F, Quantification and comparison of CaV2.1 protein half-life values derived from different shRNA infection conditions. The estimated CaV2.1 protein half-life values are ∼6.4 ± 1.0 h (with shGFP; n = 9; black) and 10.3 ± 1.4 h (with shRNF138–1; n = 9; red). The protein half-life value of CaV2.1 in the presence of shGFP is not statistically different (p > 0.05) from that of CaV2.1 with vector in D. Asterisks denote significant difference from the control (*p < 0.05).

Article Snippet: Samples were then separated by 7.5–10% SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and detected using mouse anti-β-actin (1:5000; Millipore catalog #MAB1501, RRID:AB_2223041), rabbit anti-human Ca V 2.1-CT [1:2500; the C-terminal region (aa 2369–2473) of human Ca V 2.1 long-isoform], rabbit anti-human Ca V 2.1-(II-III) [1:2500; the linker region (aa 888–904) between transmembrane domains II and III of human Ca V 2.1], rabbit anti-rat Ca V 2.1 (1:200; Alomone Laboratories catalog #ACC-001, RRID:AB_2039764), rabbit anti-Flag (1:5000; Sigma-Aldrich catalog #F7425, RRID:AB_439687), rabbit anti-GAPDH (1:8000; GeneTex catalog #GTX100118, RRID:AB_1080976), rabbit anti-GFP (1:2500, Abcam catalog #ab290, RRID:AB_303395), mouse anti-GST tag (1:5000; Eno-Gene catalog #E12-007), rat anti-HA (1:5000; Roche catalog #11867423001, RRID:AB_10094468), rabbit anti-LexA (1:5000; Millipore catalog #06-719, RRID:AB_310223), mouse anti-Myc (1:5000; clone 9E10), mouse anti-postsynaptic density-95 (anti-PSD-95, 1:5000; UC Davis/NIH NeuroMab Facility catalog #75-028, RRID:AB_2307331), rabbit anti-RNF138 (1:200; Abcam catalog #ab92730, RRID:AB_2238719), mouse anti-synaptophysin (1:5000; clone 6D5AC4), rabbit anti-α-tubulin (1:5000; GeneTex catalog #GTX112141, RRID:AB_10722892), or mouse anti-ubiquitin (1:1000; Enzo Life Sciences catalog #BML-PW8810, RRID:AB_10541840) antibodies.

Techniques: Over Expression, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Cell Culture, shRNA, Infection, Western Blot, Transformation Assay, Derivative Assay

Journal: The Journal of Neuroscience

Article Title: Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Ca v 2.1 (P/Q-Type) Calcium Channels

doi: 10.1523/JNEUROSCI.3070-16.2017

Figure Lengend Snippet: RNF138 promotes CaV2.1 polyubiquitination. Biochemical demonstration of CaV2.1 polyubiquitination in HEK293T cells. A, Top, Representative immunoblots showing the effect of HA-tagged human lysine-less ubiquitin mutant (HA-Ub-K0) or ubiquitin wild-type (HA-Ub-WT) coexpression on CaV2.1 protein level. Coexpression with the HA vector was used as the control experiment. The relative molar ratio to CaV2.1 was 1:1. Bottom, Quantification of relative CaV2.1 protein level. Standardized CaV2.1 signals were normalized to the HA vector control (n = 6–9). B, Effect of coexpressing Flag-RNF138 or Flag-RNF138-H36E on Myc-tagged CaV2.1 WT (CaV2.1-WT-6myc) polyubiquitination [CaV2.1-(Ub)n] by HA-Ub-WT. Left, Cell lysates were immunoprecipitated with α-Myc, and protein ubiquitination was recognized by immunoblotting with the anti-HA antibody (α-HA). The no-CaV2.1 lane exemplifies the background HA-Ub-WT signal in the absence of CaV2.1. Corresponding expression levels of CaV2.1 and RNF138 in the lysates are shown in the “input” lane. Ubiquitinated CaV2.1 is visualized as high-molecular-weight protein smears. Open triangles refer to the expected location of the protein band corresponding to nonubiquitinated CaV2.1. The numbers shown on the immunoblots denote densitometric quantification of relative CaV2.1 ubiquitination with respect to the HA-Ub-WT control. Right, Normalized CaV2.1 ubiquitination signals from multiple experiments were compiled for statistical analyses (n = 4–6), with the mean relative values 1.4 ± 0.2 (with RNF138) and 0.7 ± 0.1 (with RNF138-H36E). C, Effect of Flag-RNF138 and Flag-RNF1388-H36E on CaV2.1 polyubiquitination by endogenous ubiquitin in HEK293T cells. Protein ubiquitination was identified by immunoblotting with the anti-ubiquitin antibody (α-Ub). CaV2.1 ubiquitination signals were normalized to the Flag vector control (n = 7–8), with the mean relative values 1.3 ± 0.1 (with RNF138) and 0.6 ± 0.1 (with RNF138-H36E). Asterisks denote significant difference from the control (*p < 0.05).

