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Santa Cruz Biotechnology mouse anti αii spectrin
Mouse Anti αii Spectrin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mouse+anti+%CE%B1ii+spectrin/pm35040026-73-48-52?v=Santa+Cruz+Biotechnology
Average 93 stars, based on 106 article reviews

mouse anti αii spectrin - by Bioz Stars, 2026-07

93/100 stars

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Anti-sortilin Western blot, age and body weight of examined Sort +/+ and Sort −/− mice, and study design. A protein band of about 90 kDa was detected specifically in sortilin-deficient (Sort1 −/− ) mice, consistent with the presence of a truncated protein after disruption of the reading frame in Exon 14 (A). Age (B) and body weight (C) were heterogenous without statistical significance (one way ANOVA, Šídák's multiple comparisons test). The flow chart shows the experimental time-course (D). Arrows represent days pre or post injury (dpi) after controlled cortical impact (CCI). Text above indicates interventions, text below indicates outcome parameters. Neurological severity score (NSS) and Rotarod (RR) performance were assessed one day before as well as 1 dpi (cohort 1) and 5 dpi (cohort 2). Lesion volume (LV), quantitative polymerase chain reaction (qPCR), terminal desoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and spectrin breakdown products (SBDPs) were analysed at the respective time points.

Journal: Heliyon

Article Title: Sortilin is dispensable for secondary injury processes following traumatic brain injury in mice

doi: 10.1016/j.heliyon.2024.e35198

Figure Lengend Snippet: Anti-sortilin Western blot, age and body weight of examined Sort +/+ and Sort −/− mice, and study design. A protein band of about 90 kDa was detected specifically in sortilin-deficient (Sort1 −/− ) mice, consistent with the presence of a truncated protein after disruption of the reading frame in Exon 14 (A). Age (B) and body weight (C) were heterogenous without statistical significance (one way ANOVA, Šídák's multiple comparisons test). The flow chart shows the experimental time-course (D). Arrows represent days pre or post injury (dpi) after controlled cortical impact (CCI). Text above indicates interventions, text below indicates outcome parameters. Neurological severity score (NSS) and Rotarod (RR) performance were assessed one day before as well as 1 dpi (cohort 1) and 5 dpi (cohort 2). Lesion volume (LV), quantitative polymerase chain reaction (qPCR), terminal desoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and spectrin breakdown products (SBDPs) were analysed at the respective time points.

Article Snippet: The following primary antibodies were used: mouse anti-αII-spectrin (1:1000, BML-FG6090, Enzo Life Science, Farmingdale, USA), mouse anti-GAPDH (1:2000, ACR001PS, Acris Antibodies Inc., San Diego, USA), rabbit anti-sortilin (1:500, ANT-009, Alomone Labs, Jerusalem, USA), followed by incubation with appropriate species-specific secondary infrared dye-conjugated antibodies: goat anti-rabbit IgG (1:15000, IRDye 800CW, Li-Cor Bioscience, Bad Homburg, Germany), goat anti-mouse IgG (1:15000, IRDye 680RD, Li-Cor Bioscience, Bad Homburg, Germany).

Techniques: Western Blot, Disruption, Real-time Polymerase Chain Reaction, End Labeling, TUNEL Assay

Sortilin-deficiency does not affect CCI-induced cell death and production of SBDPs. (A–C) Images illustrating the location of cell death examination by TUNEL staining at 1 dpi (A) Ipsilesional hemisphere of a cresyl violet stained brain cryosection. The brain lesion does not show cresyl violet staining and the orange box includes lesional and perilesional regions. (B) Low-magnification fluoresence image showing TUNEL (green) and DAPI (blue) staining corresponding to the location of the orange box. The lesion area can be identified by a high-intensity TUNEL staining and extensive loss of DAPI staining due to tissue destruction. The perilesional area contains intact cell nuclei identified by DAPI, which partially co-localize with TUNEL stainining. (C) High-magnification fluorescence image corresponding to the location of the red box shows the region of interest in the deep layers of the perilesional cortex for TUNEL/DAPI staining analysis. (D) Representative images of TUNEL/DAPI staining in brain cryosections from Sort +/+ mice and Sort −/− mice. Some TUNEL/DAPI co-stained cells are marked by arrows. (E) Quantification of the TUNEL/DAPI ratio. Data are expressed as mean ± SD, individual data is shown as dots, Student's t -test. (F) Western blot showing spectrin breakdown products (SBDPs) at ∼150 kDa at 1 dpi in protein lysates of the ipsilesional brain tissue from Sort +/+ mice (lane 2) and Sort −/− mice (lane 3) as well as in naïve Sort +/+ mice (lane 1; C). (G) Quantification of SBDPs in ipsilesional brain tissue. Data are expressed as mean ± SD, individual data is shown as dots, Mann Whitney test.

