primary antibodies targeting crmp2  (Cell Signaling Technology Inc)

Journal: Frontiers in Molecular Neuroscience

Article Title: Collapsin Response Mediator Protein 2 and Endophilin2 Coordinate Regulation of AMPA Receptor GluA1 Subunit Recycling

doi: 10.3389/fnmol.2020.00128

Figure Lengend Snippet: Collapsin response mediator protein 2 (CRMP2) interacts with endophilin2. (A) Electrostatic surface potential maps of CRMP2 (left) and endophilin2 SH3 domain (right). The blue color represents the positive potential, and red color represents the negative potential. The electrostatic potential energy of the protein surface is calculated by APBS (figures are produced by using PyMOL). (B) The structures of the SH3 domain of endophilin2 (PDB code: 3C0C) and CRMP2 (5X1A) were downloaded from the Protein Data Bank www.rcsb.org . (C) Purified glutathione-S-transferase (GST) and GST-Endo2 proteins were incubated with 1-month-old Sprague–Dawley rat brain lysates. Input and bound proteins were analyzed by immunoblotting, using antibodies against CRMP2. (D) Purified GST and GST-CRMP2 proteins were incubated with 1-month-old Sprague–Dawley rat brain lysates. Input and bound proteins were analyzed by immunoblotting, using antibodies against Endo2. (E,F) HEK293 cells were cotransfected with green fluorescence protein (GFP)-Endo2 and Flag-CRMP2, followed by the co-IP assay. Protein complexes coeluted with the anti-Flag antibody were detected using the anti-GFP antibody (E) and protein complexes coeluted with the anti-GFP antibody were detected using the anti-Flag antibody (F) . All experiments were repeated independently at least three times. (G) Representative image of DIV12 cultured hippocampal neurons stained for endophilin2 and CRMP2. White arrowheads indicate endophilin2 puncta that colocalized with CRMP2. Scale bars of 20 and 5 μm in the magnified dendrite. (H) Intensity trace of the endophilin2 and CRMP2 along the dendrite.

Article Snippet: Primary antibodies targeting CRMP2 (1:100; Cell Signaling Technology, Danvers, MA, USA) and endophilin2 (E-15; 1:100; Santa Cruz Biotechnology, Dallas, TX, USA) were used for immunocytochemistry.

Techniques: Produced, Purification, Incubation, Western Blot, Fluorescence, Co-Immunoprecipitation Assay, Cell Culture, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Collapsin Response Mediator Protein 2 and Endophilin2 Coordinate Regulation of AMPA Receptor GluA1 Subunit Recycling

doi: 10.3389/fnmol.2020.00128

Figure Lengend Snippet: C-terminal residues 303–368 of endophilin2 interact with C-terminal residues 381–562 of CRMP2. (A,B) Schematic diagrams of endophilin2 and CRMP2 truncation mutants are shown according to their different domains. (C) Purification of glutathione-S-transferase (GST) and truncated segments of GST-endophilin2 (GST-Endo2) fusion proteins. Bovine serum albumin (BSA) standard protein: 4, 8, and 12 μg. (D) Purification of GST and truncated segments of GST-CRMP2 fusion proteins. BSA standard protein: 4, 8, and 12 μg. (E) Pull-down assays were conducted with GST-Endo2 and its truncation mutants using 1-month-old Sprague–Dawley rat brain lysates. Western blotting was performed with an anti-CRMP2 antibody. (F) Pull-down assays were conducted with GST-CRMP2 and its truncation mutants using 1-month-old Sprague–Dawley rat brain lysates. Western blotting was performed with an anti-endophilin2 antibody. All experiments were repeated independently at least three times.

Article Snippet: Primary antibodies targeting CRMP2 (1:100; Cell Signaling Technology, Danvers, MA, USA) and endophilin2 (E-15; 1:100; Santa Cruz Biotechnology, Dallas, TX, USA) were used for immunocytochemistry.

Techniques: Purification, Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: Collapsin Response Mediator Protein 2 and Endophilin2 Coordinate Regulation of AMPA Receptor GluA1 Subunit Recycling

doi: 10.3389/fnmol.2020.00128

Figure Lengend Snippet: CRMP2 and endophilin2 coordinate with each other to promote the current level of AMPA receptors (AMPARs). (A) Representative tracings of miniature excitatory synaptic current (mEPSC) recorded from cultured hippocampal neurons transfected with mCherry-Endo2 and GFP, mCherry-Endo2 and GFP-CRMP2, and mCherry-Endo2 and GFP-CRMP2 ΔC381 . (B,C) Cumulative probabilities of the mEPSC amplitude and interevent interval of neurons in these groups. Insets: histogram plots of mEPSC amplitude and frequency of neurons in these groups, n = 15 cells. * p < 0.05, as compared to the mCherry-Endo2 and GFP group; # p < 0.05, as compared to the mCherry-Endo2 and GFP-CRMP2 group. (D) Representative tracings of mEPSC recorded from cultured hippocampal neurons transfected GFP-CRMP2 and mCherry, GFP-CRMP2 and mCherry-Endo2, and GFP-CRMP2 and mCherry-Endo2 ΔC303 . (E,F) Cumulative probabilities of the mEPSC amplitude and interevent interval of neurons in these groups. Insets: Histogram plots of mEPSC amplitude and frequency of neurons in these groups, n = 15 cells. * p < 0.05, as compared to the GFP-CRMP2 and mCherry group, # p < 0.05, as compared to the GFP-CRMP2 and mCherry-Endo2 group.

Article Snippet: Primary antibodies targeting CRMP2 (1:100; Cell Signaling Technology, Danvers, MA, USA) and endophilin2 (E-15; 1:100; Santa Cruz Biotechnology, Dallas, TX, USA) were used for immunocytochemistry.