Article Snippet: Samples were then separated by 7.5–10% SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and detected using mouse anti-β-actin (1:5000; Millipore catalog #MAB1501, RRID:AB_2223041), rabbit anti-human Ca V 2.1-CT [1:2500; the C-terminal region (aa 2369–2473) of human Ca V 2.1 long-isoform], rabbit anti-human Ca V 2.1-(II-III) [1:2500; the linker region (aa 888–904) between transmembrane domains II and III of human Ca V 2.1], rabbit anti-rat Ca V 2.1 (1:200; Alomone Laboratories catalog #ACC-001, RRID:AB_2039764), rabbit anti-Flag (1:5000; Sigma-Aldrich catalog #F7425, RRID:AB_439687), rabbit anti-GAPDH (1:8000; GeneTex catalog #GTX100118, RRID:AB_1080976), rabbit anti-GFP (1:2500, Abcam catalog #ab290, RRID:AB_303395), mouse anti-GST tag (1:5000; Eno-Gene catalog #E12-007), rat anti-HA (1:5000; Roche catalog #11867423001, RRID:AB_10094468), rabbit anti-LexA (1:5000; Millipore catalog #06-719, RRID:AB_310223), mouse anti-Myc (1:5000; clone 9E10), mouse anti-postsynaptic density-95 (anti-PSD-95, 1:5000; UC Davis/NIH NeuroMab Facility catalog #75-028, RRID:AB_2307331), rabbit anti-RNF138 (1:200; Abcam catalog #ab92730, RRID:AB_2238719), mouse anti-synaptophysin (1:5000; clone 6D5AC4), rabbit anti-α-tubulin (1:5000; GeneTex catalog #GTX112141, RRID:AB_10722892), or mouse anti-ubiquitin (1:1000; Enzo Life Sciences catalog #BML-PW8810, RRID:AB_10541840) antibodies.

Techniques: Western Blot, Mutagenesis, Plasmid Preparation, Immunoprecipitation, Expressing, Molecular Weight

Journal: The Journal of Neuroscience

Article Title: Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Ca v 2.1 (P/Q-Type) Calcium Channels

doi: 10.1523/JNEUROSCI.3070-16.2017

Figure Lengend Snippet: Auxiliary subunits protect CaV2.1 from RNF138-mediated protein degradation. A, Effect of coexpression with Flag-tagged human β4a, bovine β1b, or bovine β2a subunits on Myc-RNF138 regulation of CaV2.1 in HEK293T cells. Top, Representative immunoblots. Coexpression with the Flag vector was used as the control experiment. Bottom, Quantification of relative CaV2.1 protein level. Data were normalized to the Flag vector control (n = 6–9). B, Effect of coexpression with human α2δ or α2δ-β4a subunits on Myc-RNF138 regulation of CaV2.1 protein level (n = 7–9). C, Coexpression with auxiliary α2δ and β4a subunits diminishes the effect of Myc-RNF138 on CaV2.1 turnover kinetics. The estimated CaV2.1 protein half-life value in the presence of Myc-RNF138 and auxiliary α2δ and β4a subunits is ∼6.2 ± 1.0 (n = 8; red) h. For comparison, also shown is the effect of Myc-RNF138 on CaV2.1 protein half-life in the absence of the auxiliary subunits (from Fig. 4D; black), which is subject to either single or double linear-regression analyses. D, Coexpression with auxiliary α2δ and β4a subunits attenuates the effect of Flag-RNF138 on CaV2.1 polyubiquitination (n = 5). In the presence of the auxiliary subunits, the mean relative value is 0.5 ± 0.2. E–G, Effect of α2δ and β4a subunits on CaV2.1 in HEK293T cells in the absence of Myc/Flag-RNF138 overexpression. E, α2δ-β4a overexpression enhances CaV2.1 protein level (n = 6). F, α2δ-β4a overexpression decelerates CaV2.1 turnover kinetics. The estimated CaV2.1 protein half-life values are ∼8.1 ± 0.3 (with vector; n = 7; black) and 12.0 ± 1.6 (with α2δ and β4a; n = 5; red) h. G, α2δ-β4a overexpression reduces CaV2.1 polyubiquitination (n = 5). In the presence of the auxiliary subunits, the mean relative value is 0.7 ± 0.2. Open triangle refers to the expected location of the protein band corresponding to nonubiquitinated CaV2.1. CaV2.1, α2δ, and β subunits were coexpressed in the molar ratio 1:2:1. Asterisks denote significant difference from the control (*p < 0.05).

