Journal: Molecular & Cellular Proteomics : MCP

Article Title: Application of Mass Spectrometry Profiling to Establish Brusatol as an Inhibitor of Global Protein Synthesis

doi: 10.1074/mcp.M115.055509

Figure Lengend Snippet: Effects of brusatol on total protein synthesis. (A) Scatterplot showing the melting temperatures of all proteins detected in both lysates and intact cells under the DMSO condition. The solid line shows the best fit with an R2 concordance of 0.43, while the red dashed line indicates a hypothetical unity response, to demonstrate that most proteins are more thermostable when heated in lysis buffer compared with intact cells. (B) The ratios of protein abundance of 3499 proteins at the 38 °C temperature comparing brusatol-treated to DMSO-treated A549 cells. Protein abundance was quantified from summed TMT reporter ion intensities of peptide-spectrum-matchings. The scatter plot shows data from two replicate measurements indicating that the majority of proteins show reduced abundance following brusatol treatment. cystatin C (red), IGFBP4 (blue), PAF (purple), and NRCAM (brown) are indicated. (C) MS abundance data for cystatin C, IGFBP4, and PAF over the temperature ranges used are shown. X axis: temperature points to heat intact cells in the CETSA assay. Y axis: protein abundance at each temperature point normalized to the average of DMSO and brusatol-treated protein abundance at 38 °C. Green: DMSO treatment; red: brusatol treatment. (D) The same lysates used for MS analysis were used for Western blotting using antibodies specific for cystatin C, IGFBP4, and PAF.

Article Snippet: RNA was harvested via RNEasy Mini kits (Qiagen), cDNA synthesized with High Capacity cDNA reverse transcription kit (Life Technologies, Foster City, CA), and Taqman assays run using the following primer/probe sets (Life Technologies): Nrf2 (Hs00975961_g1), cystatin C (Hs00264679_m1), PAF1 (Hs00219496_m1), NRCAM (Hs01031598_m1), GCN1L (Hs00412445_m1), RPL11 (Hs00831112_s1), and RL22 (Hs01865331_s1).

Techniques: Lysis, Western Blot

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Application of Mass Spectrometry Profiling to Establish Brusatol as an Inhibitor of Global Protein Synthesis

doi: 10.1074/mcp.M115.055509

Figure Lengend Snippet: Brusatol-induced effects on protein abundance are independent of changes in mRNA. (A) A549 cells were treated with 0.1% DMSO, 50 nm or 500 nm brusatol or 5 or 50 μg/ml cycloheximide for the indicated times before harvesting the cells. Lysates were separated by SDS-PAGE and Western blotted using the indicated antibodies. (B) mRNA levels of Nrf2, cystatin C, PAF, NRCAM, GCN1L, RL22, and RL11 were analyzed at the 30 min and 4 h time points of brusatol and cycloheximide treated cells compared with no treatment control. NTC = nontargeted control siRNA. Error bars show standard deviation (n = 2). (C) Protein abundance change quantified after 4-h brusatol treatment or 6-h cycloheximide treatment (data from (24)). To filter out noise and look at more profound changes, a minimum protein level change of 0.6 (2σ) was required for the brusatol dataset. Proteins showing greater than 3σ changes were highlighted. Pearson correlation was calculated. (D) A549 cells were treated with 0.1% DMSO, 50 nm or 500 nm brusatol or 5 or 50 μg/ml cycloheximide for the indicated times before harvesting the cells. Lysates were separated by SDS-PAGE and Western blotted using the indicated antibodies.

Article Snippet: RNA was harvested via RNEasy Mini kits (Qiagen), cDNA synthesized with High Capacity cDNA reverse transcription kit (Life Technologies, Foster City, CA), and Taqman assays run using the following primer/probe sets (Life Technologies): Nrf2 (Hs00975961_g1), cystatin C (Hs00264679_m1), PAF1 (Hs00219496_m1), NRCAM (Hs01031598_m1), GCN1L (Hs00412445_m1), RPL11 (Hs00831112_s1), and RL22 (Hs01865331_s1).