Techniques: Cell Culture, Transfection

Journal: Frontiers in Molecular Neuroscience

Article Title: Collapsin Response Mediator Protein 2 and Endophilin2 Coordinate Regulation of AMPA Receptor GluA1 Subunit Recycling

doi: 10.3389/fnmol.2020.00128

Figure Lengend Snippet: CRMP2 and endophilin2 coordinate to promote the surface expression of AMPA receptor (AMPAR) GluA1 subunit. (A) Cultured hippocampal neurons transfected with mCherry-Endo2 and GFP, mCherry-Endo2 and GFP-CRMP2, and mCherry-Endo2 and GFP-CRMP2 ΔC381 were immunostained with surface GluA1. Scale bar, 50 and 10 μm in the magnified dendrite. (B) Quantitative analysis of fluorescence intensity of surface GluA1 of neurons in these groups, n = 15 cells per group. * p < 0.05, as compared to the mCherry-Endo2 and GFP group; # p < 0.05, as compared to the mCherry-Endo2 and GFP-CRMP2 group. (C) Cultured hippocampal neurons transfected with GFP-CRMP2 and mCherry, GFP-CRMP2 and mCherry-Endo2, and GFP-CRMP2 and mCherry-Endo2 ΔC303 were immunostained with surface GluA1. Scale bar, 50 and 10 μm in the magnified dendrite. (D) Quantitative analysis of fluorescence intensity of surface GluA1 of neurons in these groups, n = 15 cells per group. * p < 0.05, as compared to the GFP-CRMP2 and mCherry group; # p < 0.05, as compared to the mCherry-Endo2 and GFP-CRMP2 group.

Article Snippet: Primary antibodies targeting CRMP2 (1:100; Cell Signaling Technology, Danvers, MA, USA) and endophilin2 (E-15; 1:100; Santa Cruz Biotechnology, Dallas, TX, USA) were used for immunocytochemistry.

Techniques: Expressing, Cell Culture, Transfection, Fluorescence

Journal: Frontiers in Molecular Neuroscience

Article Title: Collapsin Response Mediator Protein 2 and Endophilin2 Coordinate Regulation of AMPA Receptor GluA1 Subunit Recycling

doi: 10.3389/fnmol.2020.00128

Figure Lengend Snippet: The phosphorylation of CRMP2 decreases its interaction with endophilin2. (A) Schematic of GSK3β and Cdk5 and RhoK phosphorylation sites within the rat CRMP2 sequence. Numbers represent amino acid residues within the CRMP2 sequence. (B) The expression and purification of glutathione-S-transferase (GST), GST-CRMP2, and its phosphomimetic and dephosphomimetic mutants. BSA standard protein: 2, 4, and 8 μg. (C) Pull-down assays were conducted using GST-CRMP2 and its phosphomimetic and dephosphomimetic mutants in brain lysates of 1-month-old Sprague–Dawley rats. Western blotting was performed using an anti-endophilin2 antibody. (D) Pull-down assays were conducted with GST-CRMP2 and its phosphomimetic and dephosphomimetic mutants in brain lysates of 1-month-old Sprague–Dawley rats. Western blotting was performed with an anti-tubulin antibody. Each result is representative of three separate experiments with similar results.

Article Snippet: Primary antibodies targeting CRMP2 (1:100; Cell Signaling Technology, Danvers, MA, USA) and endophilin2 (E-15; 1:100; Santa Cruz Biotechnology, Dallas, TX, USA) were used for immunocytochemistry.

Techniques: Phospho-proteomics, Sequencing, Expressing, Purification, Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: Collapsin Response Mediator Protein 2 and Endophilin2 Coordinate Regulation of AMPA Receptor GluA1 Subunit Recycling

doi: 10.3389/fnmol.2020.00128

Figure Lengend Snippet: CRMP2 phosphorylation reduces the surface expression of GluA1 in hippocampal neurons. (A) Representative tracings of mEPSC recorded from cultured hippocampal neurons transfected with mCherry-Endo2 and GFP, mCherry-Endo2 and GFP-CRMP2, mCherry-Endo2 and GFP-CRMP2 QmA , and mCherry-Endo2 and GFP-CRMP2 QmD . (B,C) Cumulative probabilities of the mEPSC amplitude and interevent interval of neurons in these groups. Insets: histogram plots of mEPSC amplitude and frequency of neurons in these groups, n = 15 cells per group. * p < 0.05, as compared to the mCherry-Endo2 and GFP group, # p < 0.05, as compared to the mCherry-Endo2 and GFP-CRMP2 QmD group. (D) Cultured hippocampal neurons transfected with mCherry-Endo2 and GFP, mCherry-Endo2 and GFP-CRMP2, mCherry-Endo2 and GFP-CRMP2 QmA , and mCherry-Endo2 and GFP-CRMP2 QmD were immunostained with surface GluA1. Scale bar, 50 and 10 μm in the magnified dendrite. (E) Quantitative analysis of fluorescence intensity of surface GluA1 of neurons in these groups, n = 15 cells per group. * p < 0.05, as compared to the mCherry-Endo2 and GFP group, # p < 0.05, as compared to the mCherry-Endo2 and GFP-CRMP2 QmD group.

Article Snippet: Primary antibodies targeting CRMP2 (1:100; Cell Signaling Technology, Danvers, MA, USA) and endophilin2 (E-15; 1:100; Santa Cruz Biotechnology, Dallas, TX, USA) were used for immunocytochemistry.