Journal: Heliyon

Article Title: Sortilin is dispensable for secondary injury processes following traumatic brain injury in mice

doi: 10.1016/j.heliyon.2024.e35198

Figure Lengend Snippet: Sortilin-deficiency does not affect CCI-induced cell death and production of SBDPs. (A–C) Images illustrating the location of cell death examination by TUNEL staining at 1 dpi (A) Ipsilesional hemisphere of a cresyl violet stained brain cryosection. The brain lesion does not show cresyl violet staining and the orange box includes lesional and perilesional regions. (B) Low-magnification fluoresence image showing TUNEL (green) and DAPI (blue) staining corresponding to the location of the orange box. The lesion area can be identified by a high-intensity TUNEL staining and extensive loss of DAPI staining due to tissue destruction. The perilesional area contains intact cell nuclei identified by DAPI, which partially co-localize with TUNEL stainining. (C) High-magnification fluorescence image corresponding to the location of the red box shows the region of interest in the deep layers of the perilesional cortex for TUNEL/DAPI staining analysis. (D) Representative images of TUNEL/DAPI staining in brain cryosections from Sort +/+ mice and Sort −/− mice. Some TUNEL/DAPI co-stained cells are marked by arrows. (E) Quantification of the TUNEL/DAPI ratio. Data are expressed as mean ± SD, individual data is shown as dots, Student's t -test. (F) Western blot showing spectrin breakdown products (SBDPs) at ∼150 kDa at 1 dpi in protein lysates of the ipsilesional brain tissue from Sort +/+ mice (lane 2) and Sort −/− mice (lane 3) as well as in naïve Sort +/+ mice (lane 1; C). (G) Quantification of SBDPs in ipsilesional brain tissue. Data are expressed as mean ± SD, individual data is shown as dots, Mann Whitney test.

Techniques: TUNEL Assay, Staining, Fluorescence, Western Blot, MANN-WHITNEY

CSF biomarker (αII-spectrin, SBDP150/145, GFAP, GBDP 38/44K) levels measured using immunoblotting in the diagnosis of SCI and comparison between SCI and healthy control groups: ( A ) representative images from immunoblotting for αII-spectrin, SBDP 150/145, GFAP, and GBDP38/44K ( B – E ); quantification shown as the median, interquartile range (box), and upper and lower values (whiskers) for αII-spectrin ( B ), SBDP150/145 K ( C ), GFAP ( D ), and GBDP 38/44K ( E ). ** Denotes statistical results compared between patients with SCI ( n = 15) and healthy control ( n = 10) groups. Note: **, *** p values < 0.05 and 0.005 vs. control.

Journal: Diagnostics

Article Title: Association between Cerebrospinal Fluid and Serum Biomarker Levels and Diagnosis, Injury Severity, and Short-Term Outcomes in Patients with Acute Traumatic Spinal Cord Injury

doi: 10.3390/diagnostics13101814

Figure Lengend Snippet: CSF biomarker (αII-spectrin, SBDP150/145, GFAP, GBDP 38/44K) levels measured using immunoblotting in the diagnosis of SCI and comparison between SCI and healthy control groups: ( A ) representative images from immunoblotting for αII-spectrin, SBDP 150/145, GFAP, and GBDP38/44K ( B – E ); quantification shown as the median, interquartile range (box), and upper and lower values (whiskers) for αII-spectrin ( B ), SBDP150/145 K ( C ), GFAP ( D ), and GBDP 38/44K ( E ). ** Denotes statistical results compared between patients with SCI ( n = 15) and healthy control ( n = 10) groups. Note: **, *** p values < 0.05 and 0.005 vs. control.

Article Snippet: Monoclonal anti-mouse αII-spectrin (Enzo Life Sciences, NY, USA), anti-spectrin antibody, polyclonal anti-rabbit GFAP (Abcam, Walham, MA, USA) (capable of detecting major GFAP breakdown products GBDP 38/44K), and SDBP 150/145 were incubated with immunoblotting membranes at a dilution of 1:1000 in 5% milk at 4 °C overnight.