Article Snippet: Samples were then separated by 7.5–10% SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and detected using mouse anti-β-actin (1:5000; Millipore catalog #MAB1501, RRID:AB_2223041), rabbit anti-human Ca V 2.1-CT [1:2500; the C-terminal region (aa 2369–2473) of human Ca V 2.1 long-isoform], rabbit anti-human Ca V 2.1-(II-III) [1:2500; the linker region (aa 888–904) between transmembrane domains II and III of human Ca V 2.1], rabbit anti-rat Ca V 2.1 (1:200; Alomone Laboratories catalog #ACC-001, RRID:AB_2039764), rabbit anti-Flag (1:5000; Sigma-Aldrich catalog #F7425, RRID:AB_439687), rabbit anti-GAPDH (1:8000; GeneTex catalog #GTX100118, RRID:AB_1080976), rabbit anti-GFP (1:2500, Abcam catalog #ab290, RRID:AB_303395), mouse anti-GST tag (1:5000; Eno-Gene catalog #E12-007), rat anti-HA (1:5000; Roche catalog #11867423001, RRID:AB_10094468), rabbit anti-LexA (1:5000; Millipore catalog #06-719, RRID:AB_310223), mouse anti-Myc (1:5000; clone 9E10), mouse anti-postsynaptic density-95 (anti-PSD-95, 1:5000; UC Davis/NIH NeuroMab Facility catalog #75-028, RRID:AB_2307331), rabbit anti-RNF138 (1:200; Abcam catalog #ab92730, RRID:AB_2238719), mouse anti-synaptophysin (1:5000; clone 6D5AC4), rabbit anti-α-tubulin (1:5000; GeneTex catalog #GTX112141, RRID:AB_10722892), or mouse anti-ubiquitin (1:1000; Enzo Life Sciences catalog #BML-PW8810, RRID:AB_10541840) antibodies.

Techniques: Western Blot, Plasmid Preparation, Over Expression

Journal: The Journal of Neuroscience

Article Title: Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Ca v 2.1 (P/Q-Type) Calcium Channels

doi: 10.1523/JNEUROSCI.3070-16.2017

Figure Lengend Snippet: RNF138 regulates protein level of EA2-causing CaV2.1 mutants. A, Left, Comparison of CaV2.1 WT and EA2 mutants (R1281X, F1406C) expression in HEK293T cells in the absence (top left) or presence (bottom left) of auxiliary α2δ and β4a subunits. Immunoblotting of CaV2.1 proteins was performed with an antibody that recognizes the transmembrane domains II-III linker region of human CaV2.1 subunit [α-CaV2.1-(II–III)]. Relative CaV2.1 protein level was normalized to the corresponding WT control (n = 7). Right, Treatment with 10 μm MG132 for 24 h enhances protein level of EA2 mutants. CaV2.1 subunits were expressed in the absence of the auxiliary subunits. Data were normalized to the DMSO treatment (−) control (n = 9–15). B, RNF138 (left) and the H36E mutant (right) regulate protein expression of EA2 mutants. Coexpression with the Myc vector was used as the control experiment. Data in each coexpression condition were normalized to the vector control (n = 4–10). C, shRNA suppression of endogenous RNF138 expression in HEK293T cells increases protein level of EA2 mutants. Data were normalized to the shGFP infection control (n = 4–8). Asterisks denote significant difference from the control (*p < 0.05).

Article Snippet: Samples were then separated by 7.5–10% SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and detected using mouse anti-β-actin (1:5000; Millipore catalog #MAB1501, RRID:AB_2223041), rabbit anti-human Ca V 2.1-CT [1:2500; the C-terminal region (aa 2369–2473) of human Ca V 2.1 long-isoform], rabbit anti-human Ca V 2.1-(II-III) [1:2500; the linker region (aa 888–904) between transmembrane domains II and III of human Ca V 2.1], rabbit anti-rat Ca V 2.1 (1:200; Alomone Laboratories catalog #ACC-001, RRID:AB_2039764), rabbit anti-Flag (1:5000; Sigma-Aldrich catalog #F7425, RRID:AB_439687), rabbit anti-GAPDH (1:8000; GeneTex catalog #GTX100118, RRID:AB_1080976), rabbit anti-GFP (1:2500, Abcam catalog #ab290, RRID:AB_303395), mouse anti-GST tag (1:5000; Eno-Gene catalog #E12-007), rat anti-HA (1:5000; Roche catalog #11867423001, RRID:AB_10094468), rabbit anti-LexA (1:5000; Millipore catalog #06-719, RRID:AB_310223), mouse anti-Myc (1:5000; clone 9E10), mouse anti-postsynaptic density-95 (anti-PSD-95, 1:5000; UC Davis/NIH NeuroMab Facility catalog #75-028, RRID:AB_2307331), rabbit anti-RNF138 (1:200; Abcam catalog #ab92730, RRID:AB_2238719), mouse anti-synaptophysin (1:5000; clone 6D5AC4), rabbit anti-α-tubulin (1:5000; GeneTex catalog #GTX112141, RRID:AB_10722892), or mouse anti-ubiquitin (1:1000; Enzo Life Sciences catalog #BML-PW8810, RRID:AB_10541840) antibodies.