Techniques: Phospho-proteomics, Expressing, Cell Culture, Transfection, Fluorescence

Journal: Frontiers in Molecular Neuroscience

Article Title: Collapsin Response Mediator Protein 2 and Endophilin2 Coordinate Regulation of AMPA Receptor GluA1 Subunit Recycling

doi: 10.3389/fnmol.2020.00128

Figure Lengend Snippet: The phosphorylation of CRMP2 at Thr514/Ser518 reduces its interaction with endophilin2. (A) The expression and purification of glutathione-S-transferase (GST)-CRMP2 and its phosphorylated and dephosphorylated mutants. (B) Pull-down assays were conducted with GST-CRMP2 and its phosphorylated and dephosphorylated mutants in brain lysates of 1-month-old rats. Western blotting was performed with an anti-endophilin2 antibody (up); quantitative analysis of the relative binding of Endo2 to CRMP2 was performed (down), n = 3, ** p < 0.01, *** p < 0.001 compared to GST-CRMP2 group. (C) HEK293 cells were cotransfected with Flag-Endo2 and GFP, GFP-CRMP2, and its dephosphomimetic and dephosphomimetic mutants, after which the co-IP assay was performed. Protein complexes that were coeluted with the anti-GFP antibody were detected using the anti-Flag antibody (up); quantitative analysis of the relative binding of Endo2 to CRMP2 was performed (down), n = 3, ** p < 0.01 compared to the CRMP2 group. (D) Cultured hippocampal neurons were infected with Ad-HA vector and Ad-HA-GSK3β S9A (constitutively active GSK3β mutant containing a serine-to-alanine substitution at residue 9) for 24 h. The cell lysates were immunoprecipitated with anti-CRMP2 or the control immunoglobulin G (IgG) antibody, followed by the immunoblotting of immunoprecipitated samples with anti-endophilin2 antibody. (E) Quantification of Endo2-immureactive bands after normalization with coprecipitated CRMP2 levels, n = 3, ** p < 0.01.

Article Snippet: Primary antibodies targeting CRMP2 (1:100; Cell Signaling Technology, Danvers, MA, USA) and endophilin2 (E-15; 1:100; Santa Cruz Biotechnology, Dallas, TX, USA) were used for immunocytochemistry.

Techniques: Phospho-proteomics, Expressing, Purification, Western Blot, Binding Assay, Co-Immunoprecipitation Assay, Cell Culture, Infection, Plasmid Preparation, Mutagenesis, Residue, Immunoprecipitation, Control

Journal: Frontiers in Molecular Neuroscience

Article Title: Collapsin Response Mediator Protein 2 and Endophilin2 Coordinate Regulation of AMPA Receptor GluA1 Subunit Recycling

doi: 10.3389/fnmol.2020.00128

Figure Lengend Snippet: The phosphorylation of CRMP2 at Thr514/Ser518 reduces the surface expression and increases degradation of GluA1. (A) Cultured hippocampal neurons transfected with mCherry-Endo2 and GFP, and mCherry-Endo2 and GFP (incubated with CHIR99021), mCherry-Endo2 and GFP-CRMP2, mCherry-Endo2 and GFP-CRMP2 T514A/S518A , and mCherry-Endo2 and GFP-CRMP2 T514D/S518D ; then, they were immunostained with surface GluA1. Scale bar, 50 and 10 μm in the magnified dendrite. (B) Quantitative analysis of fluorescence intensity of surface GluA1 of neurons in these groups, n = 15 cells per group, * p < 0.05, as compared to the mCherry-Endo2 and GFP group; # p < 0.05, as compared to the mCherry-Endo2 and GFP-CRMP2 group. (C,D) Representative images (C) and quantitative analysis (D) show the colocalization of EEA1 and internalized GluA1 in hippocampal neurons. Neurons were coimmunostained at DIV12, using antibodies against GluA1 and EEA1. Scale bar, 10 μm. (E,F) Representative images (E) and quantitative analysis (F) show the colocalization of LAMP1 and internalized GluA1 in hippocampal neurons. Neurons were coimmunostained at DIV12, using antibodies against GluA1 and LAMP1. Scale bar, 10 μm. n = 15 cells per group. * p < 0.05, as compared to the mCherry-Endo2 and GFP group; # p < 0.05, as compared to the mCherry-Endo2 and GFP-CRMP2 group.

Article Snippet: Primary antibodies targeting CRMP2 (1:100; Cell Signaling Technology, Danvers, MA, USA) and endophilin2 (E-15; 1:100; Santa Cruz Biotechnology, Dallas, TX, USA) were used for immunocytochemistry.

Techniques: Phospho-proteomics, Expressing, Cell Culture, Transfection, Incubation, Fluorescence

Journal: Communications Biology

Article Title: GSK3-CRMP2 signaling mediates axonal regeneration induced by Pten knockout

doi: 10.1038/s42003-019-0524-1

Figure Lengend Snippet: PTEN −/− releases CRMP2 from GSK3 inhibition. a Schematic illustration of results shown in experiments of ( b – j ). b , e Western blots of T514-phosphorylated (pCRMP2) and total CRMP2 in optic nerve lysates from PTEN +/+ and PTEN −/− mice without and with Gsk3α S/A (α S/A ), or Gsk3β S/A (β S/A ) knockins. Animals were either left untreated (con, b ) or subjected to ONC ( e ) 5 days before tissue isolation. Optic nerve segments proximal to the lesion site were used for the analysis. βIII-tubulin (tubulin) was used as a loading control. c , d , f , g Densitometric quantification of total CRMP2 and pCRMP2 relative to tubulin and normalized to PTEN +/+ on Western blots as depicted in ( b ) and ( e ). Levels of pCRMP2 were considerably compromised upon PTEN −/− . Decreased levels were reversed in α S/A and β S/A genotypes, while total CRMP2 remained unaltered in all experimental groups. Values represent means ± SEM of three optic nerves ( n = 3) per group. h Retinal flat mounts isolated from animals treated as described in ( b ) and ( e ). Retinae were stained for T 514 -pCRMP2 (red) and βIII-tubulin (tubulin, green). ONC and to a stronger extent PTEN −/− reduced pCRMP2 levels in RGC somas and axons compared with the PTEN +/+ , while levels remained high in PTEN −/− /GSK3 S/A mice. Scale bar: 50 μm. i Retinal flat mounts from PTEN +/+ mice either untreated or 5 days upon ONC that were injected with AAV-HA-CRMP2 T514/A 3 weeks before the injury. The activity of mTOR was analyzed by pS6 levels (red), while HA-staining (HA-CRMP2 T/A , green) identified transduced βIII-tubulin-positive RGCs (tubulin, cyan). Scale bar: 25 µm. j Quantification of pS6-positive RGCs in retinal flat mounts as described in ( i ). Values represent means ± SEM of three retinae per group ( n = 3). Significances of intergroup differences were evaluated using one-way analysis of variance (ANOVA) with Holm-Sidak post hoc test ( c , d , f , g ), or student's t -test ( j ). Treatment effects were compared with PTEN +/+ con: ## p < 0.01, n.s. = non-significant; and PTEN +/+ ONC: *** p < 0.001, n.s. = non-significant. Treatment effect in ( j ): * p < 0.05