Techniques: SDS Page, Western Blot, Standard Deviation

Journal: bioRxiv

Article Title: Semaphorin3F Drives Dendritic Spine Pruning through Rho-GTPase Signaling

doi: 10.1101/2021.03.05.433425

Figure Lengend Snippet: Structural Interactions of NrCAM in the Sema3F Holoreceptor Complex. A. Domain structure of the Sema3F holoreceptor complex comprising NrCAM (Ig1-6 and fibronectin III repeats), Npn2 (a1, a2, b1, b2 and MAM) domains, and PlexA3 with intrinsic Rap-GAP activity. B. Structural model for NrCAM Ig1 (blue) binding to Npn2 a1 (pink) Critical residues in the domain are rendered in ball-and-stick. Residue R 120 in NrCAM TAR 120 NER motif is shown interacting with Npn2 E56. Residues D82, D84, and K85 in NrCAM Ig1 represent the “DDK” network, which is predicted to interact with R31 in the Npn2 a1 domain. The residue numbering represents the mouse sequences. C. Co-immunoprecipitation of WT NrCAM and NrCAM mutants Asp 82 Arg, Asp 84 Ala, and Lys 85 Glu with WT Npn2 from transfected HEK293T cells (equal amounts of extract protein), shown by immunoprecipitation (IP) of NrCAM and immunoblotting for Npn2 (IB). Blots were reprobed for NrCAM (lower panels). Inputs show equivalent levels of Npn2 and NrCAM in cell extracts. The blots shown are representative of 3 experiments. D. Co-immunoprecipitation of WT NrCAM with either WT Npn2 or Npn2 mutant Arg 31 Glu from transfected HEK293T cells (equal amounts extract protein). Blots were reprobed for NrCAM (lower panels). Equivalent levels of NrCAM and Npn2 were detected in input blots. The blots shown are representative of 3 experiments. E. Representative images showing NrCAM null neurons transfected with NrCAM Lys 85 Glu mutant or WT NrCAM plasmids in pCAGG-IRES-EGFP, treated with Fc or Sema3F-Fc for 30 min, immunostained for EGFP, and apical dendrites imaged confocally. Scale bar = 2µm. F. Quantification of mean spine density ± SEM on apical dendrites (n > 500 spines from ≥ 11 images per condition). Each point represents the mean spine density per image. 2-way ANOVA with Tukey post-hoc pairwise comparisons showed statistical significance (Control Fc vs. Sema3F-Fc, p = 0.021; Control Sema3F-Fc vs. K91E Fc, p = 0.008; Control Sema3F-Fc vs. K91E Sema3F-Fc, p = 0.004). G. NrCAM immunofluorescence staining (red) of unpermeabilized cortical neurons in cultures from NrCAM null mice transfected with WT NrCAM or NrCAM Lys 85 Glu plasmids. Plasmids included EGFP reporter to allow visualization of spines. Scale bar = 2µm. H. HEK293T cells were transfected with Npn2 and PlexA3 with or without NrCAM and cell lysates were assayed using co-immunoprecipitation (IP) of Npn2. Immunoblotting (IB) showed greater levels of PlexA3 in Npn2 IPs in the presence of WT NrCAM. Input blots show equivalent protein levels. The blots and quantification shown are representative of 3 experiments.

Article Snippet: Western blotting was carried out using mouse monoclonal antibody clone M2 directed against the FLAG-tag on Npn2 (SigmaAldrich #F3165) or rabbit anti-NrCAM antibody (R&D Systems AF8538) and Avansta enhanced chemiluminescence.

Techniques: Activity Assay, Binding Assay, Residue, Immunoprecipitation, Transfection, Western Blot, Mutagenesis, Control, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Semaphorin3F Drives Dendritic Spine Pruning through Rho-GTPase Signaling