Techniques: Biomarker Assay, Western Blot

Ability of CSF αII-spectrin, SBDP150/145, GFAP, and GBDP 38/44K levels to predict injury severity (AIS grade) measured by immunoblotting. * Denotes statistical results compared to initial AIS grade A ( n = 11) and AIS grade B ( n = 6) patients in SCI group. Quantification is shown as the median, interquartile range (box), and upper and lower values (whiskers). Note: *, **, *** p values < 0.05, 0.01 and 0.001 between groups.

Journal: Diagnostics

Article Title: Association between Cerebrospinal Fluid and Serum Biomarker Levels and Diagnosis, Injury Severity, and Short-Term Outcomes in Patients with Acute Traumatic Spinal Cord Injury

doi: 10.3390/diagnostics13101814

Figure Lengend Snippet: Ability of CSF αII-spectrin, SBDP150/145, GFAP, and GBDP 38/44K levels to predict injury severity (AIS grade) measured by immunoblotting. * Denotes statistical results compared to initial AIS grade A ( n = 11) and AIS grade B ( n = 6) patients in SCI group. Quantification is shown as the median, interquartile range (box), and upper and lower values (whiskers). Note: *, **, *** p values < 0.05, 0.01 and 0.001 between groups.

Techniques: Western Blot

a Schematic for co-immunoprecipitation (co-IP)-based mass spectrometry identification of candidate MPS-interacting proteins. The antibody against a bait protein, βII-spectrin, αII-spectrin, or α-adducin, was attached to protein-G-coated beads. The antibody-coated beads were then used to capture the bait protein and the co-immunoprecipitated proteins from the cultured hippocampal neuron lysate or adult mouse whole-brain lysate. The co-immunoprecipitated proteins were identified using mass spectrometry. b Top: Venn diagrams showing the overlap of the identified proteins in three co-IP experiments using βII-spectrin, αII-spectrin or α-adducin as the bait, either from cultured hippocampal neuron (DIV 20) lysates (left) or from mouse whole-brain lysates (right). Two biological replicates were performed for each co-IP condition. Bottom: Venn diagram showing the overlap between the 480 identified proteins from cultured hippocampal neurons and the 670 identified proteins from mouse whole-brain lysates (See Supplementary Data for the full list of the candidate MPS-interacting proteins). c Functional annotation clustering of the enriched Gene Ontology (GO) terms in the biological process (BP) category for the 480 candidate MPS-interacting proteins identified in cultured hippocampal neurons. A selected subset of GO BP terms are shown (See Supplementary Data for the full list of clustered GO BP terms enriched in the candidate MPS-interacting proteins). The corresponding p -values (bars) and protein numbers (next to the bars) for each enriched GO BP term are shown on the right. d Venn diagrams showing the overlap between the enriched GO BP terms of the 480 candidate MPS-interacting proteins identified in cultured mouse hippocampal neurons (DIV 20) and the enriched GO BP terms of the 670 candidate MPS-interacting proteins identified in the adult mouse whole brain (left). Also shown in comparison is the overlap between the enriched GO BP terms of the 480 candidate MPS-interacting proteins in cultured mouse hippocampal neurons (DIV 20) and the enriched GO BP terms of 670 randomly selected genes from the mouse genome (right). e Same as d but for GO MF terms instead of GO BP terms.

Journal: Nature Communications

Article Title: Proteomic and functional analyses of the periodic membrane skeleton in neurons

