nerve conduction velocity module Search Results


na v 1 2  (TargetMol)
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TargetMol na v 1 2
PTPRN interacts with Na V 1.2 channels and negatively modulates Na V 1.2‐mediated current. A) Workflow showing experimental procedures for identification of PTPRN interactors and whole‐cell patch‐clamp recordings. B) Top panel: Classification of the interactors identified in IP‐MS experiments with GO enrichment analysis. Bottom panel: Classification of the interactors identified in IP‐MS experiments with KEGG enrichment analysis. C) Tandem mass spectrometric spectra of a unique peptide of Na V 1.2 identified in the IP‐PTPRN sample. D) Staining for Na V 1.2 (top) and PTPRN (bottom) in hippocampal DG region of adult mice. Scale bar, 50 µm. E) Immunoblot analysis of Na V 1.2 and PTPRN in IP‐PTPRN (left) and IP‐Na V 1.2 (right) samples prepared from mouse hippocampal lysate. F) Immunoblot analysis of HA‐Na V 1.2 and PTPRN‐Flag in IP‐Flag (Left) and IP‐HA (Right) samples prepared from HEK‐293T cell lysate. G) Representative whole‐cell currents recorded from HEK‐293T cells expressing Na V 1.2 and empty vector ( n = 18), Na V 1.2 and PTPRN in a 1:1 ratio ( n = 18), or Na V 1.2 and PTPRN in a 1:2 ratio ( n = 20). Currents were evoked by 5 mV steps depolarization from −90 to 45 mV and cells were held at ‐90 mV. H) Current density versus voltage relationship for the cells described in (G). Currents in all figures were normalized to cell capacitance. I) Graph depicting the voltage dependence of activation for Na V 1.2 channels described in (G). The lines are the best‐fitted Boltzmann curves. J) Current density versus voltage relationship for the primary cortical neurons isolated from PTPRN‐KO ( n = 13) or PTPRN‐WT mice ( n = 16). Currents in all figures were normalized to cell capacitance. K) Validation of Scn8a deletion by western blot analysis using primary cortical neurons. L) Validation of PTPRN knockdown efficiency by western blot analysis using primary cortical neurons infected with lentivirus containing shRNA‐PTPRN or nonsilencing lentivirus for 7 days. M) RT‐PCR analysis of PTPRN in primary cortical neurons described in (L). The levels of mRNA were normalized to those of β‐Actin. n = 3, **** p < 0.0001, #### p < 0.0001, $$$$ p < 0.0001, one‐way ANOVA with Bonferroni's multiple‐comparisons test. N) Current density versus voltage relationship for the primary cortical neurons isolated from Scn8a‐KO mice infected with lentivirus containing shRNA‐PTPRN#3 ( n = 15) or nonsilencing lentivirus ( n = 15) for 7 days. Currents in all figures were normalized to cell capacitance. Data are represented as mean ± s.e.m.
Na V 1 2, supplied by TargetMol, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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nerve conduction velocity module  (ADInstruments)
95
ADInstruments nerve conduction velocity module
PTPRN interacts with Na V 1.2 channels and negatively modulates Na V 1.2‐mediated current. A) Workflow showing experimental procedures for identification of PTPRN interactors and whole‐cell patch‐clamp recordings. B) Top panel: Classification of the interactors identified in IP‐MS experiments with GO enrichment analysis. Bottom panel: Classification of the interactors identified in IP‐MS experiments with KEGG enrichment analysis. C) Tandem mass spectrometric spectra of a unique peptide of Na V 1.2 identified in the IP‐PTPRN sample. D) Staining for Na V 1.2 (top) and PTPRN (bottom) in hippocampal DG region of adult mice. Scale bar, 50 µm. E) Immunoblot analysis of Na V 1.2 and PTPRN in IP‐PTPRN (left) and IP‐Na V 1.2 (right) samples prepared from mouse hippocampal lysate. F) Immunoblot analysis of HA‐Na V 1.2 and PTPRN‐Flag in IP‐Flag (Left) and IP‐HA (Right) samples prepared from HEK‐293T cell lysate. G) Representative whole‐cell currents recorded from HEK‐293T cells expressing Na V 1.2 and empty vector ( n = 18), Na V 1.2 and PTPRN in a 1:1 ratio ( n = 18), or Na V 1.2 and PTPRN in a 1:2 ratio ( n = 20). Currents were evoked by 5 mV steps depolarization from −90 to 45 mV and cells were held at ‐90 mV. H) Current density versus voltage relationship for the cells described in (G). Currents in all figures were normalized to cell capacitance. I) Graph depicting the voltage dependence of activation for Na V 1.2 channels described in (G). The lines are the best‐fitted Boltzmann curves. J) Current density versus voltage relationship for the primary cortical neurons isolated from PTPRN‐KO ( n = 13) or PTPRN‐WT mice ( n = 16). Currents in all figures were normalized to cell capacitance. K) Validation of Scn8a deletion by western blot analysis using primary cortical neurons. L) Validation of PTPRN knockdown efficiency by western blot analysis using primary cortical neurons infected with lentivirus containing shRNA‐PTPRN or nonsilencing lentivirus for 7 days. M) RT‐PCR analysis of PTPRN in primary cortical neurons described in (L). The levels of mRNA were normalized to those of β‐Actin. n = 3, **** p < 0.0001, #### p < 0.0001, $$$$ p < 0.0001, one‐way ANOVA with Bonferroni's multiple‐comparisons test. N) Current density versus voltage relationship for the primary cortical neurons isolated from Scn8a‐KO mice infected with lentivirus containing shRNA‐PTPRN#3 ( n = 15) or nonsilencing lentivirus ( n = 15) for 7 days. Currents in all figures were normalized to cell capacitance. Data are represented as mean ± s.e.m.
Nerve Conduction Velocity Module, supplied by ADInstruments, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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emg monitoring set-up medelec vickers/modul  (Medelec Ltd)
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Medelec Ltd emg monitoring set-up medelec vickers/modul
PTPRN interacts with Na V 1.2 channels and negatively modulates Na V 1.2‐mediated current. A) Workflow showing experimental procedures for identification of PTPRN interactors and whole‐cell patch‐clamp recordings. B) Top panel: Classification of the interactors identified in IP‐MS experiments with GO enrichment analysis. Bottom panel: Classification of the interactors identified in IP‐MS experiments with KEGG enrichment analysis. C) Tandem mass spectrometric spectra of a unique peptide of Na V 1.2 identified in the IP‐PTPRN sample. D) Staining for Na V 1.2 (top) and PTPRN (bottom) in hippocampal DG region of adult mice. Scale bar, 50 µm. E) Immunoblot analysis of Na V 1.2 and PTPRN in IP‐PTPRN (left) and IP‐Na V 1.2 (right) samples prepared from mouse hippocampal lysate. F) Immunoblot analysis of HA‐Na V 1.2 and PTPRN‐Flag in IP‐Flag (Left) and IP‐HA (Right) samples prepared from HEK‐293T cell lysate. G) Representative whole‐cell currents recorded from HEK‐293T cells expressing Na V 1.2 and empty vector ( n = 18), Na V 1.2 and PTPRN in a 1:1 ratio ( n = 18), or Na V 1.2 and PTPRN in a 1:2 ratio ( n = 20). Currents were evoked by 5 mV steps depolarization from −90 to 45 mV and cells were held at ‐90 mV. H) Current density versus voltage relationship for the cells described in (G). Currents in all figures were normalized to cell capacitance. I) Graph depicting the voltage dependence of activation for Na V 1.2 channels described in (G). The lines are the best‐fitted Boltzmann curves. J) Current density versus voltage relationship for the primary cortical neurons isolated from PTPRN‐KO ( n = 13) or PTPRN‐WT mice ( n = 16). Currents in all figures were normalized to cell capacitance. K) Validation of Scn8a deletion by western blot analysis using primary cortical neurons. L) Validation of PTPRN knockdown efficiency by western blot analysis using primary cortical neurons infected with lentivirus containing shRNA‐PTPRN or nonsilencing lentivirus for 7 days. M) RT‐PCR analysis of PTPRN in primary cortical neurons described in (L). The levels of mRNA were normalized to those of β‐Actin. n = 3, **** p < 0.0001, #### p < 0.0001, $$$$ p < 0.0001, one‐way ANOVA with Bonferroni's multiple‐comparisons test. N) Current density versus voltage relationship for the primary cortical neurons isolated from Scn8a‐KO mice infected with lentivirus containing shRNA‐PTPRN#3 ( n = 15) or nonsilencing lentivirus ( n = 15) for 7 days. Currents in all figures were normalized to cell capacitance. Data are represented as mean ± s.e.m.
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Image Search Results