Article Snippet: Phospho-GSK3α and CRMP2 primary antibodies were detected using conformation-specific horseradish peroxidase conjugated anti-rabbit (IgG) secondary antibody (1:4000, Cell Signaling Technologies).

Techniques: Inhibition, Western Blot, Isolation, Control, Staining, Injection, Activity Assay

Journal: Communications Biology

Article Title: GSK3-CRMP2 signaling mediates axonal regeneration induced by Pten knockout

doi: 10.1038/s42003-019-0524-1

Figure Lengend Snippet: PTEN −/− stimulated neurite growth is CRMP2 dependent. a Representative pictures of retinal cell cultures plated on PDL. PTEN +/+ and PTEN −/− mice were subjected to ONC to transform RGCs into a regenerative state. Cells were cultured 5 days after that. Cultures were either untreated (−) or incubated with rapamycin (rap; 10 nM), lacosamide (LCM, 5 µM), or a combination of both. After 2 days in culture, RGCs were stained for phosphorylated S6 (pS6, red) and βIII-tubulin (tubulin, green). b Quantification of neurite growth of RGCs as described in ( a ) in the absence (PDL) or presence of CNS myelin (myelin). Some of the cultures were treated with the ROCK inhibitor Y27632 (Y27, 10 µM). Values represent means ± SEM of three ( n = 3) independent experiments with four wells as technical replicate per group and were normalized to the PTEN −/− , PDL group with an average neurite length of 6.12 µm per RGC of in total ~500 RGCs with ~30 RGCs showing neurite growth. c Quantification of the percentage of pS6-positive RGCs in cultures as described in ( a ). Values represent means ± SEM of 4 independent experiments ( n = 4). d ) Quantification of neurite length of βIII-tubulin-positive RGCs from PTEN +/+ , PTEN −/− , or PTEN −/− mice with a Gsk3α S/A (PTEN −/− /α S/A ) or Gsk3β S/A (PTEN −/− /β S/A ) knockin. Animals were subjected to ONC 5 days before culture preparation. Cultures were grown in the in the absence or presence of CNS myelin and either left untreated (−) or incubated with lacosamide (LCM, 5 µM) or Y27632 (Y27, 10 µM) and fixed after 48 h. Values represent means ± SEM of n = 4 independent experiments with four wells as technical replicate per group. Significances of intergroup differences were evaluated using two-way ( b , c ) or three-way ( d ) analysis of variance (ANOVA) with Holm-Sidak post hoc test. Treatment effects: compared with PTEN −/− , PDL: +++ p < 0.001; PTEN −/− , myelin: ### p < 0.001; or as indicated: * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. = non-significant

Article Snippet: Phospho-GSK3α and CRMP2 primary antibodies were detected using conformation-specific horseradish peroxidase conjugated anti-rabbit (IgG) secondary antibody (1:4000, Cell Signaling Technologies).

Techniques: Cell Culture, Incubation, Staining, Knock-In

Journal: Communications Biology

Article Title: GSK3-CRMP2 signaling mediates axonal regeneration induced by Pten knockout

doi: 10.1038/s42003-019-0524-1

Figure Lengend Snippet: CRMP2 T/A overcomes GSK3β S/A -mediated inhibition on nerve regeneration. a Retinal flat-mounts from PTEN −/− mouse 3 weeks after intravitreal injection of AAV-Cre + AAV-HA-CRMP2 T/A , immunohistochemically stained for Cre (red), the HA-tag of CRMP2 T/A (HA-CRMP2 T/A , green), and βIII-tubulin (tubulin, blue). Scale bar: 50 μm. b Representative longitudinal optic nerve sections with regenerating, CTB-labeled axons 3 weeks after ONC. PTEN −/− or PTEN −/− /GSK3β S/A mice had received treatment with either AAV-GFP (GFP) or AAV-HA-CRMP2 T/A (CRMP2 T/A ) 3 weeks before ONC. Asterisks indicate the lesion site. Scale bar: 200 µm. c – f Magnifications of optic nerve sections as indicated in ( b ) at 2.5 mm beyond the lesion site. Scale bar: 100 µm. g Quantification of regenerating axons at indicated distances beyond the crush site in optic nerves as described in ( b ). CRMP2 T/A rescued adverse effect of Gsk3β S/A knockin on PTEN −/− -induced optic nerve regeneration. Values represent means ± SEM of at least seven animals per group (PTEN −/− /GFP, n = 13; PTEN −/− /β S/A /GFP, n = 7; PTEN −/− /CRMP2 T/A , n = 12; PTEN −/− /β S/A /CRMP2 T/A , n = 8). h βIII-tubulin-stained whole-mount retinae from AAV-HA-CRMP2 T/A treated PTEN −/− and PTEN −/− /GSK3β S/A mice as described in ( b ). Scale bar: 50 µm. i Quantification of RGCs per mm 2 in retinal whole-mounts as described in ( h ). The number of surviving RGCs 21 days after ONC were similar for the respective genotypes. The dashed bar represents values from PTEN −/− mice as shown in Fig. for comparison. All values represent means ± SEM of 4–10 retinae per group (PTEN −/− , n = 10; PTEN −/− /CRMP2 T/A , n = 6; PTEN −/− /β S/A /CRMP2 T/A , n = 4). Significances of intergroup differences were evaluated using two-way ( g ) or one-way ( i ) analysis of variance (ANOVA) followed by Holm-Sidak post hoc test. Treatment effects: ** p < 0.01, *** p < 0.001, n.s. = non-significant

Article Snippet: Phospho-GSK3α and CRMP2 primary antibodies were detected using conformation-specific horseradish peroxidase conjugated anti-rabbit (IgG) secondary antibody (1:4000, Cell Signaling Technologies).