doi: 10.1038/s41467-022-30720-x

Figure Lengend Snippet: a Schematic for co-immunoprecipitation (co-IP)-based mass spectrometry identification of candidate MPS-interacting proteins. The antibody against a bait protein, βII-spectrin, αII-spectrin, or α-adducin, was attached to protein-G-coated beads. The antibody-coated beads were then used to capture the bait protein and the co-immunoprecipitated proteins from the cultured hippocampal neuron lysate or adult mouse whole-brain lysate. The co-immunoprecipitated proteins were identified using mass spectrometry. b Top: Venn diagrams showing the overlap of the identified proteins in three co-IP experiments using βII-spectrin, αII-spectrin or α-adducin as the bait, either from cultured hippocampal neuron (DIV 20) lysates (left) or from mouse whole-brain lysates (right). Two biological replicates were performed for each co-IP condition. Bottom: Venn diagram showing the overlap between the 480 identified proteins from cultured hippocampal neurons and the 670 identified proteins from mouse whole-brain lysates (See Supplementary Data for the full list of the candidate MPS-interacting proteins). c Functional annotation clustering of the enriched Gene Ontology (GO) terms in the biological process (BP) category for the 480 candidate MPS-interacting proteins identified in cultured hippocampal neurons. A selected subset of GO BP terms are shown (See Supplementary Data for the full list of clustered GO BP terms enriched in the candidate MPS-interacting proteins). The corresponding p -values (bars) and protein numbers (next to the bars) for each enriched GO BP term are shown on the right. d Venn diagrams showing the overlap between the enriched GO BP terms of the 480 candidate MPS-interacting proteins identified in cultured mouse hippocampal neurons (DIV 20) and the enriched GO BP terms of the 670 candidate MPS-interacting proteins identified in the adult mouse whole brain (left). Also shown in comparison is the overlap between the enriched GO BP terms of the 480 candidate MPS-interacting proteins in cultured mouse hippocampal neurons (DIV 20) and the enriched GO BP terms of 670 randomly selected genes from the mouse genome (right). e Same as d but for GO MF terms instead of GO BP terms.

Article Snippet: The following primary antibodies were used in this study: guinea pig anti-MAP2 antibody 1:500 dilution for immunofluorescence (IF) (Synaptic Systems, 188004), rabbit anti-MAP2 antibody 1:500 for IF (Synaptic Systems, 188002), mouse anti-αII spectrin antibody 1:400 for IF (Biolegend, 803201, Clone D8B7), mouse anti-αII spectrin antibody 1:200 for IF (Encor Biotechnology, MCA-3D7, Clone 3D7), rabbit anti-αII spectrin antibody 1:200 for IF (Encor Biotechnology, RPCA-aII-Spec), mouse anti-αII spectrin antibody 1:200 for IF (EMD Millipore, MAB1622, Clone AA6), mouse anti-βII spectrin antibody 1:200 for IF (BD Biosciences, 612563, Clone 42), mouse anti-dematin antibody 1:50 for IF (Santa Cruz Biotechnology, sc-135881, Clone 18), rabbit anti-coronin 2B antibody 1:200 for IF (Novus Biologicals, NBP 1-85567), mouse anti-tubulin antibody 1:100 for IF (Santa Cruz Biotechnology, sc-5286, Clone B7), rabbit anti-Tau antibody 1:500 for IF (Synaptic Systems, 314002), mouse anti-K v 1.2 channel antibody 1:200 for IF (Neuromab, 75-008, Clone K14/16), rabbit anti-neurofascin antibody 1:200 for IF (Neuromab, 75–172, Clone A12/18), rabbit anti-NrCAM 1:200 for IF (Abcam, ab24344), goat anti-CHL1 antibody 1:200 for IF (R&D systems, AF2147), rabbit anti-NCAM1 antibody 1:200 for IF (EMD Millipore, AB5032), mouse anti-ankyrin G antibody 1:100 for IF (Santa Cruz Biotechnology, sc-12719, Clone 463), mouse anti-bassoon antibody 1:400 for IF (Enzo, ADI-VAM-PS003-F, Clone SAP7F407), rabbit anti-homer antibody 1:500 for IF (Synaptic Systems, 160003), rabbit anti-L1CAM antibody 1:500 for Western blot (WB) (ABclonal, A8555), rat anti-L1CAM antibody 1:200 for IF (R&D Systems, MAB5674, Clone 555), rabbit anti-NMIIB (Myh10) (N-terminus) antibody 1:200 for IF (GeneTex, GTX133378), rabbit anti-NMIIA (Myh9) (N-terminus) antibody 1:200 for IF (GeneTex, GTX101751), rabbit anti-NMIIB (Myh10) (C-terminus) antibody 1:200 for IF (Biolegend, 909901), rabbit anti-Glutamate Receptor 2 & 3 antibody 1:200 for IF (EMD Millipore, AB1506), rabbit anti-GFP antibody 1:400 for IF (Thermo Fisher Scientific, A11122). rabbit anti-β-actin antibody 1:1000 for WB (Proteintech, 20536-1-AP).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Mass Spectrometry, Cell Culture, Functional Assay, Comparison