PTPRN interacts with Na V 1.2 channels and negatively modulates Na V 1.2‐mediated current. A) Workflow showing experimental procedures for identification of PTPRN interactors and whole‐cell patch‐clamp recordings. B) Top panel: Classification of the interactors identified in IP‐MS experiments with GO enrichment analysis. Bottom panel: Classification of the interactors identified in IP‐MS experiments with KEGG enrichment analysis. C) Tandem mass spectrometric spectra of a unique peptide of Na V 1.2 identified in the IP‐PTPRN sample. D) Staining for Na V 1.2 (top) and PTPRN (bottom) in hippocampal DG region of adult mice. Scale bar, 50 µm. E) Immunoblot analysis of Na V 1.2 and PTPRN in IP‐PTPRN (left) and IP‐Na V 1.2 (right) samples prepared from mouse hippocampal lysate. F) Immunoblot analysis of HA‐Na V 1.2 and PTPRN‐Flag in IP‐Flag (Left) and IP‐HA (Right) samples prepared from HEK‐293T cell lysate. G) Representative whole‐cell currents recorded from HEK‐293T cells expressing Na V 1.2 and empty vector ( n = 18), Na V 1.2 and PTPRN in a 1:1 ratio ( n = 18), or Na V 1.2 and PTPRN in a 1:2 ratio ( n = 20). Currents were evoked by 5 mV steps depolarization from −90 to 45 mV and cells were held at ‐90 mV. H) Current density versus voltage relationship for the cells described in (G). Currents in all figures were normalized to cell capacitance. I) Graph depicting the voltage dependence of activation for Na V 1.2 channels described in (G). The lines are the best‐fitted Boltzmann curves. J) Current density versus voltage relationship for the primary cortical neurons isolated from PTPRN‐KO ( n = 13) or PTPRN‐WT mice ( n = 16). Currents in all figures were normalized to cell capacitance. K) Validation of Scn8a deletion by western blot analysis using primary cortical neurons. L) Validation of PTPRN knockdown efficiency by western blot analysis using primary cortical neurons infected with lentivirus containing shRNA‐PTPRN or nonsilencing lentivirus for 7 days. M) RT‐PCR analysis of PTPRN in primary cortical neurons described in (L). The levels of mRNA were normalized to those of β‐Actin. n = 3, **** p < 0.0001, #### p < 0.0001, $$$$ p < 0.0001, one‐way ANOVA with Bonferroni's multiple‐comparisons test. N) Current density versus voltage relationship for the primary cortical neurons isolated from Scn8a‐KO mice infected with lentivirus containing shRNA‐PTPRN#3 ( n = 15) or nonsilencing lentivirus ( n = 15) for 7 days. Currents in all figures were normalized to cell capacitance. Data are represented as mean ± s.e.m.