Techniques: Inhibition, Injection, Staining, Labeling, Knock-In, Comparison

Journal: Pain

Article Title: A membrane-delimited N-myristoylated CRMP2 peptide aptamer inhibits CaV2.2 trafficking and reverses inflammatory and postoperative pain behaviors

doi: 10.1097/j.pain.0000000000000147

Figure Lengend Snippet: N-myristate-tat-CBD3 peptide inhibits the CaV2.2–CRMP2 interaction and augments CRMP2–tubulin interaction without effects on CRMP2 phosphorylation or oligomerization. (A), Spinal cord lysates were incubated with recombinant CRMP2-GST protein (0.4 μM) in the absence (no peptide, lane 2) or presence of increasing concentrations (as indicated) of control peptides (myr-tat, myr-tat-CBD3scr1, myr-tat-CBD3scr2) or tat-CBD3 or myr-tat-CBD3 peptides. The CRMP2-GST–bound proteins were recovered by incubation with glutathione sepharose beads and immunoblotted with CaV2.2 (top) and CRMP2 (bottom). Representative blots from 3 separate experiments are shown. (B), Summary of mean relative binding of CaV2.2 to CRMP2. CaV2.2–CRMP2 binding was reduced by myr-tat-CBD3 in a concentration-dependent manner (*P < 0.01). n = 3 separate, individual experiments. (C), Representative Western blots of lysates prepared from DRG incubated for 2 hours with vehicle (no peptide) or the indicated peptides (20 μM) probed with the indicated CRMP2 and phospho-specific CRMP2 antibodies. The positions of molecular weight markers (in kilodaltons, kD) are illustrated on the right. (D), Summary of the mean relative levels of phospho-CRMP2 (normalized to total CRMP2) in arbitrary units (au). None of the peptides affected the expression of phospho-CRMP2 forms (n = 3–6 wells per condition). (E), 96-well plates coated with 200 ng tubulin were incubated with increasing concentrations of peptides. The Y-axis displays the OD450 absorbance of the ELISA using CRMP2-specific antibodies. CRMP2 bound to tubulin with half-saturation concentration of ~607 nM.86 Binding of heat-denatured CRMP2 demonstrated nonsaturable background binding to tubulin. A control peptide, tat-CBD3rev, did not affect CRMP2-tubulin binding. Competitive binding assay revealed that the tat-CBD3 and myr-tat-CBD3 peptides increase the binding of CRMP2 to tubulin. All measurements were performed in sextuplicate and error bars indicate SEM. Most of the error bars are smaller than the symbols. (F), Covalent cross-linking of purified CRMP2 was induced with increasing concentrations of glutaraldehyde (0.1–10 mM) in the absence or presence of 50 mM NaCl. The analyzed molecular weights of the purified CRMP2 protein are 65.5 kDa and 59.3 kDa. Silver-stained sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of a typical cross-linking experiment is shown. The positions of monomer ( ), dimer ( ), and tetramer ( ) are indicated on the right; the positions of molecular weight markers (kD) are on the left. Increasing the amount of cross-linker increases the amount of cross-linked oligomers with dimers and tetramers forming at 1 mM and predominantly tetramers at 10 mM or higher. The inclusion of tat-CBD3 or myr-tat-CBD3 or the control peptides (myr-tat, myr-tat-CBD3scr1, myr-tat-CBD3scr2) does not alter the extent of dimer or tetramer induced by 1 mM glutaraldehyde. A small amount of octameric CRMP2 is also observed (arrow). High-molecular weight aggregates that do not enter the gel are also observed (arrowhead). The peptides do not directly bind CRMP2 as the oligomeric profiles are similar irrespective of the presence of NaCl which tests if peptides disrupt the structure during the oligomerization of CRMP2. (G), The amount of protein found in oligomers (monomers, dimers, and tetramers) under 1 mM glutaraldehyde cross-linking condition was divided by the total CRMP2 in the same lane and plotted as a percentage of oligomers. n = 3 separate, individual experiments; the total number of samples analyzed is 4 to 6 per condition.

Article Snippet: The bound CRMP2 was detected by CRMP2 primary antibody (150 ng/mL, Sigma) followed by HRP-conjugated secondary antibody (GE Healthcare).

Techniques: Incubation, Recombinant, Binding Assay, Concentration Assay, Western Blot, Molecular Weight, Expressing, Enzyme-linked Immunosorbent Assay, Competitive Binding Assay, Purification, Staining, Polyacrylamide Gel Electrophoresis, SDS Page

Journal: Pain

Article Title: A membrane-delimited N-myristoylated CRMP2 peptide aptamer inhibits CaV2.2 trafficking and reverses inflammatory and postoperative pain behaviors

doi: 10.1097/j.pain.0000000000000147

Figure Lengend Snippet: tat-CBD3 and N-myristate-tat-CBD3 peptides induce changes in localization of CaV2.2 and CRMP2 in primary sensory neurons. Representative micrographs of DRG cells immunolabeled with CaV2.2 (green) and CRMP2 (red). The neurons are treated with (A) tat-CBD3 (20 μM) and (B) myr-tat-CBD3 (20 μM) for 5 minutes, 20 minutes, 2 hours and 48 hours. Control cells not treated with the peptides or treated with the control peptides myr-tat or myr-tat-CBD3scr1 (20 μM) display an overlap in membrane labeling of both CaV2.2 and CRMP2. Treatment with the peptides for 5 minutes, 30 minutes, and 2 hours reduces the membrane labeling of the CaV2.2 while retaining CRMP2 labeling at the membrane. At 48 hours, CaV2.2 and CRMP2 labeling is scattered in distinct puncta within the cytosol. Scale bar length is 20 μm. n = 2 separate, individual experiments; the total number of cells analyzed is 18 to 26 per time point.