a – e Left: 3D STORM images of αII-spectrin ( a ), tropomodulin 1 ( b ), tropomodulin 2 ( c ), dematin ( d ), and coronin 2B ( e ) in axonal regions of cultured hippocampal neurons. Middle: One-dimensional (1D) autocorrelation of the imaged molecules for the axon region indicated by the dashed line in the left panels. Signals were projected to the longitudinal axis of the axon segment, and 1D autocorrelation was calculated using the projected signals. Right: Average 1D autocorrelation of the imaged molecules over 20–80 randomly selected axon regions. Scale bars: 1 μm. Colored scale bar indicates the z-coordinates. f Average 1D autocorrelation amplitudes of the βII-spectrin distribution, indicating the degree of periodicity of the MPS, of untreated neurons, neurons transfected with adenoviruses expressing αII-spectrin shRNA, α-adducin shRNA, ankyrin B shRNA, or coronin 2B shRNA, and neurons cultured from tropomodulin 1, tropomodulin 2 or dematin knockout mice. Data are mean ± s.e.m. ( n = 3 biological replicates for each condition; 40–80 axonal regions were examined for each condition). ** p < 0.005 (two-sided unpaired Student’s t-test); p-value s (from left to right): 6.0 × 10 −4 , 4.9 × 10 −3 , 4.0 × 10 −3 and 1.1 × 10 −3 . g Domain organization of βII-spectrin. h – j Left: 3D STORM image of axon regions of βII-spectrin knockout neurons transfected with plasmid expressing GFP-tagged full length βII-spectrin ( h ), βII-spectrin-ΔCH mutant ( i ) or βII-spectrin-ΔPH mutant ( j ). The proteins were visualized by immunostaining with anti-GFP antibody. Scale bars: 1 µm. Right: average 1D autocorrelation of the imaged proteins. k Average 1D auto-correlation amplitudes for the distributions of GFP-tagged full length βII-spectrin and two GFP-tagged βII-spectrin truncation mutants, as described in ( h – j ), calculated from GFP-positive axon segments. Data are mean ± s.e.m. ( n = 3 biological replicates for each condition; 30–70 axonal regions were examined for each condition). ** p < 0.005 (two-sided unpaired Student’s t-test); p -values (from left to right): 1.3 × 10 −3 and 1.4 × 10 −4 . STORM images in a – e and h – j are representative examples from three independent experiments with similar results. Source data are provided in the Source Data file.

Journal: Nature Communications

Article Title: Proteomic and functional analyses of the periodic membrane skeleton in neurons

doi: 10.1038/s41467-022-30720-x

Figure Lengend Snippet: a – e Left: 3D STORM images of αII-spectrin ( a ), tropomodulin 1 ( b ), tropomodulin 2 ( c ), dematin ( d ), and coronin 2B ( e ) in axonal regions of cultured hippocampal neurons. Middle: One-dimensional (1D) autocorrelation of the imaged molecules for the axon region indicated by the dashed line in the left panels. Signals were projected to the longitudinal axis of the axon segment, and 1D autocorrelation was calculated using the projected signals. Right: Average 1D autocorrelation of the imaged molecules over 20–80 randomly selected axon regions. Scale bars: 1 μm. Colored scale bar indicates the z-coordinates. f Average 1D autocorrelation amplitudes of the βII-spectrin distribution, indicating the degree of periodicity of the MPS, of untreated neurons, neurons transfected with adenoviruses expressing αII-spectrin shRNA, α-adducin shRNA, ankyrin B shRNA, or coronin 2B shRNA, and neurons cultured from tropomodulin 1, tropomodulin 2 or dematin knockout mice. Data are mean ± s.e.m. ( n = 3 biological replicates for each condition; 40–80 axonal regions were examined for each condition). ** p < 0.005 (two-sided unpaired Student’s t-test); p-value s (from left to right): 6.0 × 10 −4 , 4.9 × 10 −3 , 4.0 × 10 −3 and 1.1 × 10 −3 . g Domain organization of βII-spectrin. h – j Left: 3D STORM image of axon regions of βII-spectrin knockout neurons transfected with plasmid expressing GFP-tagged full length βII-spectrin ( h ), βII-spectrin-ΔCH mutant ( i ) or βII-spectrin-ΔPH mutant ( j ). The proteins were visualized by immunostaining with anti-GFP antibody. Scale bars: 1 µm. Right: average 1D autocorrelation of the imaged proteins. k Average 1D auto-correlation amplitudes for the distributions of GFP-tagged full length βII-spectrin and two GFP-tagged βII-spectrin truncation mutants, as described in ( h – j ), calculated from GFP-positive axon segments. Data are mean ± s.e.m. ( n = 3 biological replicates for each condition; 30–70 axonal regions were examined for each condition). ** p < 0.005 (two-sided unpaired Student’s t-test); p -values (from left to right): 1.3 × 10 −3 and 1.4 × 10 −4 . STORM images in a – e and h – j are representative examples from three independent experiments with similar results. Source data are provided in the Source Data file.

