Journal: International Journal of Molecular Sciences

Article Title: Mouse Ataxin-2 Expansion Downregulates CamKII and Other Calcium Signaling Factors, Impairing Granule—Purkinje Neuron Synaptic Strength

doi: 10.3390/ijms21186673

Figure Lengend Snippet: Expression analyses of Ca 2+ associated subcellular signaling pathways in Atxn2 -CAG100-KIN cerebellum throughout disease course. ( A ) Transcript levels of cytoplasmic IP 3 metabolism components ( Gria3, Grm1 , Grm4 , Plcb3 , Plcb4 , Plcg1 , Inpp5a, Itpka, Prkcd ), Ca 2+ -CaM signaling components ( Pcp4 , Camkk1 , Camkk2 , Camk2a , Camk2b , Camk2d , Camk2g , Camk4 ) and downstream CaMKIV targets ( Khdrbs1-3 ) were quantified by RT-qPCR in Atxn2 -KO (blue bars) and Atxn2 -CAG100-KIN mouse cerebellum at pre-onset (pink bars) and terminal (red bars) disease stages; ( B ) Protein levels of GluA3, CaMKIIα, CaMKIV and Sam68 in soluble (RIPA) and insoluble (SDS) fractions of Atxn2 -CAG100-KIN mouse cerebellum at pre-onset and final disease stages were determined by quantitative immunoblots. Student’s t -test with Welch’s correction; 0.05 < p < 0.1 T , p < 0.05 *, p < 0.01 **, p < 0.001 ***, p < 0.0001 ****. Further information regarding individual fold changes and p -values can be found in .

Article Snippet: The primary antibodies utilized in this study are: ACTB (Sigma Aldrich, St. Louis, MO, USA, A5441, 1:10,000), ATP2A2 (Cell Signaling, Danvers, MA, USA, #9580, 1:1000), CaMKIIα (Cell Signaling, Danvers, MA, USA, #3357, 1:1000), CaMKIV (Cell Signaling, Danvers, MA, USA, #4032, 1:500), GluA3 (Cell Signaling, Danvers, MA, USA, #4676, 1:1000), ITPR1 (Abcam, Cambridge, UK, ab5804, 1:500), and Sam68 (Abclonal, Woburn, MA, USA, A6101, 1:1000).

Techniques: Expressing, Protein-Protein interactions, Quantitative RT-PCR, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Spinal α 2 δ -1 induces GluA3 degradation to regulate assembly of calcium-permeable AMPA receptors and pain hypersensitivity

doi: 10.1172/JCI193349

Figure Lengend Snippet: ( A – C ) Representative current-voltage (I-V) plots of glutamate-elicited currents ( A and B ) and quantification ( C ) show the differential effect of coexpression of α2δ-1, α2δ-2, or α2δ-3 on GluA3 currents in HEK29 cells ( n = 11 cells for GluA3/empty vector [pcDNA], GluA3/α2δ-1, and GluA3/α2δ-2; n = 12 cells for GluA3/α2δ-3). ( D – F ) Representative I-V plots of glutamate-elicited currents ( D ) and mean current density ( E ) and rectification index ( F ) in HEK293 cells transfected with GluA2/A3 with either pcDNA or α2δ-1 ( n = 12 cells for GluA2/GluA3; n = 13 cells for GluA2/GluA3/α2δ-1). * P < 0.05, *** P < 0.001; 1-way ANOVA followed by Dunnett’s post hoc test in C ; 2-tailed Student’s t test in E and F . Data are expressed as means ± SEM. ( G and H ) Original confocal immunofluorescence images show the distribution of GluA3 (red) and GFP-tagged α2δ-1 (green) in HEK293 cells transfected with either GluA3/pcDNA or GluA3/α2δ-1-GFP. Areas in yellow boxes in G are magnified in H . Scale bars: 50 μm ( G ), 10 μm ( H ).

Article Snippet: The rabbit anti-GluA3 antibody (mAb5117, Cell Signaling Technology) or rabbit anti-GluA2 antibody (AB10529, EMD Millipore) was preincubated with protein G agarose beads at 25°C for 1 h, and the protein samples were then exposed to the antibody-conjugated beads at 4°C overnight.

Techniques: Plasmid Preparation, Transfection, Immunofluorescence

Journal: The Journal of Clinical Investigation

Article Title: Spinal α 2 δ -1 induces GluA3 degradation to regulate assembly of calcium-permeable AMPA receptors and pain hypersensitivity

doi: 10.1172/JCI193349

Figure Lengend Snippet: ( A and B ) Representative immunoblot images ( A ) and quantification ( B ) show the distinct effect of coexpression of HA-tagged α2δ-1, α2δ-2, or α2δ-3 on GluA3 protein levels in HEK293 cells ( n = 8 independent experiments per group). GAPDH was used as the internal control for normalizing the protein levels on the same gel. *** P < 0.001; 1-way ANOVA followed by Dunnett’s post hoc test.