Journal: Advanced Science

Article Title: A Novel Ubiquitin Ligase Adaptor PTPRN Suppresses Seizure Susceptibility through Endocytosis of Na V 1.2 Sodium Channels

doi: 10.1002/advs.202400560

Figure Lengend Snippet: PTPRN interacts with Na V 1.2 channels and negatively modulates Na V 1.2‐mediated current. A) Workflow showing experimental procedures for identification of PTPRN interactors and whole‐cell patch‐clamp recordings. B) Top panel: Classification of the interactors identified in IP‐MS experiments with GO enrichment analysis. Bottom panel: Classification of the interactors identified in IP‐MS experiments with KEGG enrichment analysis. C) Tandem mass spectrometric spectra of a unique peptide of Na V 1.2 identified in the IP‐PTPRN sample. D) Staining for Na V 1.2 (top) and PTPRN (bottom) in hippocampal DG region of adult mice. Scale bar, 50 µm. E) Immunoblot analysis of Na V 1.2 and PTPRN in IP‐PTPRN (left) and IP‐Na V 1.2 (right) samples prepared from mouse hippocampal lysate. F) Immunoblot analysis of HA‐Na V 1.2 and PTPRN‐Flag in IP‐Flag (Left) and IP‐HA (Right) samples prepared from HEK‐293T cell lysate. G) Representative whole‐cell currents recorded from HEK‐293T cells expressing Na V 1.2 and empty vector ( n = 18), Na V 1.2 and PTPRN in a 1:1 ratio ( n = 18), or Na V 1.2 and PTPRN in a 1:2 ratio ( n = 20). Currents were evoked by 5 mV steps depolarization from −90 to 45 mV and cells were held at ‐90 mV. H) Current density versus voltage relationship for the cells described in (G). Currents in all figures were normalized to cell capacitance. I) Graph depicting the voltage dependence of activation for Na V 1.2 channels described in (G). The lines are the best‐fitted Boltzmann curves. J) Current density versus voltage relationship for the primary cortical neurons isolated from PTPRN‐KO ( n = 13) or PTPRN‐WT mice ( n = 16). Currents in all figures were normalized to cell capacitance. K) Validation of Scn8a deletion by western blot analysis using primary cortical neurons. L) Validation of PTPRN knockdown efficiency by western blot analysis using primary cortical neurons infected with lentivirus containing shRNA‐PTPRN or nonsilencing lentivirus for 7 days. M) RT‐PCR analysis of PTPRN in primary cortical neurons described in (L). The levels of mRNA were normalized to those of β‐Actin. n = 3, **** p < 0.0001, #### p < 0.0001, $$$$ p < 0.0001, one‐way ANOVA with Bonferroni's multiple‐comparisons test. N) Current density versus voltage relationship for the primary cortical neurons isolated from Scn8a‐KO mice infected with lentivirus containing shRNA‐PTPRN#3 ( n = 15) or nonsilencing lentivirus ( n = 15) for 7 days. Currents in all figures were normalized to cell capacitance. Data are represented as mean ± s.e.m.

Article Snippet: To investigate the mechanism of the regulation on Na V 1.2 by PTPRN, 80 μ m Dynasore (Abcam), [ ] 1 μ m TAK‐243 (TargetMol), [ ] 20 μ m Pitstop2 (GLPBIO), [ ] or 50 μ m Heclin (GLPBIO) [ ] were added into the medium 2 h before the electrophysiology recording.