Techniques: Cell Culture, Transfection, Expressing, shRNA, Knock-Out, Plasmid Preparation, Mutagenesis, Immunostaining

a Antibody-binding epitopes at the C- or N-terminus of a NMII bipolar filament (left), and the antibody-binding epitope at the C-terminus of βII-spectrin located near the center of the spectrin tetramer. b Left: Two-color STORM image of βII-spectrin (C-terminus, green) and NMIIB (N-terminus, magenta) in axons of cultured hippocampal neurons. Right: Average 1D distributions of βII-spectrin (C-terminus, green) and NMIIB (N-terminus, magenta) signals projected to the longitudinal axon axis from many axon segments. Scale bar: 1 μm. c Similar to ( b ) but for the C-terminus of NMIIB. d Average 1D cross-correlation between the distributions of βII-spectrin (C-terminus) and NMIIB (N-terminus) (magenta) and between the distributions of βII-spectrin (C-terminus) and NMIIB (C-terminus) (green) along the axons of cultured neurons, derived from 20–50 axon segments. The average 1D auto-correlation of βII-spectrin distribution (black) is shown as a reference. e Average 1D autocorrelation amplitudes of βII-spectrin distribution (left) and average periodic spacing of the MPS (right), for axons of untreated neurons, Blebbistatin (Bleb)-treated neurons, and neurons transfected with adenoviruses expressing shRNAs against NMIIA, NMIIB, or both NMIIA and NMIIB heavy chains. f Average diameter of axons (measured by βII spectrin immunostainining) for untreated neurons, Bleb-treated neurons, and neurons treated with shRNAs against NMIIA, NMIIB, or both NMIIA and NMIIB heavy chains. * p < 0.05 (two-sided unpaired student’s t-test); p-value s (from left to right): 4.7 × 10 −2 and 4.7 × 10 −2 . g Average diameter of axons (measured by CTB staining) for neurons treated with control (scramble) shRNA, neurons treated with control shRNA and Bleb, neurons treated with βII-spectrin shRNA, and neurons treated with βII-spectrin shRNA and Bleb. * p < 0.05 (two-sided unpaired student’s t-test); p-value s (from left to right): 3.2 × 10 −2 , 1.0 × 10 −2 and 3.3 × 10 −2 . Data are mean ± s.e.m ( n = 3 biological replicates; 50–130 axonal regions per condition). STORM images in b – c are representative examples from three independent experiments with similar results. Source data are provided in the Source Data file.

Journal: Nature Communications

Article Title: Proteomic and functional analyses of the periodic membrane skeleton in neurons

doi: 10.1038/s41467-022-30720-x

Figure Lengend Snippet: a Antibody-binding epitopes at the C- or N-terminus of a NMII bipolar filament (left), and the antibody-binding epitope at the C-terminus of βII-spectrin located near the center of the spectrin tetramer. b Left: Two-color STORM image of βII-spectrin (C-terminus, green) and NMIIB (N-terminus, magenta) in axons of cultured hippocampal neurons. Right: Average 1D distributions of βII-spectrin (C-terminus, green) and NMIIB (N-terminus, magenta) signals projected to the longitudinal axon axis from many axon segments. Scale bar: 1 μm. c Similar to ( b ) but for the C-terminus of NMIIB. d Average 1D cross-correlation between the distributions of βII-spectrin (C-terminus) and NMIIB (N-terminus) (magenta) and between the distributions of βII-spectrin (C-terminus) and NMIIB (C-terminus) (green) along the axons of cultured neurons, derived from 20–50 axon segments. The average 1D auto-correlation of βII-spectrin distribution (black) is shown as a reference. e Average 1D autocorrelation amplitudes of βII-spectrin distribution (left) and average periodic spacing of the MPS (right), for axons of untreated neurons, Blebbistatin (Bleb)-treated neurons, and neurons transfected with adenoviruses expressing shRNAs against NMIIA, NMIIB, or both NMIIA and NMIIB heavy chains. f Average diameter of axons (measured by βII spectrin immunostainining) for untreated neurons, Bleb-treated neurons, and neurons treated with shRNAs against NMIIA, NMIIB, or both NMIIA and NMIIB heavy chains. * p < 0.05 (two-sided unpaired student’s t-test); p-value s (from left to right): 4.7 × 10 −2 and 4.7 × 10 −2 . g Average diameter of axons (measured by CTB staining) for neurons treated with control (scramble) shRNA, neurons treated with control shRNA and Bleb, neurons treated with βII-spectrin shRNA, and neurons treated with βII-spectrin shRNA and Bleb. * p < 0.05 (two-sided unpaired student’s t-test); p-value s (from left to right): 3.2 × 10 −2 , 1.0 × 10 −2 and 3.3 × 10 −2 . Data are mean ± s.e.m ( n = 3 biological replicates; 50–130 axonal regions per condition). STORM images in b – c are representative examples from three independent experiments with similar results. Source data are provided in the Source Data file.