Techniques: Expressing, Western Blot, Mutagenesis, Plasmid Preparation, shRNA, Infection

Journal: The Journal of Neuroscience

Article Title: Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Ca v 2.1 (P/Q-Type) Calcium Channels

doi: 10.1523/JNEUROSCI.3070-16.2017

Figure Lengend Snippet: RNF138 mediates protein degradation of EA2 mutants. A, Comparable mRNA levels for CaV2.1 WT, R1281X, and F1406C in HEK293T cells subject to the indicated transfection condition (p > 0.05; n = 3). RT-PCR was performed in the absence (top) or presence (bottom) of DNase I treatment before reverse transcription reaction. Vertical arrows refer to the blank control involving identical PCR in the absence of cDNA template. Standardized CaV2.1 signals were normalized with respect to CaV2.1 WT. B, RNF138-H36E reverses the impaired protein stability of EA2 mutants. CaV2.1 subunits were expressed in the absence of auxiliary subunits. The estimated protein half-life values for R1281X are ∼1.6 ± 0.3 h (with vector; n = 7; red), 1.4 ± 0.3 h (with RNF138; n = 6; green), and 6.0 ± 0.7 h (with RNF138-H36E; n = 9; blue). The estimated protein half-life values for F1406C are ∼2.2 ± 0.2 h (with vector; n = 7; red), 1.8 ± 0.2 h (with RNF138; n = 7; green), and 8.3 ± 1.0 h (with RNF138-H36E; n = 10; blue). For comparison, also shown is the protein half-life value of CaV2.1 WT (with vector) from Figure 4D (black). C, Coexpression with HA-Ub-K0 upregulates the protein level of EA2 mutants. Coexpression with the HA vector was used as the control experiment. The relative molar ratio to CaV2.1 is 1:1. Data were normalized to the HA vector control (n = 5–8). D, EA2 mutants display enhanced polyubiquitination that is regulated by RNF138 (n = 4–10). Compared with CaV2.1 WT, the mean relative values are 1.7 ± 0.3 (R1281X) and 1.3 ± 0.1 (F1406C). For R1281X, the mean relative values are 1.5 ± 0.2 (with RNF138) and 0.8 ± 0.1 (with RNF138-H36E). For F1406C, the mean relative values are 1.3 ± 0.1 (with RNF138) and 0.7 ± 0.1 (with RNF138-H36E). Protein ubiquitination by endogenous ubiquitin in HEK293T cells was identified by immunoblotting with α-Ub. Open and filled triangles refer to the expected location of the protein band corresponding to nonubiquitinated CaV2.1 WT/F1406C and R1281X, respectively. Asterisks denote significant difference from the control (*p < 0.05).

Article Snippet: Samples were then separated by 7.5–10% SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and detected using mouse anti-β-actin (1:5000; Millipore catalog #MAB1501, RRID:AB_2223041), rabbit anti-human Ca V 2.1-CT [1:2500; the C-terminal region (aa 2369–2473) of human Ca V 2.1 long-isoform], rabbit anti-human Ca V 2.1-(II-III) [1:2500; the linker region (aa 888–904) between transmembrane domains II and III of human Ca V 2.1], rabbit anti-rat Ca V 2.1 (1:200; Alomone Laboratories catalog #ACC-001, RRID:AB_2039764), rabbit anti-Flag (1:5000; Sigma-Aldrich catalog #F7425, RRID:AB_439687), rabbit anti-GAPDH (1:8000; GeneTex catalog #GTX100118, RRID:AB_1080976), rabbit anti-GFP (1:2500, Abcam catalog #ab290, RRID:AB_303395), mouse anti-GST tag (1:5000; Eno-Gene catalog #E12-007), rat anti-HA (1:5000; Roche catalog #11867423001, RRID:AB_10094468), rabbit anti-LexA (1:5000; Millipore catalog #06-719, RRID:AB_310223), mouse anti-Myc (1:5000; clone 9E10), mouse anti-postsynaptic density-95 (anti-PSD-95, 1:5000; UC Davis/NIH NeuroMab Facility catalog #75-028, RRID:AB_2307331), rabbit anti-RNF138 (1:200; Abcam catalog #ab92730, RRID:AB_2238719), mouse anti-synaptophysin (1:5000; clone 6D5AC4), rabbit anti-α-tubulin (1:5000; GeneTex catalog #GTX112141, RRID:AB_10722892), or mouse anti-ubiquitin (1:1000; Enzo Life Sciences catalog #BML-PW8810, RRID:AB_10541840) antibodies.