Techniques: Patch Clamp, Protein-Protein interactions, Staining, Western Blot, Expressing, Plasmid Preparation, Activation Assay, Isolation, Biomarker Discovery, Knockdown, Infection, shRNA, Reverse Transcription Polymerase Chain Reaction

PTPRN promotes Na V 1.2 channel internalization through ubiquitin‐dependent endocytosis. A) Schematic of the effect of inhibitors on the trafficking and ubiquitination of proteins. B) Left panel: Immunoblot analysis of cell surface biotinylation performed in primary cortical neurons infected with lentivirus containing shRNA‐PTPRN#3 or nonsilencing lentivirus for 7 days. Total lysates (total) and biotinylated fractions (membrane) were analyzed by western blot analysis. Right panel: Quantification of Na V 1.2 total expression and surface expression. n = 3, * p < 0.05, two‐way ANOVA with Bonferroni's multiple‐comparisons test. C) Current density versus voltage relationship for HEK‐293T cells expressing Na V 1.2 and PTPRN in a 1:2 ratio (Na V 1.2 + PTPRN) or Na V 1.2 and empty vector (Na V 1.2), with or without treatment using dynasore (80 µ m , 2 h). n = 21 for all samples except Na V 1.2 + PTPRN plus dynasore ( n = 24). D) Graph depicting voltage‐dependence of activation for Na V 1.2 channels described in (C). The lines are the best‐fitted Boltzmann curves. E) Current density versus voltage relationship for HEK‐293T cells expressing Na V 1.2 and PTPRN in a 1:2 ratio (Na V 1.2 + PTPRN) or Na V 1.2 and empty vector (Na V 1.2), with or without treatment using TAK‐243 (1 µ m , 2 h). n = 18 and 26 for untreated Na V 1.2 and Na V 1.2 + PTPRN, respectively, and n = 20 and 26 for Na V 1.2 and Na V 1.2 + PTPRN plus TAK‐243, respectively. F) Graph depicting voltage dependence of activation for Na V 1.2 channels described in (E). The lines are the best‐fitted Boltzmann curves. G) Current density versus voltage relationship for HEK‐293T cells expressing Na V 1.2 and PTPRN in a 1:2 ratio (Na V 1.2 + PTPRN) or Na V 1.2 and empty vector (Na V 1.2), with or without treatment with Heclin (20 µ m , 2 h). n = 18 and 26 for Na V 1.2 and Na V 1.2 + PTPRN plus Heclin, respectively, and n = 19 for both samples without treatment. H) Graph depicting voltage dependence of activation for Na V 1.2 channels described in (G). The lines are the best‐fitted Boltzmann curves. I) Workflow showing experimental procedures for purification of PTPRN intracellular fragment (aa 601–979, PTPRN‐cyto) and TagRFP and whole cell patch‐clamp recordings. J) Left panel: Coomassie blue staining of PTPRN‐cyto (left) or TagRFP (right) after purification and separation by SDS‐PAGE. Right panel: Representative images showing peptide delivery through a pipette. Scale bar, 50 µm. K) Current density versus voltage relationship for HEK‐293T cells expressing Na V 1.2 after intracellular equilibration with 1 µ m PTPRN‐cyto ( n = 25) or TagRFP ( n = 23). L) Graph depicting voltage dependence of activation for Na V 1.2 channels described in (K). The lines are the best‐fitted Boltzmann curves. Data are represented as mean ± s.e.m.

Journal: Advanced Science

Article Title: A Novel Ubiquitin Ligase Adaptor PTPRN Suppresses Seizure Susceptibility through Endocytosis of Na V 1.2 Sodium Channels