Techniques: Binding Assay, Cell Culture, Derivative Assay, Transfection, Expressing, Staining, shRNA

$a Top panels: Conventional fluorescence images of tubulin (green) and dendrite marker MAP2 (magenta) for neurons transfected with adenoviruses expressing scrambled (control) shRNA, βII-spectrin shRNA, ankyrin B shRNA, L1CAM shRNA, NCAM1 shRNA, CHL1 shRNA, or shRNAs against all three adhesion molecules (L1CAM, NCAM1 and CHL1). The tubulin-positive neurites lacking MAP2 signal are axons. Bottom panel: Average diameter of axon bundles (or single axons) quantified for the conditions described in the top panels. The last bar and dashed line represent the average single-axon diameter obtained from images of sparsely cultured neurons. * indicates p < 0.05 (two-sided unpaired student’s t-test); p-value s (from left to right): 3.9 × 10 −2 , 1.3 × 10 −2 , 1.7 × 10 −2 , 3.7 × 10 −2 , 1.8 × 10 −2 and 9.3 × 10 −3 . b Top: Conventional fluorescence images of cultured neurons immunostained for axon marker Tau (green) and dendrite marker MAP2 (magenta) under the conditions described in ( a ). Bottom: Average fraction of the total length of dendrites that are bundled with axons, quantified for the conditions indicated in the top panel. * p < 0.05 (two-sided unpaired student’s t-test); p-value s (from left to right): 1.7 × 10 −2 , 1.8 × 10 −2 , 4.9 × 10 −2 , 2.4 × 10 −2 and 1.5 × 10 −2 . c Top: Conventional fluorescence images of cultured neurons immunostained for the presynaptic marker Bassoon (green) and postsynaptic marker Homer1 (magenta) under the conditions described in ( a ). Bottom: Average synapse density per unit area on dendrites, quantified for the conditions as described in the top panel. Only puncta showing both presynaptic and postsynaptic marker signals were counted as synapses. * p < 0.05 and ** p < 0.005 (two-sided unpaired student’s t-test); p-value s (from left to right): 3.8 × 10 −2 , 6.4 × 10 −3 , 9.6 × 10 −3 and 3.3 × 10 −3 . Scale bars: 5 μm. Data are mean ± s.e.m ( n = 3 biological replicates; 15–25 imaged regions per condition). Images in a – c are representative examples from three independent experiments with similar results. Source data are provided in the Source Data file.$

Journal: Nature Communications

Article Title: Proteomic and functional analyses of the periodic membrane skeleton in neurons