doi: 10.1101/2021.03.05.433425

Figure Lengend Snippet: Postulated Dual Signaling Pathway for Sema3F-induced Spine Pruning through Rac1 and RhoA. Sema3F induced signaling begins with binding between the NrCAM-Npn2-PlexA3 holoreceptor and ligand Sema3F. Sema3F induces clustering and conformational changes that initiates the RapGAP activity of the intracellular portion of PlexA3, leading to the inactivation of Rap1. It is speculated that a Rap-dependent RhoGAP is inactivated allowing RhoA-GTP to be active. GTP-bound RhoA binds ROCK1/2 which then activates Myosin II by phosphorylating the myosin light chain (MLC). Phosphorylated MLC II induces actomyosin contraction which may create tension that leads to actin depolymerization. To provide the framework necessary for contractile force, Tiam1 is recruited to exchange GDP for GTP on Rac1. Rac1 then activates PAK1-3. PAK can then phosphorylate LIMK1/2, which phosphorylates Cofilin1, inactivating it. Cofilin inactivation leads to elongation of actin filaments providing the framework for tension generation.

Article Snippet: Western blotting was carried out using mouse monoclonal antibody clone M2 directed against the FLAG-tag on Npn2 (SigmaAldrich #F3165) or rabbit anti-NrCAM antibody (R&D Systems AF8538) and Avansta enhanced chemiluminescence.

Techniques: Binding Assay, Activity Assay

Journal: Science Advances

Article Title: Secretomics reveals gelatinase substrates at the blood-brain barrier that are implicated in astroglial barrier function

doi: 10.1126/sciadv.adg0686

Figure Lengend Snippet: ( A ) Immunofluorescence staining for MMP substrates, VCAM-1, NrCAM, agrin, NOTCH3, together with GFAP to mark astrocytes and DAPI; boxed areas are shown to the right at higher magnifications. Scale bars, 100 μm. ( B ) Silver-stained gels showing cleavage products of gelatinase substrates after overnight incubation without (0) or with 1:10 or 1:100 ratios of MMP-9:substrate or ADAM10:substrate. Arrows mark the positions of ADAM10 in samples. Asterisks mark specific cleavage products. Data are representative of two to three experiments.

Article Snippet: To check the ability of recombinant mouse MMP-9 and MMP-2 (R&D Systems) or recombinant mouse ADAM10 (R&D Systems) to in vitro cleave targets identified in the secretome analyses, mouse recombinant VCAM-1 (His-Tag) (Biozol), N-cadherin Fc-chimera (R&D Systems), cadherin-4 (R&D Systems), cadherin-11 Fc-chimera (R&D Systems), mouse recombinant NrCAM (R&D Systems), and recombinant rat agrin (R&D Systems) were diluted in 50 mM tris-HCl (pH 7.4), 200 mM NaCl, 5 mM CaCl 2 , 1 mM APMA, and 0.05% Brij35 to a final concentration of 40 μg/ml, and MMP-9 or MMP-2 was added to a final concentration of 4 μg/ml (10:1 ratio) or 400 ng/ml (100:1 ratio).

Techniques: Immunofluorescence, Staining, Incubation

Journal: Science Advances

Article Title: Secretomics reveals gelatinase substrates at the blood-brain barrier that are implicated in astroglial barrier function

doi: 10.1126/sciadv.adg0686

Figure Lengend Snippet: WT brain sections were immunofluorescently stained for ( A ) GFAP to mark astrocytes, anti–laminin-γ1 chain antibody to mark BMs and perivascular cuffs, and NrCAM; DAPI marks all nuclei; scale bars, 100 μm; areas marked by the dotted lines are shown at higher magnifications in boxed areas. ( B ) Immunofluorescence staining for VCAM-1 and pan-laminin (Pan LM) in naïve and in early- and late-stage EAE brains; arrow marks VCAM-1 in CNS parenchyma at late-stage EAE; DAPI marks nuclei; scale bars, 100 μm (naïve) and 50 μm (early/late cuff). ( C and D ) Triple immunofluorescence staining for CD45, Pan LM, and VCAM-1 shows up-regulation of VCAM-1 around inflammatory cuffs, and its loss at this site where CD45 infiltration occurs (arrows), which correlates with sites of gelatinase activity as shown by in situ zymography (C, bottom) performed on consecutive sections; images to the far right in (C) show in situ hybridizations performed in the presence of the MMP inhibitor, 1,10-phenantrolin. Boxed area in (D) is shown at higher magnification in bottom panels; yellow asterisks mark vessel lumen. Scale bars, 100 μm (C) and 50 μm (D).