Article Snippet: The bound CRMP2 was detected by CRMP2 primary antibody (150 ng/mL, Sigma) followed by HRP-conjugated secondary antibody (GE Healthcare).

Techniques: Immunolabeling, Labeling

Journal: Pain

Article Title: A membrane-delimited N-myristoylated CRMP2 peptide aptamer inhibits CaV2.2 trafficking and reverses inflammatory and postoperative pain behaviors

doi: 10.1097/j.pain.0000000000000147

Figure Lengend Snippet: tat-CBD3 and N-myristate-tat-CBD3 peptides induce changes in colocalization of CRMP2 and CaV2.2. Quantification of colocalization of CaV2.2 with CRMP2 was carried using Pearson (A) and Manders (B) overlap method. The values in the graph are the overlap coefficients. Control cells not treated with a peptide or treated with myr-tat or myr-tat-CBD3scr1 peptides display high level of overlap as demonstrated by the high coefficient values. However, treatment with tat-CBD3 and myr-tat-CBD3 for 5 minutes, 30 minutes, 2 hours, and 48 hours reduces the overlap as indicated by the reduction of the coefficient values. *P < 0.05 compared with control. #P < 0.05 comparing tat-CBD3-treated vs myr-tat-CBD3-treated or with control peptides myr-tat-treated or myr-tat-CBD3scr1-treated DRG. n = 2 separate, individual experiments; the total number of cells analyzed is 18 to 26 per time point.

Article Snippet: The bound CRMP2 was detected by CRMP2 primary antibody (150 ng/mL, Sigma) followed by HRP-conjugated secondary antibody (GE Healthcare).

Techniques:

Journal: Molecular neurodegeneration

Article Title: Phosphorylation of collapsin response mediator protein-2 disrupts neuronal maturation in a model of adult neurogenesis: Implications for neurodegenerative disorders.

doi: 10.1186/1750-1326-6-67

Figure Lengend Snippet: Figure 2 Down-regulation of CDK5 reduces CRMP2 phosphorylation in NPC-derived neural progeny. Differentiating NPCs were treated on day 2 with the pharmacological inhibitor Roscovitine (Rosco) or siRNA against CDK5 (siCDK5), followed by infection with p35-adenovirus. On day 4, NPC-derived neural progeny were fixed for immunofluorescence or lysed for immunoblot analysis. (A-C) Immunocytochemical analysis with an antibody against pSer522-CRMP2 showed low levels of endogenous immunoreactivity in uninfected, vehicle-treated cells (A), with a slight reduction in cells treated with Roscovitine (B) or siCDK5 (C). (D-F) NPC-derived neural progeny expressing p35 showed a notable increase in CRMP2 phosphorylation (D) that was reversed by treatment with Rosco (E) or siCDK5 (F). (G) Reversal of CRMP2 hyperphosphorylation with CDK5 down-regulation. (H) Immunoblot analysis of pSer522-CRMP2, total (t)CRMP2, CDK5, and actin as a loading control. (I) Semi-quantitative image analysis of levels of CRMP2 phosphorylation in NPCs infected with p35-adv with or without Rosco or siCDK5. * p < 0.05 compared to vehicle-treated controls by one-way ANOVA with post-hoc Dunnett’s test. # p < 0.05 compared to p35-expressing NPCs by one-way ANOVA with post-hoc Tukey-Kramer test. Scale bar = 10 μm.

Article Snippet: For double-labeling analysis, coverslips or brain sections were incubated with a rabbit polyclonal primary antibody against phospho-CRMP2 (Ser522, 1:1500, ECM Biosciences) or CRMP2 (1:1500, Millipore) detected with Tyramide Red.

Techniques: Phospho-proteomics, Derivative Assay, Infection, Immunofluorescence, Western Blot, Expressing, Control

Journal: Molecular neurodegeneration

Article Title: Phosphorylation of collapsin response mediator protein-2 disrupts neuronal maturation in a model of adult neurogenesis: Implications for neurodegenerative disorders.

doi: 10.1186/1750-1326-6-67

Figure Lengend Snippet: Figure 3 Expression of S522A-CRMP2 mutant construct rescues neurite deficits in p35-expressing NPC-derived neural progeny. Differentiating NPCs were transfected on day 2 with wild-type (WT) human CRMP2 or mutated S522A-CRMP2 plasmids, followed by infection with p35 adenovirus. Cells were fixed on day 4 of differentiation with glutaraldehyde for b-tubulin immunofluorescence, or lysed for immunoblot analysis. (A-F) Immunocytochemical analysis of b-tubulin immunoreactivity showed reduced b-tubulin-positive neurites in NPC-derived neural progeny infected with p35-adv with or without co-expression of WT-CRMP2, and rescue with co-expression of S522A-CRMP2 plasmid. (G) Image analysis showing reduced b-tubulin-positive neurite lengths in p35-expressing and p35+WT-CRMP2 NPC-derived neural progeny that was recovered after co-expression of p35 and S522A-CRMP2. (H) Immunoblot analysis showing levels of CRMP2 phosphorylation in p35-expressing NPC-derived neural progeny transfected with S522A-CRMP2 or WT-CRMP2 plasmid. (I) Semi-quantitative image analysis of ratios of pSer522- CRMP2 (pCRMP2)/total CRMP2 (tCRMP2) levels in NPC-derived neural progeny expressing p35 with and without WT-CRMP2 or S522A-CRMP2 co- expression. * p < 0.05 compared to vehicle-treated controls by one-way ANOVA with post-hoc Dunnett’s test. # p < 0.05 compared to WT- CRMP2-transfected NPCs by one-way ANOVA with post-hoc Tukey-Kramer test. Scale bar = 25 μm, 10 μm (insets).