doi: 10.1038/s41467-022-30720-x

Figure Lengend Snippet: a Top panels: Conventional fluorescence images of tubulin (green) and dendrite marker MAP2 (magenta) for neurons transfected with adenoviruses expressing scrambled (control) shRNA, βII-spectrin shRNA, ankyrin B shRNA, L1CAM shRNA, NCAM1 shRNA, CHL1 shRNA, or shRNAs against all three adhesion molecules (L1CAM, NCAM1 and CHL1). The tubulin-positive neurites lacking MAP2 signal are axons. Bottom panel: Average diameter of axon bundles (or single axons) quantified for the conditions described in the top panels. The last bar and dashed line represent the average single-axon diameter obtained from images of sparsely cultured neurons. * indicates p < 0.05 (two-sided unpaired student’s t-test); p-value s (from left to right): 3.9 × 10 −2 , 1.3 × 10 −2 , 1.7 × 10 −2 , 3.7 × 10 −2 , 1.8 × 10 −2 and 9.3 × 10 −3 . b Top: Conventional fluorescence images of cultured neurons immunostained for axon marker Tau (green) and dendrite marker MAP2 (magenta) under the conditions described in ( a ). Bottom: Average fraction of the total length of dendrites that are bundled with axons, quantified for the conditions indicated in the top panel. * p < 0.05 (two-sided unpaired student’s t-test); p-value s (from left to right): 1.7 × 10 −2 , 1.8 × 10 −2 , 4.9 × 10 −2 , 2.4 × 10 −2 and 1.5 × 10 −2 . c Top: Conventional fluorescence images of cultured neurons immunostained for the presynaptic marker Bassoon (green) and postsynaptic marker Homer1 (magenta) under the conditions described in ( a ). Bottom: Average synapse density per unit area on dendrites, quantified for the conditions as described in the top panel. Only puncta showing both presynaptic and postsynaptic marker signals were counted as synapses. * p < 0.05 and ** p < 0.005 (two-sided unpaired student’s t-test); p-value s (from left to right): 3.8 × 10 −2 , 6.4 × 10 −3 , 9.6 × 10 −3 and 3.3 × 10 −3 . Scale bars: 5 μm. Data are mean ± s.e.m ( n = 3 biological replicates; 15–25 imaged regions per condition). Images in a – c are representative examples from three independent experiments with similar results. Source data are provided in the Source Data file.

Techniques: Fluorescence, Marker, Transfection, Expressing, shRNA, Cell Culture

$In neurites, actin filaments form ring-like structures that are connected by spectrin tetramers, and actin filaments in these rings are likely capped by adducin and tropomodulin at their fast- and slow-growing ends, respectively. The length and structure of the actin filaments within the actin rings remains an open question and a recent electron microscopy study suggested the possibility that actin rings in the AIS are made of long, intertwined actin filaments . Dematin and coronin bind to the actin filaments, and ankyrin B and ankyrin G bind to the site near the center of each spectrin tetramer. The NMII bipolar filaments bind preferentially to actin filaments within the same actin rings and may regulate the diameter of neurites by exerting radial contractile forces. It is possible that a smaller fraction of NMII bipolar filaments may connect adjacent actin rings. Membrane proteins, such as ion channels, cell adhesion molecules, receptors and signaling molecules, are associated either with the actin filaments through adaptor proteins such as dematin, or with the center positions of spectrin tetramers through adaptor proteins such as ankyrin. Recruitment of cell adhesion molecules to periodic sites on the MPS and interactions of cell adhesion molecules between two abutting neurites bring their respective MPS structures in phase, which could in turn bring additional MPS-bound cell adhesion molecules from the two neurites into proximity, enhancing neurite-neurite interactions. Moreover, the MPS could serve as a structural platform that organizes transmembrane proteins and membrane-associated signaling molecules, potentially facilitating a variety of signaling pathways in neurons.$

Journal: Nature Communications

Article Title: Proteomic and functional analyses of the periodic membrane skeleton in neurons

doi: 10.1038/s41467-022-30720-x

Figure Lengend Snippet: In neurites, actin filaments form ring-like structures that are connected by spectrin tetramers, and actin filaments in these rings are likely capped by adducin and tropomodulin at their fast- and slow-growing ends, respectively. The length and structure of the actin filaments within the actin rings remains an open question and a recent electron microscopy study suggested the possibility that actin rings in the AIS are made of long, intertwined actin filaments . Dematin and coronin bind to the actin filaments, and ankyrin B and ankyrin G bind to the site near the center of each spectrin tetramer. The NMII bipolar filaments bind preferentially to actin filaments within the same actin rings and may regulate the diameter of neurites by exerting radial contractile forces. It is possible that a smaller fraction of NMII bipolar filaments may connect adjacent actin rings. Membrane proteins, such as ion channels, cell adhesion molecules, receptors and signaling molecules, are associated either with the actin filaments through adaptor proteins such as dematin, or with the center positions of spectrin tetramers through adaptor proteins such as ankyrin. Recruitment of cell adhesion molecules to periodic sites on the MPS and interactions of cell adhesion molecules between two abutting neurites bring their respective MPS structures in phase, which could in turn bring additional MPS-bound cell adhesion molecules from the two neurites into proximity, enhancing neurite-neurite interactions. Moreover, the MPS could serve as a structural platform that organizes transmembrane proteins and membrane-associated signaling molecules, potentially facilitating a variety of signaling pathways in neurons.

Techniques: Electron Microscopy, Membrane

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