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Western Blot

Journal: The Journal of Neuroscience

Article Title: Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Ca v 2.1 (P/Q-Type) Calcium Channels

doi: 10.1523/JNEUROSCI.3070-16.2017

Figure Lengend Snippet: RNF138 attenuates surface expression of CaV2.1. A, Surface biotinylation experiments on HEK293T cells expressing Myc-tagged CaV2.1 WT, R1281X, or F1406C (CaV2.1–6myc). Left, Representative immunoblots. Cell lysates from biotinylated intact cells were either used directly for immunoblotting analyses (Total) or subject to streptavidin pull-down before immunoblotting analyses (Surface). CaV2.1–6myc, α2δ, and β4a subunits were coexpressed in the molar ratio 1:2:1. GAPDH expression is displayed as the loading control. Right, Quantification of surface protein level (surface signal) and membrane trafficking efficiency (surface/total ratio; n = 4). The surface protein density was standardized as the ratio of surface signal to cognate total GAPDH signal, followed by normalization to that of CaV2.1-WT. The total protein density was standardized as the ratio of input signal to GAPDH signal. The membrane-trafficking efficiency was quantified as surface protein density divided by the corresponding standardized total protein density, followed by normalization to that of CaV2.1-WT. B, Surface biotinylation experiments on HEK293T cells coexpressing CaV2.1–6myc channel with HA-Ub-K0 (n = 3–8). Coexpression with the HA vector (−) was used as the control experiment. CaV2.1–6myc and HA-Ub-K0 were coexpressed in the molar ratio 1:1. C, D, Surface biotinylation experiments on HEK293T cells coexpressing CaV2.1–6myc channel with Flag-RNF138 (n = 4) or Flag-RNF138-H36E (n = 3–6). Coexpression with the Flag vector (−) was used as the control experiment. CaV2.1–6myc and Flag-RNF138 were coexpressed in the molar ratio 1:3. Asterisks denote significant difference from the control (*p < 0.05).

Article Snippet: Samples were then separated by 7.5–10% SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and detected using mouse anti-β-actin (1:5000; Millipore catalog #MAB1501, RRID:AB_2223041), rabbit anti-human Ca V 2.1-CT [1:2500; the C-terminal region (aa 2369–2473) of human Ca V 2.1 long-isoform], rabbit anti-human Ca V 2.1-(II-III) [1:2500; the linker region (aa 888–904) between transmembrane domains II and III of human Ca V 2.1], rabbit anti-rat Ca V 2.1 (1:200; Alomone Laboratories catalog #ACC-001, RRID:AB_2039764), rabbit anti-Flag (1:5000; Sigma-Aldrich catalog #F7425, RRID:AB_439687), rabbit anti-GAPDH (1:8000; GeneTex catalog #GTX100118, RRID:AB_1080976), rabbit anti-GFP (1:2500, Abcam catalog #ab290, RRID:AB_303395), mouse anti-GST tag (1:5000; Eno-Gene catalog #E12-007), rat anti-HA (1:5000; Roche catalog #11867423001, RRID:AB_10094468), rabbit anti-LexA (1:5000; Millipore catalog #06-719, RRID:AB_310223), mouse anti-Myc (1:5000; clone 9E10), mouse anti-postsynaptic density-95 (anti-PSD-95, 1:5000; UC Davis/NIH NeuroMab Facility catalog #75-028, RRID:AB_2307331), rabbit anti-RNF138 (1:200; Abcam catalog #ab92730, RRID:AB_2238719), mouse anti-synaptophysin (1:5000; clone 6D5AC4), rabbit anti-α-tubulin (1:5000; GeneTex catalog #GTX112141, RRID:AB_10722892), or mouse anti-ubiquitin (1:1000; Enzo Life Sciences catalog #BML-PW8810, RRID:AB_10541840) antibodies.

Techniques: Expressing, Western Blot, Plasmid Preparation

Journal: The Journal of Neuroscience

Article Title: Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Ca v 2.1 (P/Q-Type) Calcium Channels

doi: 10.1523/JNEUROSCI.3070-16.2017

Figure Lengend Snippet: RNF138 suppresses functional expression of CaV2.1. Representative Ba2+ current traces through CaV2.1 WT (A, B) and F1406C (C, D) channels under different coexpression conditions in Xenopus oocytes are shown. The holding potential was −90 mV and 70 ms test pulses ranged from −80 mV to +60 mV in +10 mV steps. The peak Ba2+ current values at +20 mV were measured for comparing relative current amplitudes. Data were normalized to the corresponding water coinjection control. E, F, Representative Ba2+ current traces through CaV2.1 WT channels under different coexpression conditions in HEK293T cells. The peak Ba2+ current amplitudes at +10 mV were measured for comparing relative current densities. Data were normalized to the corresponding vector control. CaV2.1, α2δ, and β4a subunits were coexpressed in the molar ratio 1:2:2. The number of observation is shown in parentheses. Asterisks denote a significant difference from the control condition (*p < 0.05).