Article Snippet: The rabbit anti-GluA3 antibody (mAb5117, Cell Signaling Technology) or rabbit anti-GluA2 antibody (AB10529, EMD Millipore) was preincubated with protein G agarose beads at 25°C for 1 h, and the protein samples were then exposed to the antibody-conjugated beads at 4°C overnight.

Techniques: Western Blot, Control

Journal: The Journal of Clinical Investigation

Article Title: Spinal α 2 δ -1 induces GluA3 degradation to regulate assembly of calcium-permeable AMPA receptors and pain hypersensitivity

doi: 10.1172/JCI193349

Figure Lengend Snippet: ( A ) Representative immunoblot images and quantification show the concentration-dependent reduction in GluA3 protein levels induced by α2δ-1 coexpression in HEK293 cells ( n = 7 independent experiments per group). ( B ) Representative immunoblot images and quantification show the effect of coexpression with GFP on GluA3 protein levels in HEK293 cells ( n = 7 independent experiments per group). ( C ) Representative immunoblot images and quantification show the protein levels of GluA2 and GluA3 in HEK293 cells expressing GluA2/GluA3 with either empty vectors or α2δ-1 ( n = 6 independent experiments per group). ( D ) Representative immunoblot images and quantification show the protein levels of GluA3 and α2δ-1 in HEK293 cells expressing GluA3 with either empty vectors or α2δ-1 ( n = 6 independent experiments per group). GAPDH was used as the internal control for normalizing the protein levels on the same gel. ** P < 0.01, *** P < 0.001; 1-way ANOVA followed by Dunnett’s post hoc test in A and B ; 2-tailed Student’s t test in C and D . Data are expressed as means ± SEM.

Article Snippet: The rabbit anti-GluA3 antibody (mAb5117, Cell Signaling Technology) or rabbit anti-GluA2 antibody (AB10529, EMD Millipore) was preincubated with protein G agarose beads at 25°C for 1 h, and the protein samples were then exposed to the antibody-conjugated beads at 4°C overnight.

Techniques: Western Blot, Concentration Assay, Expressing, Control

Journal: The Journal of Clinical Investigation

Article Title: Spinal α 2 δ -1 induces GluA3 degradation to regulate assembly of calcium-permeable AMPA receptors and pain hypersensitivity

doi: 10.1172/JCI193349

Figure Lengend Snippet: ( A ) Original confocal images show the distribution of GluA3 (green), IB4 (red), and NeuN (blue) in the spinal dorsal horn of sham control and SNL rats. Scale bars: 100 μm (upper panels), 50 μm (lower panels). ( B and C ) Representative immunoblot images ( B ) and quantification ( C ) show the protein levels of α2δ-1 and GluA3 in the dorsal spinal cord of sham control and SNL rats. β-Actin served as the internal control for normalizing the protein levels on the same gel ( n = 8 mice per group). ( D and E ) Representative immunoblot images ( D ) and quantification ( E ) show the protein levels of GluA3 and GluA2/GluA3 complexes in the dorsal spinal cord from sham and SNL rats treated intrathecally with vehicle or 10 μg pregabalin (PGB; n = 9 rats per group) 3 weeks after surgery. Protein extracts from rat spinal cord tissues were immunoprecipitated using a rabbit GluA2 antibody or IgG. Immunoblotting was then performed using mouse GluA2, mouse GluA3, and mouse β-actin antibodies. β-Actin served as the internal control for normalizing GluA3 protein levels in the input. The corresponding immunoprecipitated GluA2 protein bands were used for normalizing GluA2/GluA3 protein complex levels. * P < 0.05, ** P < 0.01, *** P < 0.001; 2-tailed Student’s t test in C ; 2-way ANOVA followed by Tukey’s post hoc test in E . Data are expressed as means ± SEM.

Article Snippet: The rabbit anti-GluA3 antibody (mAb5117, Cell Signaling Technology) or rabbit anti-GluA2 antibody (AB10529, EMD Millipore) was preincubated with protein G agarose beads at 25°C for 1 h, and the protein samples were then exposed to the antibody-conjugated beads at 4°C overnight.