doi: 10.1002/advs.202400560

Figure Lengend Snippet: PTPRN promotes Na V 1.2 channel internalization through ubiquitin‐dependent endocytosis. A) Schematic of the effect of inhibitors on the trafficking and ubiquitination of proteins. B) Left panel: Immunoblot analysis of cell surface biotinylation performed in primary cortical neurons infected with lentivirus containing shRNA‐PTPRN#3 or nonsilencing lentivirus for 7 days. Total lysates (total) and biotinylated fractions (membrane) were analyzed by western blot analysis. Right panel: Quantification of Na V 1.2 total expression and surface expression. n = 3, * p < 0.05, two‐way ANOVA with Bonferroni's multiple‐comparisons test. C) Current density versus voltage relationship for HEK‐293T cells expressing Na V 1.2 and PTPRN in a 1:2 ratio (Na V 1.2 + PTPRN) or Na V 1.2 and empty vector (Na V 1.2), with or without treatment using dynasore (80 µ m , 2 h). n = 21 for all samples except Na V 1.2 + PTPRN plus dynasore ( n = 24). D) Graph depicting voltage‐dependence of activation for Na V 1.2 channels described in (C). The lines are the best‐fitted Boltzmann curves. E) Current density versus voltage relationship for HEK‐293T cells expressing Na V 1.2 and PTPRN in a 1:2 ratio (Na V 1.2 + PTPRN) or Na V 1.2 and empty vector (Na V 1.2), with or without treatment using TAK‐243 (1 µ m , 2 h). n = 18 and 26 for untreated Na V 1.2 and Na V 1.2 + PTPRN, respectively, and n = 20 and 26 for Na V 1.2 and Na V 1.2 + PTPRN plus TAK‐243, respectively. F) Graph depicting voltage dependence of activation for Na V 1.2 channels described in (E). The lines are the best‐fitted Boltzmann curves. G) Current density versus voltage relationship for HEK‐293T cells expressing Na V 1.2 and PTPRN in a 1:2 ratio (Na V 1.2 + PTPRN) or Na V 1.2 and empty vector (Na V 1.2), with or without treatment with Heclin (20 µ m , 2 h). n = 18 and 26 for Na V 1.2 and Na V 1.2 + PTPRN plus Heclin, respectively, and n = 19 for both samples without treatment. H) Graph depicting voltage dependence of activation for Na V 1.2 channels described in (G). The lines are the best‐fitted Boltzmann curves. I) Workflow showing experimental procedures for purification of PTPRN intracellular fragment (aa 601–979, PTPRN‐cyto) and TagRFP and whole cell patch‐clamp recordings. J) Left panel: Coomassie blue staining of PTPRN‐cyto (left) or TagRFP (right) after purification and separation by SDS‐PAGE. Right panel: Representative images showing peptide delivery through a pipette. Scale bar, 50 µm. K) Current density versus voltage relationship for HEK‐293T cells expressing Na V 1.2 after intracellular equilibration with 1 µ m PTPRN‐cyto ( n = 25) or TagRFP ( n = 23). L) Graph depicting voltage dependence of activation for Na V 1.2 channels described in (K). The lines are the best‐fitted Boltzmann curves. Data are represented as mean ± s.e.m.

Article Snippet: To investigate the mechanism of the regulation on Na V 1.2 by PTPRN, 80 μ m Dynasore (Abcam), [ ] 1 μ m TAK‐243 (TargetMol), [ ] 20 μ m Pitstop2 (GLPBIO), [ ] or 50 μ m Heclin (GLPBIO) [ ] were added into the medium 2 h before the electrophysiology recording.

Techniques: Ubiquitin Proteomics, Western Blot, Infection, shRNA, Membrane, Expressing, Plasmid Preparation, Activation Assay, Purification, Patch Clamp, Staining, SDS Page, Transferring

PTPRN facilitates ubiquitination of Na V 1.2 channels by recruiting NEDD4L. A) Schematic diagram of Na V 1.2 intracellular regions and Na V 1.2‐1.5 chimeric channels (Na V 1.2/5C and Na V 1.5/2C). B) Immunoblot analysis of HA‐tagged Na V 1.2 intracellular regions and PTPRN‐Flag in IP‐Flag samples prepared from HEK‐293T cell lysate. C) Current density versus voltage relationship for HEK‐293T cells expressing the Na V 1.2/5C chimera and PTPRN in a 1:2 ratio (Na V 1.2/5C + PTPRN) ( n = 26) or the Na V 1.2/5C chimera and empty vector in a 1:2 ratio (Na V 1.2/5C, n = 19). D) Graph depicting voltage dependence of activation for the Na V 1.2/5C chimera described in (C). The lines are the best‐fitted Boltzmann curves. E) Current density versus voltage relationship for HEK‐293T cells expressing the Na V 1.5/2C chimera and PTPRN in a 1:2 ratio (Na V 1.5/2C + PTPRN) or the Na V 1.2/5C chimera and empty vector in a 1:2 ratio (Na V 1.5/2C), n = 20. F) Graph depicting voltage‐dependence of activation for the Na V 1.2/5C chimera described in (E). The lines are the best‐fitted Boltzmann curves. G) Current density versus voltage relationship for HEK‐293T cells expressing wild‐type (WT) Na V 1.2, Na V 1.2 IL‐AA , or Na V 1.2 PPSY‐AAAA mutations with PTPRN or empty vector in a 1:2 ratio. n = 20, 22, 23, 22, and 22 (from up to down in legend). H) Graph depicting voltage dependence of activation for Na V 1.2 channels described in (G). The lines are the best‐fitted Boltzmann curves. I) Immunoblot analysis of HA‐tagged NEDD4/NEDD4L and PTPRN‐Flag in IP‐Flag samples prepared from HEK‐293T cell lysate. J) Immunoblot analysis of Na V 1.2, NEDD4L and PTPRN in the IP‐PTPRN sample prepared from human temporal lobe lysate. K) Current density versus voltage relationship for HEK‐293T cells expressing WT Na V 1.2 with or without PTPRN/NEDD4L CS mutation. n = 18, 24, 24, and 23 (from up to down in legend). L) Graph depicting voltage dependence of activation for Na V 1.2 channels described in (K). The lines are the best‐fitted Boltzmann curves. M) Validation of NEDD4L knockdown efficiency by western blot analysis using primary cortical neurons infected with lentivirus containing shRNA‐NEDD4L or nonsilencing lentivirus for 7 days. N) Current density versus voltage relationship for the primary cortical neurons isolated from Scn8a‐KO mice infected with lentivirus containing shRNA‐PTPRN#3 and shRNA‐NEDD4L ( n = 15) or nonsilencing lentivirus ( n = 15) for 7 days. Currents in all figures were normalized to cell capacitance. O) Western blot analysis of indicated proteins in IP‐Na V 1.2 samples prepared from hippocampus of PTPRN‐KO or PTPRN‐WT mice. P) Quantification of Na V 1.2 ubiquitination level (top) and the interaction between Na V 1.2 and NEDD4L (bottom). n = 3, * p < 0.05, *** p < 0.001, unpaired two‐tailed Student's t ‐test. Q) Western blot analysis of indicated proteins in IP‐Na V 1.2 samples prepared from hippocampus of mice infected with control AAV or AAV expressing PTPRN. R) Quantification of Na V 1.2 ubiquitination level (top) and the interaction between Na V 1.2 and NEDD4L (bottom). n = 3, * p < 0.05, ** p < 0.01, unpaired two‐tailed Student's t ‐test. Data are represented as mean ± s.e.m.