Article Snippet: Samples were then separated by 7.5–10% SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and detected using mouse anti-β-actin (1:5000; Millipore catalog #MAB1501, RRID:AB_2223041), rabbit anti-human Ca V 2.1-CT [1:2500; the C-terminal region (aa 2369–2473) of human Ca V 2.1 long-isoform], rabbit anti-human Ca V 2.1-(II-III) [1:2500; the linker region (aa 888–904) between transmembrane domains II and III of human Ca V 2.1], rabbit anti-rat Ca V 2.1 (1:200; Alomone Laboratories catalog #ACC-001, RRID:AB_2039764), rabbit anti-Flag (1:5000; Sigma-Aldrich catalog #F7425, RRID:AB_439687), rabbit anti-GAPDH (1:8000; GeneTex catalog #GTX100118, RRID:AB_1080976), rabbit anti-GFP (1:2500, Abcam catalog #ab290, RRID:AB_303395), mouse anti-GST tag (1:5000; Eno-Gene catalog #E12-007), rat anti-HA (1:5000; Roche catalog #11867423001, RRID:AB_10094468), rabbit anti-LexA (1:5000; Millipore catalog #06-719, RRID:AB_310223), mouse anti-Myc (1:5000; clone 9E10), mouse anti-postsynaptic density-95 (anti-PSD-95, 1:5000; UC Davis/NIH NeuroMab Facility catalog #75-028, RRID:AB_2307331), rabbit anti-RNF138 (1:200; Abcam catalog #ab92730, RRID:AB_2238719), mouse anti-synaptophysin (1:5000; clone 6D5AC4), rabbit anti-α-tubulin (1:5000; GeneTex catalog #GTX112141, RRID:AB_10722892), or mouse anti-ubiquitin (1:1000; Enzo Life Sciences catalog #BML-PW8810, RRID:AB_10541840) antibodies.

Techniques: Functional Assay, Expressing, Plasmid Preparation

Journal: The Journal of Neuroscience

Article Title: Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Ca v 2.1 (P/Q-Type) Calcium Channels

doi: 10.1523/JNEUROSCI.3070-16.2017

Figure Lengend Snippet: RNF138 promotes EA2 mutant-induced degradation of CaV2.1 WT subunits. A, Biochemical demonstration of the dominant-negative effect of untagged R1281X and GFP-tagged F1406C on CaV2.1-WT-6myc protein expression (n = 4–8). EA2 mutant was coexpressed with CaV2.1-WT in HEK293T cells in the molar ratio 1:1. Auxiliary α2δ and β4a subunits were also coexpressed together. B, Treatment with 10 μm MG132 for 24 h fully reverses the dominant-negative effect of EA2 mutants on CaV2.1-WT-6myc protein expression (n = 5–7). C, Coexpression with RNF138-H36E fully reverses the dominant-negative effect of EA2 mutants on CaV2.1-WT-6myc protein expression (n = 4–5). Quantitative data collected in the presence of RNF138-H36E are illustrated in red. D, Coexpression with RNF138-H36E reverses the dominant-negative effect of EA2 mutants on CaV2.1-WT-6myc turnover kinetics. The estimated protein half-life values for CaV2.1-WT-6myc in the absence of RNF138-H36E (black) are ∼8.1 ± 0.3 h (with vector; n = 9), 2.3 ± 0.4 h (with R1281X; n = 7), and 2.4 ± 0.4 h (with F1406C; n = 10). The estimated protein half-life values for CaV2.1-WT-6myc in the presence of RNF138-H36E (red) are ∼10.1 ± 0.7 h (with vector; n = 6), 9.2 ± 1.7 h (with R1281X; n = 5), and 9.3 ± 2.2 h (with F1406C; n = 5). E, Coexpression with RNF138-H36E partially reverses the dominant-negative effect of EA2 mutants on CaV2.1 WT current density. The number of observations is shown in parentheses. F, Biotinylation analyses of the dominant-negative effect of EA2 mutants on CaV2.1 WT membrane trafficking in the absence (left) or presence (right) of RNF138-H36E. Asterisks denote significant difference from the control (*p < 0.05).

Article Snippet: Samples were then separated by 7.5–10% SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and detected using mouse anti-β-actin (1:5000; Millipore catalog #MAB1501, RRID:AB_2223041), rabbit anti-human Ca V 2.1-CT [1:2500; the C-terminal region (aa 2369–2473) of human Ca V 2.1 long-isoform], rabbit anti-human Ca V 2.1-(II-III) [1:2500; the linker region (aa 888–904) between transmembrane domains II and III of human Ca V 2.1], rabbit anti-rat Ca V 2.1 (1:200; Alomone Laboratories catalog #ACC-001, RRID:AB_2039764), rabbit anti-Flag (1:5000; Sigma-Aldrich catalog #F7425, RRID:AB_439687), rabbit anti-GAPDH (1:8000; GeneTex catalog #GTX100118, RRID:AB_1080976), rabbit anti-GFP (1:2500, Abcam catalog #ab290, RRID:AB_303395), mouse anti-GST tag (1:5000; Eno-Gene catalog #E12-007), rat anti-HA (1:5000; Roche catalog #11867423001, RRID:AB_10094468), rabbit anti-LexA (1:5000; Millipore catalog #06-719, RRID:AB_310223), mouse anti-Myc (1:5000; clone 9E10), mouse anti-postsynaptic density-95 (anti-PSD-95, 1:5000; UC Davis/NIH NeuroMab Facility catalog #75-028, RRID:AB_2307331), rabbit anti-RNF138 (1:200; Abcam catalog #ab92730, RRID:AB_2238719), mouse anti-synaptophysin (1:5000; clone 6D5AC4), rabbit anti-α-tubulin (1:5000; GeneTex catalog #GTX112141, RRID:AB_10722892), or mouse anti-ubiquitin (1:1000; Enzo Life Sciences catalog #BML-PW8810, RRID:AB_10541840) antibodies.