Techniques: Control, Western Blot, Immunoprecipitation

Journal: The Journal of Clinical Investigation

Article Title: Spinal α 2 δ -1 induces GluA3 degradation to regulate assembly of calcium-permeable AMPA receptors and pain hypersensitivity

doi: 10.1172/JCI193349

Figure Lengend Snippet: Representative immunoblot images ( A and C ) and quantification show the total ( B ) and synaptosome ( D ) protein levels of GluA3 and α2δ-1 in the dorsal spinal cord of naive rats injected intrathecally with control lentiviruses or lentiviruses expressing Cacna2d1 ( n = 6 rats per group). β-Actin served as the internal control for normalizing the GluA3 and α2δ-1 protein levels on the same gel. PSD-95, a synaptic protein marker, served as the internal control for normalizing the GluA3 and α2δ-1 protein levels in synaptosome fractions. *** P < 0.001; 2-tailed Student’s t test. Data are expressed as means ± SEM.

Article Snippet: The rabbit anti-GluA3 antibody (mAb5117, Cell Signaling Technology) or rabbit anti-GluA2 antibody (AB10529, EMD Millipore) was preincubated with protein G agarose beads at 25°C for 1 h, and the protein samples were then exposed to the antibody-conjugated beads at 4°C overnight.

Techniques: Western Blot, Injection, Control, Expressing, Marker

Journal: The Journal of Clinical Investigation

Article Title: Spinal α 2 δ -1 induces GluA3 degradation to regulate assembly of calcium-permeable AMPA receptors and pain hypersensitivity

doi: 10.1172/JCI193349

Figure Lengend Snippet: ( A and B ) Representative immunoblot images ( A ) and quantification ( B ) show the basal protein levels of GluA3 in the dorsal spinal cord of WT and Cana2d1- KO mice ( n = 6 mice per group). ( C and D ) Representative immunoblot images ( C ) and quantification ( D ) show the protein levels of GluA3 and α2δ-1 in dorsal spinal cord tissues from WT and Cana2d1- KO mice subjected to sham or SNI surgery ( n = 11 mice per group). β-Actin served as the internal control for normalizing the GluA3 and α2δ-1 protein levels on the same gel. ** P < 0.01; 2-tailed Student’s t test. Data are expressed as means ± SEM.

Article Snippet: The rabbit anti-GluA3 antibody (mAb5117, Cell Signaling Technology) or rabbit anti-GluA2 antibody (AB10529, EMD Millipore) was preincubated with protein G agarose beads at 25°C for 1 h, and the protein samples were then exposed to the antibody-conjugated beads at 4°C overnight.

Techniques: Western Blot, Control

Journal: The Journal of Clinical Investigation

Article Title: Spinal α 2 δ -1 induces GluA3 degradation to regulate assembly of calcium-permeable AMPA receptors and pain hypersensitivity

doi: 10.1172/JCI193349

Figure Lengend Snippet: ( A and B ) Representative immunoblot images ( A ) and quantification ( B ) show GluA3 and α2δ-1 protein levels in HEK293 cells coexpressing GluA3 with YFP-tagged WT α2δ-1 or chimeric constructs [α2δ-1CT (α2δ-2) and α2δ-1CT (α2δ-3) ] ( n = 8 independent experiments per group). ( C and D ) Representative immunoblot images ( C ) and quantification ( D ) show the effects of treatment with control peptide (1 μM), α2δ-1CT peptide (1 μM), pregabalin (PGB; 20 μM), and MG132 (10 μM) on the GluA3 protein levels in HEK293 cells coexpressing α2δ-1 and GluA3 ( n = 9 independent experiments per group). PT, peptide. GAPDH was used as an internal control for normalizing the GluA3 protein levels on the same gel. *** P < 0.001; 1-way ANOVA followed by Dunnett’s post hoc test. Data are expressed as means ± SEM.

Article Snippet: The rabbit anti-GluA3 antibody (mAb5117, Cell Signaling Technology) or rabbit anti-GluA2 antibody (AB10529, EMD Millipore) was preincubated with protein G agarose beads at 25°C for 1 h, and the protein samples were then exposed to the antibody-conjugated beads at 4°C overnight.

Techniques: Western Blot, Construct, Control

Journal: The Journal of Clinical Investigation

Article Title: Spinal α 2 δ -1 induces GluA3 degradation to regulate assembly of calcium-permeable AMPA receptors and pain hypersensitivity

doi: 10.1172/JCI193349

Figure Lengend Snippet: ( A ) Time-course effects of intrathecal injection of 20 μg MG132 or vehicle (Veh) on hindpaw nociceptive thresholds in sham and SNL rats 3 weeks after surgery ( n = 9 rats per group). * P < 0.05, ** P < 0.01, *** P < 0.001 versus baseline (time 0); # P < 0.05, ### P < 0.001 versus Veh-SNL group at the same time point; 2-way ANOVA followed by Tukey’s post hoc test. ( B ) Representative immunoblot images and quantification show the effect of MG132 treatment on GluA3 protein levels in dorsal spinal cord tissues from SNL and sham rats ( n = 9 rats per group). * P < 0.05, *** P < 0.001; 1-way ANOVA followed by Tukey’s post hoc test. Data are expressed as means ± SEM.