Article Snippet: To check the ability of recombinant mouse MMP-9 and MMP-2 (R&D Systems) or recombinant mouse ADAM10 (R&D Systems) to in vitro cleave targets identified in the secretome analyses, mouse recombinant VCAM-1 (His-Tag) (Biozol), N-cadherin Fc-chimera (R&D Systems), cadherin-4 (R&D Systems), cadherin-11 Fc-chimera (R&D Systems), mouse recombinant NrCAM (R&D Systems), and recombinant rat agrin (R&D Systems) were diluted in 50 mM tris-HCl (pH 7.4), 200 mM NaCl, 5 mM CaCl 2 , 1 mM APMA, and 0.05% Brij35 to a final concentration of 40 μg/ml, and MMP-9 or MMP-2 was added to a final concentration of 4 μg/ml (10:1 ratio) or 400 ng/ml (100:1 ratio).

Techniques: Staining, Immunofluorescence, Activity Assay, In Situ, Zymography

Journal: Science Advances

Article Title: Secretomics reveals gelatinase substrates at the blood-brain barrier that are implicated in astroglial barrier function

doi: 10.1126/sciadv.adg0686

Figure Lengend Snippet: ( A ) Immunofluorescence staining of WT and DKO astrocyte-neuronal cocultures for vGlut to mark excitatory synapses and vGAT to mark inhibitory synapses, plus GFAP to mark astrocytes. Wavelet transformations of synapse stainings are shown in bottom panels; scale bars, 20 μm. ( B ) Corresponding statistical analysis of four experiments with separate culture preparations; data are expressed as relative frequency of GABAergic compared to glutamatergic synapses. Data are means ± SD with two replicates and seven to eight regions analyzed per experiment with each region comprising around 500 to 1000 synapses. Statistical analysis was Student’s t test; * P < 0.05. ( C ) Immunofluorescence staining of NrCAM, GFAP, and either MAP2 to mark neurons, vGlut, or vGAT in WT and DKO astrocyte-neuronal cocultures; scale bars, 10 μm.

Article Snippet: To check the ability of recombinant mouse MMP-9 and MMP-2 (R&D Systems) or recombinant mouse ADAM10 (R&D Systems) to in vitro cleave targets identified in the secretome analyses, mouse recombinant VCAM-1 (His-Tag) (Biozol), N-cadherin Fc-chimera (R&D Systems), cadherin-4 (R&D Systems), cadherin-11 Fc-chimera (R&D Systems), mouse recombinant NrCAM (R&D Systems), and recombinant rat agrin (R&D Systems) were diluted in 50 mM tris-HCl (pH 7.4), 200 mM NaCl, 5 mM CaCl 2 , 1 mM APMA, and 0.05% Brij35 to a final concentration of 40 μg/ml, and MMP-9 or MMP-2 was added to a final concentration of 4 μg/ml (10:1 ratio) or 400 ng/ml (100:1 ratio).

Techniques: Immunofluorescence, Staining

Journal: Science Advances

Article Title: Secretomics reveals gelatinase substrates at the blood-brain barrier that are implicated in astroglial barrier function

doi: 10.1126/sciadv.adg0686

Figure Lengend Snippet: ( A ) Representative gelatin gel zymography of CSF samples from patients with multiple sclerosis (MS1 to MS6) and age- and sex-matched somatoform controls (details in table S1). NGAL is neutrophil gelatinase–associated lipocalin. ( B ) ELISA for total MMP-9 in relapsing-remitting multiple sclerosis (RRMS) ( n = 15) and somatoform ( n = 15) CSF samples; statistical analyses were Mann-Whitney, ** P < 0.005. The same CSF samples were tested in ( C ) ELISA for soluble VCAM-1 (sVCAM-1); data are expressed as change relative to somatoform controls. AU is arbitrary units. ( D ) Western blot for soluble NrCAM (sNrCAM); bar graph shows quantification of band intensities. Statistical analyses were Student’s t test (C) and Mann-Whitney (D); ** P < 0.005 and **** P < 0.0001. ( E ) Double immunofluorescence staining for GFAP and VCAM-1 or NrCAM in normal-appearing white matter (NAWM), perivascular infiltrates in NAWM, and demyelinating lesions; scale bars, 100 μm. Arrows mark VCAM-1 expressed at the perivascular border and in the CNS parenchyma; bottom panels showing an astrocyte-expressing VCAM-1 in the demyelinating lesion at a higher magnification; scale bar, 25 μm.