Journal: Advanced Science

Article Title: A Novel Ubiquitin Ligase Adaptor PTPRN Suppresses Seizure Susceptibility through Endocytosis of Na V 1.2 Sodium Channels

doi: 10.1002/advs.202400560

Figure Lengend Snippet: PTPRN facilitates ubiquitination of Na V 1.2 channels by recruiting NEDD4L. A) Schematic diagram of Na V 1.2 intracellular regions and Na V 1.2‐1.5 chimeric channels (Na V 1.2/5C and Na V 1.5/2C). B) Immunoblot analysis of HA‐tagged Na V 1.2 intracellular regions and PTPRN‐Flag in IP‐Flag samples prepared from HEK‐293T cell lysate. C) Current density versus voltage relationship for HEK‐293T cells expressing the Na V 1.2/5C chimera and PTPRN in a 1:2 ratio (Na V 1.2/5C + PTPRN) ( n = 26) or the Na V 1.2/5C chimera and empty vector in a 1:2 ratio (Na V 1.2/5C, n = 19). D) Graph depicting voltage dependence of activation for the Na V 1.2/5C chimera described in (C). The lines are the best‐fitted Boltzmann curves. E) Current density versus voltage relationship for HEK‐293T cells expressing the Na V 1.5/2C chimera and PTPRN in a 1:2 ratio (Na V 1.5/2C + PTPRN) or the Na V 1.2/5C chimera and empty vector in a 1:2 ratio (Na V 1.5/2C), n = 20. F) Graph depicting voltage‐dependence of activation for the Na V 1.2/5C chimera described in (E). The lines are the best‐fitted Boltzmann curves. G) Current density versus voltage relationship for HEK‐293T cells expressing wild‐type (WT) Na V 1.2, Na V 1.2 IL‐AA , or Na V 1.2 PPSY‐AAAA mutations with PTPRN or empty vector in a 1:2 ratio. n = 20, 22, 23, 22, and 22 (from up to down in legend). H) Graph depicting voltage dependence of activation for Na V 1.2 channels described in (G). The lines are the best‐fitted Boltzmann curves. I) Immunoblot analysis of HA‐tagged NEDD4/NEDD4L and PTPRN‐Flag in IP‐Flag samples prepared from HEK‐293T cell lysate. J) Immunoblot analysis of Na V 1.2, NEDD4L and PTPRN in the IP‐PTPRN sample prepared from human temporal lobe lysate. K) Current density versus voltage relationship for HEK‐293T cells expressing WT Na V 1.2 with or without PTPRN/NEDD4L CS mutation. n = 18, 24, 24, and 23 (from up to down in legend). L) Graph depicting voltage dependence of activation for Na V 1.2 channels described in (K). The lines are the best‐fitted Boltzmann curves. M) Validation of NEDD4L knockdown efficiency by western blot analysis using primary cortical neurons infected with lentivirus containing shRNA‐NEDD4L or nonsilencing lentivirus for 7 days. N) Current density versus voltage relationship for the primary cortical neurons isolated from Scn8a‐KO mice infected with lentivirus containing shRNA‐PTPRN#3 and shRNA‐NEDD4L ( n = 15) or nonsilencing lentivirus ( n = 15) for 7 days. Currents in all figures were normalized to cell capacitance. O) Western blot analysis of indicated proteins in IP‐Na V 1.2 samples prepared from hippocampus of PTPRN‐KO or PTPRN‐WT mice. P) Quantification of Na V 1.2 ubiquitination level (top) and the interaction between Na V 1.2 and NEDD4L (bottom). n = 3, * p < 0.05, *** p < 0.001, unpaired two‐tailed Student's t ‐test. Q) Western blot analysis of indicated proteins in IP‐Na V 1.2 samples prepared from hippocampus of mice infected with control AAV or AAV expressing PTPRN. R) Quantification of Na V 1.2 ubiquitination level (top) and the interaction between Na V 1.2 and NEDD4L (bottom). n = 3, * p < 0.05, ** p < 0.01, unpaired two‐tailed Student's t ‐test. Data are represented as mean ± s.e.m.