Article Snippet: For double-labeling analysis, coverslips or brain sections were incubated with a rabbit polyclonal primary antibody against phospho-CRMP2 (Ser522, 1:1500, ECM Biosciences) or CRMP2 (1:1500, Millipore) detected with Tyramide Red.

Techniques: Expressing, Mutagenesis, Construct, Derivative Assay, Transfection, Infection, Immunofluorescence, Western Blot, Plasmid Preparation, Phospho-proteomics

Journal: Molecular neurodegeneration

Article Title: Phosphorylation of collapsin response mediator protein-2 disrupts neuronal maturation in a model of adult neurogenesis: Implications for neurodegenerative disorders.

doi: 10.1186/1750-1326-6-67

Figure Lengend Snippet: Figure 6 Increased CRMP2 phosphorylation in NPCs treated with HIV-gp120 protein, and rescue with CDK5 siRNA knockdown. Differentiating NPCs were transfected day 2 of differentiation with siRNA specific for CDK5 (siCDK5) or transfection reagent control, followed by treatment with recombinant HIV-gp120 protein (100 ng/mL) or vehicle control. Cells were fixed on day 4 of differentiation for double- immunolabeling analysis with antibodies against b-III Tubulin (immature neuronal marker) and pSer522-CRMP2 (pSer-CRMP2). (A-C) b-III Tubulin- positive NPC-derived neuronal progeny treated with vehicle control express background levels of pSer-CRMP2. (D-F) NPC-derived neuronal progeny treated with gp120 for 48 hrs display increased pSer-CRMP2 immunoreactivity in b-III Tubulin-positive cells. (G-I) b-III Tubulin-positive NPC-derived neuronal progeny treated with siCDK5 show low levels of pSer-CRMP2 immunoreactivity. (J-L) b-III Tubulin-positive NPC-derived neuronal progeny treated with siCDK5 and gp120 show background levels of pSer-CRMP2 immunoreactivity. (M, N) Semi-quantitative image analysis of b-III Tubulin (M) and pSer-CRMP2 (N) immunoreactivity. * p < 0.05 compared to vehicle-treated controls by one-way ANOVA with post-hoc Dunnett’s test. Scale bar = 20 μm.

Article Snippet: For double-labeling analysis, coverslips or brain sections were incubated with a rabbit polyclonal primary antibody against phospho-CRMP2 (Ser522, 1:1500, ECM Biosciences) or CRMP2 (1:1500, Millipore) detected with Tyramide Red.

Techniques: Phospho-proteomics, Knockdown, Transfection, Control, Recombinant, Immunolabeling, Marker, Derivative Assay

Journal: Molecular neurodegeneration

Article Title: Phosphorylation of collapsin response mediator protein-2 disrupts neuronal maturation in a model of adult neurogenesis: Implications for neurodegenerative disorders.

doi: 10.1186/1750-1326-6-67

Figure Lengend Snippet: Figure 7 CRMP2 is hyperphosphorylated and dendritic development is impaired in primary neurons over-expressing p35 alone or in combination with HIV-gp120 protein treatment. Primary rat hippocampal neurons were infected two days after plating with p35-adv, followed by treatment with recombinant HIV-gp120 protein or vehicle control. Cells were fixed on day 4 of differentiation for double- immunolabeling analysis with antibodies against MAP2 (dendritic marker) and pSer522-CRMP2 (pSer-CRMP2). (A-C) MAP2-positive primary neurons treated with vehicle control express background levels of pSer-CRMP2. (D-F) Primary neurons infected with p35-adv display increased pSer-CRMP2 immunoreactivity and reduced MAP2-immunoreactive dendrites. (G-I) Primary neurons treated with recombinant HIV-gp120 display increased pSer-CRMP2 immunoreactivity and reduced MAP2-immunoreactive dendrites, with an accumulation of pSer-CRMP2 immunoreactivity in varicosities along the neuronal processes (arrows). (J-L) Primary neurons infected with p35-adv and treated with recombinant HIV-gp120 display increased pSer-CRMP2 immunoreactivity and extensive damage to MAP2-immunoreactive dendrites, with an abundant accumulation of pSer-CRMP2 immunoreactivity in varicosities along the neuronal processes (arrows). (M, N) Semi-quantitative image analysis of pSer-CRMP2 (M) and MAP2 (N) immunoreactivity. (O) Immunoblot analysis of total cell lysates showing increased CRMP2 phosphorylation in primary hippocampal neurons infected with p35-adv or treated with HIV-gp120 protein. (P) Semi-quantitative image analysis of fold change in CRMP2 phosphorylation at the CDK5 epitope (Ser522) in immunoblots from cells expressing p35-adv or treated with gp120. * p < 0.05 compared to vehicle-treated controls by one-way ANOVA with post-hoc Dunnett’s test. Scale bar = 20 μm.

Article Snippet: For double-labeling analysis, coverslips or brain sections were incubated with a rabbit polyclonal primary antibody against phospho-CRMP2 (Ser522, 1:1500, ECM Biosciences) or CRMP2 (1:1500, Millipore) detected with Tyramide Red.