Article Snippet: To check the ability of recombinant mouse MMP-9 and MMP-2 (R&D Systems) or recombinant mouse ADAM10 (R&D Systems) to in vitro cleave targets identified in the secretome analyses, mouse recombinant VCAM-1 (His-Tag) (Biozol), N-cadherin Fc-chimera (R&D Systems), cadherin-4 (R&D Systems), cadherin-11 Fc-chimera (R&D Systems), mouse recombinant NrCAM (R&D Systems), and recombinant rat agrin (R&D Systems) were diluted in 50 mM tris-HCl (pH 7.4), 200 mM NaCl, 5 mM CaCl 2 , 1 mM APMA, and 0.05% Brij35 to a final concentration of 40 μg/ml, and MMP-9 or MMP-2 was added to a final concentration of 4 μg/ml (10:1 ratio) or 400 ng/ml (100:1 ratio).

Techniques: Zymography, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Western Blot, Double Immunofluorescence Staining, Expressing

Journal: bioRxiv

Article Title: Ankyrin-R regulates fast-spiking interneuron excitability through perineuronal nets and Kv3.1b K + channels

doi: 10.1101/2021.01.21.427626

Figure Lengend Snippet: AnkR is a scaffolding protein that binds to and stabilizes PNN-associated CAMs (including NrCAM and PlexinA4) and ion channels (including Kv3.1b) by linking them to the β 1- α 2 spectrin-based cytoskeleton. Loss of AnkR results in 1) altered PNN morphology including reduced WFA intensity and decreased compactness of the nets; 2) molecular changes including reduced β 1 spectrin, PNN-associated NrCAM, and Kv3.1b; 3) behavioral changes including decreased anxiety-like behaviors in the open field and elevated plus maze; and 4) electrophysiological changes including decreased AP latency and threshold, broader APs with shallower and delayed AHP, and decreased firing rate during current injection.

Article Snippet: The primary antibodies used here include: mouse monoclonal antibodies against AnkR (UC Davis/NIH NeuroMab Facility Cat# 75-380, RRID:AB_2491109), β 1 spectrin (UC Davis/NIH NeuroMab Facility Cat# 73-374, RRID:AB_2315814), AnkG (UC Davis/NIH NeuroMab Facility Cat# 73-146, RRID:AB_10697718), parvalbumin (UC Davis/NIH NeuroMab Facility Cat# 73-455, RRID:AB_2629420), actin (Millipore Cat# MAB1501, RRID:AB_2223041), tenascinR (R and D Systems Cat# MAB1624, RRID:AB_2207001), aggrecan (Millipore Cat# AB1031, RRID:AB_90460), brevican (UC Davis/NIH NeuroMab Facility Cat# 75-294, RRID:AB_2315824), NrCAM (R and D Systems Cat# MAB2034, RRID:AB_2267411), Kv3.1b (UC Davis/NIH NeuroMab Facility Cat# N16B/8, RRID:AB_2750730 and Thermo Fisher Cat# MA5-27684, RRID:AB_2735238), Kv3.3 (Antibodies-Online Cat# ABIN572016, RRID:AB_10782137), Kv7.2 (James Trimmer, University of California at Davis Cat# N26A/23, RRID:AB_2750761), Flag-tag or DDDDK-tag (MBL International Cat# M185-3L, RRID:AB_11123930); rabbit polyclonal antibodies against AnkR( ) (RRID:AB_2833096), Ank1 (Thermo Fisher Scientific Cat# PA5-63372, RRID:AB_2638015), neurofilament M (Millipore Cat# AB1987, RRID:AB_91201), parvalbumin (Novus Cat# NB120-11427, RRID:AB_791498), versican (Millipore Cat# AB1032, RRID:AB_11213831), PlexinA4 (Abcam Cat# ab39350, RRID:AB_944890), and neuropilin-1 (GeneTex Cat# GTX16786, RRID:AB_422398), Kv3.1b (Alomone Labs Cat# APC-014, RRID:AB_2040166), Kv3.3 (Alomone Labs Cat# APC-102, RRID:AB_2040170), GFP (Thermo Fisher Scientific, Cat# A-11122, RRID: AB_221569); and chicken polyclonal antibody against Neurofascin (R and D Systems Cat# AF3235, RRID:AB_10890736).