Article Snippet: To investigate the mechanism of the regulation on Na V 1.2 by PTPRN, 80 μ m Dynasore (Abcam), [ ] 1 μ m TAK‐243 (TargetMol), [ ] 20 μ m Pitstop2 (GLPBIO), [ ] or 50 μ m Heclin (GLPBIO) [ ] were added into the medium 2 h before the electrophysiology recording.

Techniques: Ubiquitin Proteomics, Western Blot, Expressing, Plasmid Preparation, Activation Assay, Mutagenesis, Biomarker Discovery, Knockdown, Infection, shRNA, Isolation, Two Tailed Test, Control

PTPRN‐Na V 1.2 axis regulates neuronal intrinsic excitability and seizure susceptibility. A) Schematic diagram showing stereotaxic injection of shRNA‐Scn2a AAV in adult PTPRN‐KO mice. B) Infection of AAV expressing shRNA‐Scn2a and EGFP in the hippocampal DG region 14 days after injection. Scale bar, 125 µm. C) Validation of Scn2a knockdown efficiency by RT‐PCR analysis of Scn2a in hippocampal tissues obtained from mice infected with AAV‐containing shRNA‐Scn2a or control AAV for 14 days. The levels of mRNA were normalized to those of β‐Actin. n = 3, ** p < 0.01, unpaired two‐tailed Student's t ‐test. D) Top panel: Validation of Scn2a knockdown efficiency by western blot analysis using hippocampi from mice infected with AAV‐containing shRNA‐Scn2a or control AAV for 14 days. Bottom panel: Quantification of the results by normalizing the protein levels of Na V 1.2 to those of β‐Actin. n = 3, * p < 0.05, unpaired two‐tailed Student's t ‐test. E) Left panel: Representative current‐clamp recordings of DG granule cells from PTPRN‐WT and PTPRN‐KO mice infected with AAV containing shRNA‐Scn2a or control AAV for 14 days. For comparison, the cells were held at ‐80 mV of membrane potential. Right panel: The mean number of APs generated in response to depolarizing current pulses. * p < 0.05, *** p < 0.001, **** p < 0.0001, #### p < 0.0001, two‐way ANOVA with Bonferroni's multiple‐comparisons test. F) Typical APs of DG granule cells from mice described in (E). G) Phase plane plots associated with APs in (F). H) The average AP threshold (left) and peak rising phase dV/dt (right). * p < 0.05, #### p < 0.0001, $$$$ p < 0.0001, one‐way ANOVA with Bonferroni's multiple‐comparisons test. I) Graph depicting the seizure progression in PTPRN‐WT and PTPRN‐KO mice infected with AAV containing shRNA‐Scn2a or control AAV for 14 days, illustrated as mean maximum seizure stage reached by 15, 30, 45, 60, and 75 min after KA administration. n = 6, $ p < 0.05, * p < 0.05, *** p < 0.001, **** p < 0.0001, ### p < 0.001, $$$ p < 0.001, two‐way ANOVA with Bonferroni's multiple‐comparisons test. J) Graph showing time taken to reach each stage of seizure after administration of KA in mice described in (I). n = 6, $$ p < 0.01, **** p < 0.0001, two‐way ANOVA with Bonferroni's multiple‐comparisons test. K) Number of class V seizures within 1 h after KA administration. n = 6, *** p < 0.001, ### p < 0.001, one‐way ANOVA with Bonferroni's multiple‐comparisons test. L) Incidence of maximum seizure stage reached during the course of the experiments in (I). n = 6, **** p < 0.0001, #### p < 0.0001, $$$$ p < 0.0001, Chi‐square test. Data are represented as mean ± s.e.m.