Techniques: Expressing, Infection, Recombinant, Control, Immunolabeling, Marker, Western Blot, Phospho-proteomics

Journal: Molecular neurodegeneration

Article Title: Phosphorylation of collapsin response mediator protein-2 disrupts neuronal maturation in a model of adult neurogenesis: Implications for neurodegenerative disorders.

doi: 10.1186/1750-1326-6-67

Figure Lengend Snippet: Figure 8 Patterns of CRMP2 immunoreactivity in the hippocampus of an HIV encephalitis brain and a gp120 transgenic mouse brain. Forty μm-thick vibratome sections from the brains of HIV encephalitis (HIVE) patients and gp120 transgenic (tg) mice were processed for double-immunolabeling analysis with antibodies against doublecortin (DCX) and CRMP2. (A-C) Intracellular CRMP2 immunoreactivity was detected throughout cells in the granular cell layer (GCL) and into the molecular layer (ML) of the hippocampal dentate gyrus in a representative section from the brain of a patient with HIVE. A subset of DCX-positive cells in the subgranular zone (SGZ) co-expressed CRMP2 (arrows). (D-F) Intracellular CRMP2 immunoreactivity was detected throughout cells in the granular cell layer (GCL) and into the molecular layer (ML) of the hippocampal dentate gyrus in a representative section from the brain of a gp120 tg mouse. A subset of DCX-positive cells in the subgranular zone (SGZ) co-expressed CRMP2 (arrows). Scale bar = 15 μm.

Article Snippet: For double-labeling analysis, coverslips or brain sections were incubated with a rabbit polyclonal primary antibody against phospho-CRMP2 (Ser522, 1:1500, ECM Biosciences) or CRMP2 (1:1500, Millipore) detected with Tyramide Red.

Techniques: Transgenic Assay, Immunolabeling

Journal: Molecular neurodegeneration

Article Title: Phosphorylation of collapsin response mediator protein-2 disrupts neuronal maturation in a model of adult neurogenesis: Implications for neurodegenerative disorders.

doi: 10.1186/1750-1326-6-67

Figure Lengend Snippet: Figure 9 Increased CRMP2 expression and phosphorylation in the hippocampus of patients with HIV encephalitis. Forty μm-thick vibratome sections from the hippocampus of HIV+ control and HIV encephalitis (HIVE) patients were processed for immunolabeling analysis with antibodies against CRMP2 and pSer522-CRMP2 (pSer-CRMP2). Low power images show overall immunoreactivity throughout the hippocampus, including the CA1-4 regions, the dentate gyrus (DG) and the subgranular zone (SGZ), and high power images show more detail of the DG including the SGZ and granular cell layer (GCL). (A-D) Patterns of total CRMP2 immunoreactivity in the hippocampus from representative HIV+ non-encephalitis (control) and HIVE brains at low (A, C) and high (B, D) power. Numerous intensely immunoreactive cell profiles were detected in the SGZ (arrows), particularly in the brains of HIVE patients. (E) Semi-quantitative image analysis of CRMP2 immunoreactivity in the hippocampal DG of HIV patients. (F-I) Patterns of pSer-CRMP2 immunoreactivity in the hippocampus from representative HIV+ non-encephalitis (control) and HIVE brains at low (F, H) and high (G, I) power. (J) Semi-quantitative image analysis of pSer-CRMP2 immunoreactivity in the hippocampal DG of HIV patients. * p < 0.05 compared to HIV+ controls by unpaired two-tailed Student’s t-test (n = 8 per group). Scale bar = 200 μm (A,C,F,H); 30 μm (B,D,G,I).

Article Snippet: For double-labeling analysis, coverslips or brain sections were incubated with a rabbit polyclonal primary antibody against phospho-CRMP2 (Ser522, 1:1500, ECM Biosciences) or CRMP2 (1:1500, Millipore) detected with Tyramide Red.

Techniques: Expressing, Phospho-proteomics, Control, Immunolabeling, Two Tailed Test

Journal: Molecular neurodegeneration

Article Title: Phosphorylation of collapsin response mediator protein-2 disrupts neuronal maturation in a model of adult neurogenesis: Implications for neurodegenerative disorders.

doi: 10.1186/1750-1326-6-67

Figure Lengend Snippet: Figure 10 Increased CRMP2 phosphorylation in gp120 transgenic mice is rescued with down-modulation of CDK5 expression. Forty μm-thick vibratome sections from the brains of 8-month old non-transgenic (nontg), gp120 transgenic (tg), CDK5 heterozygous-deficient (CDK5 +/-), and gp120 tg/CDK5+/- mice were processed for immunolabeling analysis with antibodies against CRMP2 and pSer522-CRMP2 (pSer-CRMP2). (A-D) Patterns of total CRMP2 immunoreactivity in the dentate gyrus from representative mouse brains showing a punctate expression pattern throughout the granular cell layer (GCL) and subgranular zone (SGZ), with background immunoreactivity detected in the molecular layer (ML). Intensely immunoreactive cell profiles were detected in the SGZ (arrows), particularly in the brains of gp120 tg mice. (E) Semi-quantitative image analysis of CRMP2 immunoreactivity in the hippocampal DG of nontg, gp120 tg, CDK5+/- and gp120 tg/CDK5+/- mice. (F-I) Patterns of pSer- CRMP2 immunoreactivity in the hippocampal DG from representative mouse brains showing a punctate expression pattern throughout the granular cell layer (GCL) and subgranular zone (SGZ), with background immunoreactivity detected in the molecular layer (ML). Intensely immunoreactive cell profiles were detected in the SGZ (arrows), particularly in the brains of gp120 tg mice. (J) Semi-quantitative image analysis of pSer-CRMP2 immunoreactivity in the hippocampus of nontg, gp120 tg, CDK5+/- and gp120 tg/CDK5+/- mice. * p < 0.05 compared to nontg controls by one-way ANOVA with post-hoc Dunnett’s test (n = 4 per group). Scale bar = 30 μm.

Article Snippet: For double-labeling analysis, coverslips or brain sections were incubated with a rabbit polyclonal primary antibody against phospho-CRMP2 (Ser522, 1:1500, ECM Biosciences) or CRMP2 (1:1500, Millipore) detected with Tyramide Red.

Techniques: Phospho-proteomics, Transgenic Assay, Expressing, Immunolabeling