Techniques: Scaffolding, Injection

Journal: Molecular & Cellular Proteomics

Article Title: Application of Mass Spectrometry Profiling to Establish Brusatol as an Inhibitor of Global Protein Synthesis

doi: 10.1074/mcp.m115.055509

Figure Lengend Snippet: FIG. 3. Effects of brusatol on total protein synthesis. (A) Scatterplot showing the melting temperatures of all proteins detected in both lysates and intact cells under the DMSO condition. The solid line shows the best fit with an R2 concordance of 0.43, while the red dashed line indicates a hypothetical unity response, to demonstrate that most proteins are more thermostable when heated in lysis buffer compared with intact cells. (B) The ratios of protein abundance of 3499 proteins at the 38 °C temperature comparing brusatol-treated to DMSO-treated A549 cells. Protein abundance was quantified from summed TMT reporter ion intensities of peptide-spectrum-matchings. The scatter plot shows data from two replicate measurements indicating that the majority of proteins show reduced abundance following brusatol treatment. cystatin C (red), IGFBP4 (blue), PAF (purple), and NRCAM (brown) are indicated. (C) MS abundance data for cystatin C, IGFBP4, and PAF over the temperature ranges used are shown. X axis: temperature points to heat intact cells in the CETSA assay. Y axis: protein abundance at each temperature point normalized to the average of DMSO and brusatol-treated protein abundance at 38 °C. Green: DMSO treatment; red: brusatol treatment. (D) The same lysates used for MS analysis were used for Western blotting using antibodies specific for cystatin C, IGFBP4, and PAF.

Article Snippet: Membranes were blotted with the following primary antibodies: Nrf2 (Abcam ab62352), cystatin-C (Abcam ab133495), IGFBP4 (Thermo PA5–25925), PAF1 (Bethyl A304–374A), NRCAM (ProteinTech 21608–1-AP), MCL-1 (Cell Signaling 5453), p21 (Cell Signaling 2947), Gemin-4 (Abcam ab31581), FAM96A (Acris AP51504PU-N), RL22 (Santa Cruz sc-136413), GCN1L1 (Abcam ab86139), RPL11 (Abcam ab79352), RL9 (Santa Cruz sc-292593), survivin (sc-17779), cyclin A (sc-751), and -actin (Cell Signaling 12620).

Techniques: Lysis, Quantitative Proteomics, Western Blot

Journal: Molecular & Cellular Proteomics

Article Title: Application of Mass Spectrometry Profiling to Establish Brusatol as an Inhibitor of Global Protein Synthesis

doi: 10.1074/mcp.m115.055509

Figure Lengend Snippet: FIG. 5. Brusatol-induced effects on protein abundance are independent of changes in mRNA. (A) A549 cells were treated with 0.1% DMSO, 50 nM or 500 nM brusatol or 5 or 50 g/ml cycloheximide for the indicated times before harvesting the cells. Lysates were separated by SDS-PAGE and Western blotted using the indicated antibodies. (B) mRNA levels of Nrf2, cystatin C, PAF, NRCAM, GCN1L, RL22, and RL11 were analyzed at the 30 min and 4 h time points of brusatol and cycloheximide treated cells compared with no treatment control. NTC nontargeted control siRNA. Error bars show standard deviation (n 2). (C) Protein abundance change quantified after 4-h brusatol treatment or 6-h cycloheximide treatment (data from (24)). To filter out noise and look at more profound changes, a minimum protein level change of 0.6 (2) was required for the brusatol dataset. Proteins showing greater than 3 changes were highlighted. Pearson correlation was calculated. (D) A549 cells were treated with 0.1% DMSO, 50 nM or 500 nM brusatol or 5 or 50 g/ml cycloheximide for the indicated times before harvesting the cells. Lysates were separated by SDS-PAGE and Western blotted using the indicated antibodies.