Journal: Advanced Science

Article Title: A Novel Ubiquitin Ligase Adaptor PTPRN Suppresses Seizure Susceptibility through Endocytosis of Na V 1.2 Sodium Channels

doi: 10.1002/advs.202400560

Figure Lengend Snippet: PTPRN‐Na V 1.2 axis regulates neuronal intrinsic excitability and seizure susceptibility. A) Schematic diagram showing stereotaxic injection of shRNA‐Scn2a AAV in adult PTPRN‐KO mice. B) Infection of AAV expressing shRNA‐Scn2a and EGFP in the hippocampal DG region 14 days after injection. Scale bar, 125 µm. C) Validation of Scn2a knockdown efficiency by RT‐PCR analysis of Scn2a in hippocampal tissues obtained from mice infected with AAV‐containing shRNA‐Scn2a or control AAV for 14 days. The levels of mRNA were normalized to those of β‐Actin. n = 3, ** p < 0.01, unpaired two‐tailed Student's t ‐test. D) Top panel: Validation of Scn2a knockdown efficiency by western blot analysis using hippocampi from mice infected with AAV‐containing shRNA‐Scn2a or control AAV for 14 days. Bottom panel: Quantification of the results by normalizing the protein levels of Na V 1.2 to those of β‐Actin. n = 3, * p < 0.05, unpaired two‐tailed Student's t ‐test. E) Left panel: Representative current‐clamp recordings of DG granule cells from PTPRN‐WT and PTPRN‐KO mice infected with AAV containing shRNA‐Scn2a or control AAV for 14 days. For comparison, the cells were held at ‐80 mV of membrane potential. Right panel: The mean number of APs generated in response to depolarizing current pulses. * p < 0.05, *** p < 0.001, **** p < 0.0001, #### p < 0.0001, two‐way ANOVA with Bonferroni's multiple‐comparisons test. F) Typical APs of DG granule cells from mice described in (E). G) Phase plane plots associated with APs in (F). H) The average AP threshold (left) and peak rising phase dV/dt (right). * p < 0.05, #### p < 0.0001, $$$$ p < 0.0001, one‐way ANOVA with Bonferroni's multiple‐comparisons test. I) Graph depicting the seizure progression in PTPRN‐WT and PTPRN‐KO mice infected with AAV containing shRNA‐Scn2a or control AAV for 14 days, illustrated as mean maximum seizure stage reached by 15, 30, 45, 60, and 75 min after KA administration. n = 6, $ p < 0.05, * p < 0.05, *** p < 0.001, **** p < 0.0001, ### p < 0.001, $$$ p < 0.001, two‐way ANOVA with Bonferroni's multiple‐comparisons test. J) Graph showing time taken to reach each stage of seizure after administration of KA in mice described in (I). n = 6, $$ p < 0.01, **** p < 0.0001, two‐way ANOVA with Bonferroni's multiple‐comparisons test. K) Number of class V seizures within 1 h after KA administration. n = 6, *** p < 0.001, ### p < 0.001, one‐way ANOVA with Bonferroni's multiple‐comparisons test. L) Incidence of maximum seizure stage reached during the course of the experiments in (I). n = 6, **** p < 0.0001, #### p < 0.0001, $$$$ p < 0.0001, Chi‐square test. Data are represented as mean ± s.e.m.

Article Snippet: To investigate the mechanism of the regulation on Na V 1.2 by PTPRN, 80 μ m Dynasore (Abcam), [ ] 1 μ m TAK‐243 (TargetMol), [ ] 20 μ m Pitstop2 (GLPBIO), [ ] or 50 μ m Heclin (GLPBIO) [ ] were added into the medium 2 h before the electrophysiology recording.

Techniques: Injection, shRNA, Infection, Expressing, Biomarker Discovery, Knockdown, Reverse Transcription Polymerase Chain Reaction, Control, Two Tailed Test, Western Blot, Comparison, Membrane, Generated

Proposed mechanism for PTPRN regulation of Na V 1.2 channels and neuronal intrinsic plasticity. Elevated neuronal activity augments the recruitment of NEDD4L by PTPRN to Na V 1.2 sodium channels. This interaction promotes NEDD4L‐mediated ubiquitination, subsequently leading to the endocytosis of Na V 1.2 channels. The presented regulatory mechanism provides a feedback response to heightened activity within the nervous system, thereby aiding the tuning of neuronal intrinsic plasticity.

Journal: Advanced Science

Article Title: A Novel Ubiquitin Ligase Adaptor PTPRN Suppresses Seizure Susceptibility through Endocytosis of Na V 1.2 Sodium Channels

doi: 10.1002/advs.202400560

Figure Lengend Snippet: Proposed mechanism for PTPRN regulation of Na V 1.2 channels and neuronal intrinsic plasticity. Elevated neuronal activity augments the recruitment of NEDD4L by PTPRN to Na V 1.2 sodium channels. This interaction promotes NEDD4L‐mediated ubiquitination, subsequently leading to the endocytosis of Na V 1.2 channels. The presented regulatory mechanism provides a feedback response to heightened activity within the nervous system, thereby aiding the tuning of neuronal intrinsic plasticity.

Article Snippet: To investigate the mechanism of the regulation on Na V 1.2 by PTPRN, 80 μ m Dynasore (Abcam), [ ] 1 μ m TAK‐243 (TargetMol), [ ] 20 μ m Pitstop2 (GLPBIO), [ ] or 50 μ m Heclin (GLPBIO) [ ] were added into the medium 2 h before the electrophysiology recording.

Techniques: Activity Assay, Ubiquitin Proteomics