Techniques: Mutagenesis, Dominant Negative Mutation, Expressing, Plasmid Preparation

Journal: eNeuro

Article Title: Synaptotagmin-7 Enhances Facilitation of Ca v 2.1 Calcium Channels

doi: 10.1523/ENEURO.0081-22.2022

Figure Lengend Snippet: Syt-7α accelerates the onset of facilitation of Ca v 2.1 channels. Inset top, Pulse protocol. Currents recorded with 10 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV preconditioning prepulses of the indicated durations. Inset, Example traces from control and Syt-7α transfected tsA cells following P1 and P2 pulses. A , Effect of Syt-7α on facilitation as a function of prepulse duration. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. B , in tsA-201 cells co-expressing Ca v 2.1 channel with Syt-7α, the slope is significantly increased compared with control cells. Data are represented as mean ± SEM.

Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Ca v 2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

Techniques: Transfection, Expressing

Journal: eNeuro

Article Title: Synaptotagmin-7 Enhances Facilitation of Ca v 2.1 Calcium Channels

doi: 10.1523/ENEURO.0081-22.2022

Figure Lengend Snippet: Effect of Syt-7α on prepulse facilitation of Ca v 2.1 at physiological Ca 2+ levels. Inset top, Pulse protocol. Currents recorded with 2 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV conditioning prepulses of the indicated durations. Inset, Example traces from control and Syt-7α transfected tsA cells following P1 and P2 pulses. Main panel, Graph shows the effect of Syt-7α on facilitation as a function of prepulse duration. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. Data are represented as mean ± SEM.

Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Ca v 2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

Techniques: Transfection

Journal: eNeuro

Article Title: Synaptotagmin-7 Enhances Facilitation of Ca v 2.1 Calcium Channels

doi: 10.1523/ENEURO.0081-22.2022

Figure Lengend Snippet: Effect of Syt-7α on the decay from facilitation of Ca v 2.1 channels. Inset top, Pulse protocol for measuring decay of facilitation. Ca 2+ currents are elicited by test pulses to +10 mV before (P1) and after (P2) a conditioning prepulse to +10 mV for 5 ms. Inset bottom left, Decay from facilitation measured by comparing τ between control and Syt-7α transfected cells. Inset bottom right, Comparison of P2/P1 facilitation ratio at Δ t = 0 between control and Syt-7α-expressing cells. Main panel. Effect of Syt-7α on the decay from facilitation. The facilitation ratio was obtained by normalizing the peak current from P2 to that from P1 and was plotted against the interval between the conditioning prepulse and P2. Shown are results obtained with 50-ms conditioning prepulse. Graph shows the effect of Syt-7α on decay of facilitation as a function of interpulse duration. Data are represented as mean ± SEM.

Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Ca v 2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

Techniques: Transfection, Expressing

Journal: eNeuro

Article Title: Synaptotagmin-7 Enhances Facilitation of Ca v 2.1 Calcium Channels

doi: 10.1523/ENEURO.0081-22.2022

Figure Lengend Snippet: Syt-7α potentiates Ca v 2.1 facilitation in a paired-pulse protocol following change in prepulse voltage. Inset top, Pulse protocol shown represents paired pulse protocol. Ca 2+ current was recorded using 10 m m Ca 2+ and 0.5 m m EGTA in the external and internal solutions, respectively. Pulse 1 (P1; depolarization from −80 to +10 mV) elicits the first Ca 2+ current. A second 5-ms pulse (P2) generating a second I Ca is applied 2 ms after a 50-ms conditioning prepulse with variable voltages (−40 to 60 mV). Inset bottom, Example traces from control and Syt-7α transfected tsA cells following P1 and P2 pulses. Main panel, Graph shows the effects of Syt-7α isoform on facilitation as a function of prepulse voltage. The facilitation ratio was obtained by normalizing the peak current from P2 to that from P1. Data are represented as mean ± SEM.

Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Ca v 2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

Techniques: Transfection

Journal: eNeuro

Article Title: Synaptotagmin-7 Enhances Facilitation of Ca v 2.1 Calcium Channels

doi: 10.1523/ENEURO.0081-22.2022

Figure Lengend Snippet: Effect of Syt-7α on prepulse facilitation of Ca v 2.1 channel. Facilitation of voltage-dependent activation of Ca 2+ currents. Inset, Pulse protocol to study the voltage dependence of activation before (open circle or squares; P1) and after (closed circle or squares; P2) a depolarizing prepulse from −80 to +10 mV. Tail currents were measured by holding potential at −40 mV for 5 ms after test pulses (P1, P2) to variable voltages (−40 to +80 mV). Peak tail currents were normalized to the largest tail current measured during the nonfacilitated prepulses (P1) and plotted against the test pulse voltage. A , In control tsA cells, the protocol shows an increase in facilitation P2 normalized to P1. B , Syt-7α potentiated facilitation amplitude of Ca v 2.1 and induced a right shift in prepulse facilitation curve. C , Overlaying the two graphs in A , B shows the increase in amplitude of facilitation and the right shift in voltage dependency of activation. D , Difference in voltage shift in P1 and P2 between cells co-expressing Ca v 2.1 and Syt-7α and control cells. Data are represented as mean ± SEM.

Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Ca v 2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

Techniques: Activation Assay, Expressing

Journal: eNeuro

Article Title: Synaptotagmin-7 Enhances Facilitation of Ca v 2.1 Calcium Channels

doi: 10.1523/ENEURO.0081-22.2022

Figure Lengend Snippet: Effect of Syt-7α prepulse facilitation of Ca v 2.1 channel at physiological levels. Inset, Voltage protocol. Currents recorded with 2 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV conditioning prepulses of the indicated durations. A - C , Effect of Syt-7α on facilitation as a function of prepulse voltage. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. Data are represented as mean ± SEM.

Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Ca v 2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

Techniques:

Journal: eNeuro

Article Title: Synaptotagmin-7 Enhances Facilitation of Ca v 2.1 Calcium Channels

doi: 10.1523/ENEURO.0081-22.2022

Figure Lengend Snippet: Syt-7β and Syt-7γ differentially modulate prepulse facilitation of Ca v 2.1 channels. Inset, Pulse protocol. Currents recorded with 10 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV conditioning prepulses of the indicated durations. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. A , Ca v 2.1 alone. B , Ca v 2.1 with Syt-7γ. C , Overlay of panels A , B . D , V 50 values for results in panel C . E , Ca v 2.1 alone. F , Ca v 2.1 with Syt-7γ. G , overlay of panels D , E . H , V 50 values from panel G . Data are represented as mean ± SEM additional experiments with different pulse protocols provide additional information on the effects of Syt-7β and Syt-7γ on the voltage dependence of facilitation of Ca v 2.1 channels (Extended Data ).

Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Ca v 2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

Techniques:

Journal: eNeuro

Article Title: Synaptotagmin-7 Enhances Facilitation of Ca v 2.1 Calcium Channels

doi: 10.1523/ENEURO.0081-22.2022

Figure Lengend Snippet: Syt-7 isoforms differentially modulate Ca 2+ -dependent inactivation of Ca v 2.1 channels. Ca v 2.1 currents were elicited by depolarizing from a holding potential of −80 mV to a test potential of +10 mV. A , Time courses (200 ms) of I Ba with 10 m m Ba 2+ as a permeant cation. B , Time courses (1000 ms) of I Ca with 10 m m Ca 2+ as permeant ion. Syt-7α and Syt-7β significantly slowed inactivation of the Ca v 2.1 channel in the presence of 10 m m Ca 2+ , whereas Syt-7γ had no effect. Data are represented as mean ± SEM.

Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Ca v 2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

Techniques:

Journal: eNeuro

Article Title: Synaptotagmin-7 Enhances Facilitation of Ca v 2.1 Calcium Channels

doi: 10.1523/ENEURO.0081-22.2022

Figure Lengend Snippet: Syt-7β and Syt-7γ differentially modulate facilitation of Ca v 2.1 channels. Inset, Pulse protocol. Currents recorded with 10 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV conditioning prepulses of the indicated durations. A , Left, Syt-7β increases the facilitation ratio with increasing prepulse duration. Right, Syt-7β accelerates the onset of facilitation as a function of prepulse duration. B , Left, Syt-7γ increases the facilitation ratio with increasing prepulse duration. Right, Syt-7γ does not accelerate the onset of facilitation as a function of prepulse duration. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. Data are represented as mean ± SEM additional experiments with different pulse protocols provide additional information on the effects of Syt-7β and Syt-7γ on facilitation of Ca v 2.1 channels (Extended Data ).

Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Ca v 2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

Techniques:

Journal: Cell Reports

Article Title: PRRT2 modulates presynaptic Ca 2+ influx by interacting with P/Q-type channels

doi: 10.1016/j.celrep.2021.109248

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Article Snippet: Anti - Ca v 2.1 alpha-1A Rabbit Polyclonal , Synaptic Systems , Cat. #152203; RRID: AB_2619841.

Techniques: Recombinant, Saline, Protease Inhibitor, BIA-KA, Bradford Assay, Magnetic Beads, Cell Culture, Software