Article Snippet: Membranes were blotted with the following primary antibodies: Nrf2 (Abcam ab62352), cystatin-C (Abcam ab133495), IGFBP4 (Thermo PA5–25925), PAF1 (Bethyl A304–374A), NRCAM (ProteinTech 21608–1-AP), MCL-1 (Cell Signaling 5453), p21 (Cell Signaling 2947), Gemin-4 (Abcam ab31581), FAM96A (Acris AP51504PU-N), RL22 (Santa Cruz sc-136413), GCN1L1 (Abcam ab86139), RPL11 (Abcam ab79352), RL9 (Santa Cruz sc-292593), survivin (sc-17779), cyclin A (sc-751), and -actin (Cell Signaling 12620).

Techniques: Quantitative Proteomics, SDS Page, Western Blot, Control, Standard Deviation

Journal: The Journal of Cell Biology

Article Title: Ankyrin-G coordinates assembly of the spectrin-based membrane skeleton, voltage-gated sodium channels, and L1 CAMs at Purkinje neuron initial segments

doi: 10.1083/jcb.200109026

Figure Lengend Snippet: Na v 1.6, ankyrin-G, β IV spectrin, neurofascin, and NrCAM are targeted to axon initial segments of cerebellar Purkinje neurons. Sections of adult rat cerebellum were triple labeled with antibodies against calbindin (blue), ankyrin-G (green), and either Na v 1.6 (B), βIV spectrin (E), neurofascin (H), or NrCAM (K) (red). Composite images are shown in C, F, I, and L. Arrowheads indicate Purkinje cell initial segments. Bars, 10 μm.

Article Snippet: Antibodies used include a mouse monoclonal antibody against the ankyrin-G spectrin–binding domain ( ); affinity-purified rabbit polyclonal antibodies against neurofascin , NrCAM ( ) and the peptide CIANHTGVDIHRNGDFQKNG corresponding to residues 1042–1061 of mouse or rat Na v 1.6 (Alomone Labs); a goat polyclonal antibody against calbindin (Santa Cruz Biotech), and a chicken polyclonal antibody against the βIV spectrin unique domain that has been adsorbed against brain lysate from a βIV spectrin knockout mouse (gift of Dr. M. Komada, Tokyo Institute of Technology, Tokyo, Japan).

Techniques: Labeling

Journal: The Journal of Cell Biology

Article Title: Ankyrin-G coordinates assembly of the spectrin-based membrane skeleton, voltage-gated sodium channels, and L1 CAMs at Purkinje neuron initial segments

doi: 10.1083/jcb.200109026

Figure Lengend Snippet: Targeting of β IV spectrin, Na v 1.6, NrCAM, and neurofascin to Purkinje neuron initial segments is disrupted in ankyrin-G cerebellum- specific knockout mice. Cerebellar sections from wild-type (A, D, G, J, and M) or cerebellar ankyrin-G knockout mice (B, C, E, F, H, I, K, L, N, and O) were double labeled with antibodies against calbindin (red in A–F; green in G–L) and either ankyrin-G (green in A–C), βIV spectrin (green in D–F), Na v 1.6 (red in G–I), NrCAM (red in J–L), or neurofascin (M–O). All images except M–O are composites. Bars: (A, B, and E) 5 μm; (C, D, F, G, I, and J–O) 10 μm; and (H) 25 μm.

Article Snippet: Antibodies used include a mouse monoclonal antibody against the ankyrin-G spectrin–binding domain ( ); affinity-purified rabbit polyclonal antibodies against neurofascin , NrCAM ( ) and the peptide CIANHTGVDIHRNGDFQKNG corresponding to residues 1042–1061 of mouse or rat Na v 1.6 (Alomone Labs); a goat polyclonal antibody against calbindin (Santa Cruz Biotech), and a chicken polyclonal antibody against the βIV spectrin unique domain that has been adsorbed against brain lysate from a βIV spectrin knockout mouse (gift of Dr. M. Komada, Tokyo Institute of Technology, Tokyo, Japan).

Techniques: Knock-Out, Labeling