Article Snippet: The rabbit anti-GluA3 antibody (mAb5117, Cell Signaling Technology) or rabbit anti-GluA2 antibody (AB10529, EMD Millipore) was preincubated with protein G agarose beads at 25°C for 1 h, and the protein samples were then exposed to the antibody-conjugated beads at 4°C overnight.

Techniques: Injection, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Spinal α 2 δ -1 induces GluA3 degradation to regulate assembly of calcium-permeable AMPA receptors and pain hypersensitivity

doi: 10.1172/JCI193349

Figure Lengend Snippet: ( A ) Representative immunoblot images show the ubiquitin protein levels in GluA3 precipitates from HEK293 cells expressing GluA3 with either pcDNA or α2δ-1 (similar data were obtained from 4 independent experiments). ( B ) Representative immunoblot images and quantification show the ubiquitin protein levels in GluA3 precipitates from the dorsal spinal cord of sham control and SNL rats ( n = 9 rats per group). Protein extracts from HEK293 cells or spinal cord tissues were immunoprecipitated using a rabbit GluA3 antibody or IgG. Immunoblotting was then conducted using mouse ubiquitin or mouse GluA3 antibodies. The corresponding GluA3 protein bands were used as the internal control on the same gel. ( C and D ) Representative immunoblot images ( C ) and quantification ( D ) show GluA3 protein levels in HEK293 cells expressing WT GluA3 or GluA3 mutants (K710R, K861R, and K887R) with and without α2δ-1 ( n = 12 independent experiments per group). GAPDH was used as the internal control for normalizing GluA3 and α2δ-1 protein levels on the same gel. ** P < 0.01, *** P < 0.001; 2-tailed Student’s t test in B ; 1-way ANOVA followed by Tukey’s post hoc test in D . Data are expressed as means ± SEM.

Article Snippet: The rabbit anti-GluA3 antibody (mAb5117, Cell Signaling Technology) or rabbit anti-GluA2 antibody (AB10529, EMD Millipore) was preincubated with protein G agarose beads at 25°C for 1 h, and the protein samples were then exposed to the antibody-conjugated beads at 4°C overnight.

Techniques: Western Blot, Ubiquitin Proteomics, Expressing, Control, Immunoprecipitation

Journal: The Journal of Clinical Investigation

Article Title: Spinal α 2 δ -1 induces GluA3 degradation to regulate assembly of calcium-permeable AMPA receptors and pain hypersensitivity

doi: 10.1172/JCI193349

Figure Lengend Snippet: ( A ) Changes in the hindpaw withdrawal thresholds of sham control and SNL rats 2 and 3 weeks after intrathecal injection of control (Cont) lentiviral vectors or lentiviral vectors expressing Gria3 ( n = 13 rats per group). ** P < 0.01, *** P < 0.001; 2-way ANOVA followed by Tukey’s post hoc test. ( B and C ) Representative immunoblot images ( B ) and quantification ( C ) show the protein levels of GluA3 and GluA2/GluA3 complexes in the dorsal spinal cords of sham control and SNL rats treated with intrathecal control lentiviruses or Gria3 -expressing lentiviruses ( n = 8 rats per group). Protein extracts from spinal cord tissues were immunoprecipitated (IP) using a GluA2 antibody or IgG. Immunoblotting was then conducted using GluA2, GluA3, and β-actin antibodies. β-Actin protein bands were used as the internal control on the same gel. ** P < 0.01, *** P < 0.001; 2-way ANOVA followed by Tukey’s post hoc test. ( D and E ) Representative recording traces ( D ) and quantification ( E ) show the differential effect of bath application of IEM-1460 (50 μM) on the amplitude of monosynaptic AMPAR-EPSCs in spinal lamina II neurons from SNL rats treated with intrathecal injection of control lentiviruses or Gria3 -expressing lentiviruses ( n = 18 neurons from 4 rats per group). Data were normalized to the baseline value (100%) before IEM-1460 application. * P < 0.05, ** P < 0.01 versus control vector group at the same time point; 2-way ANOVA followed by Tukey’s post hoc test. Data are presented as mean ± SEM.

Article Snippet: The rabbit anti-GluA3 antibody (mAb5117, Cell Signaling Technology) or rabbit anti-GluA2 antibody (AB10529, EMD Millipore) was preincubated with protein G agarose beads at 25°C for 1 h, and the protein samples were then exposed to the antibody-conjugated beads at 4°C overnight.

Techniques: Control, Injection, Expressing, Western Blot, Immunoprecipitation, Plasmid Preparation