astrocytic ca 2+ binding protein s100β (bovine  (Millipore)

Journal: bioRxiv

Article Title: Local Confinement within Plasma Membrane Nanodomains Drives Constitutive Activity of GPCRs

doi: 10.64898/2026.02.17.706370

Figure Lengend Snippet: Cell signaling assays. (A) For M 1 R, the intracellular Ca 2+ concentration was measured by spectral changes of the Fura-2 dye, upon treatment of live cells with agonist ( carbachol ) and antagonist ( pirenzepine ), using no ligand (DMSO vehicle-only) as control. (B) For A 2A R, changes in cAMP concentration were measured by luciferin bioluminescence mediated through ATP hydrolysis by protein kinase A (PKA), upon treatment of cells with agonist ( CGS-21680 ) and antagonist ( ZM-241385 ), with vehicle-only as control. Data are mean ± S.E.M. (standard error of the mean) of three independent experiments, normalized between 0 and 1.

Article Snippet: After overnight surface adhesion, cells were cultured in complete culture medium supplemented with 1 μg / mL doxycycline for 24 h. The medium was then replaced with Hank’s Balanced Salt Solution (HBSS) (Cytiva, SH30268.01) containing 3 μM of Fura-2 AM Ca 2+ binding dye (Invitrogen, F1201).

Techniques: Concentration Assay, Control

Journal: Protein Science : A Publication of the Protein Society

Article Title: Disruption of Ca 2+ /calmodulin: KSR1 interaction lowers ERK activation

doi: 10.1002/pro.4982

Figure Lengend Snippet: The Ca 2+ /CaM binding site on KSR1 is between amino acid residues 354–377. (a) Schematic representation of KSR1. KSR1 has five conserved area (CA) domains, termed CA1 to CA5. (b) KSR1 constructs were used for the binding analyses. The constructs are KSR1‐M (amino acids 319–433), ‐M1 (319–376), ‐M2 (349–404), and ‐M3 (377–433). The red line encompasses the Ca 2+ /CaM binding region determined based on panels (c and d). (c) GST‐tagged fragments of KSR1 generated in Escherichia coli and purified on glutathione‐Sepharose were incubated with pure CaM in the presence of 1 mM CaCl 2 . Pull‐down (PD) with GST‐coated Sepharose beads was the negative control. Input designates pure CaM not subjected to PD. Proteins bound to the beads were eluted and analyzed by SDS‐PAGE. The upper portion of the gel (above ~23 kDa) was stained with Coomassie blue; the lower portion was analyzed by Western blotting and probed for CaM. d. HEK293T cells were transiently transfected with GFP‐tagged wild‐type (WT) KSR1 or the deletion mutant construct KSR1Δ 354–377 . Equal amounts of cell lysate were subjected to CaM‐Sepharose PDs in the presence of 1 mM CaCl 2 . PDs with protein A‐Sepharose (ProA) beads were carried out in parallel as negative controls. Samples were resolved by Western blotting and probed with anti‐GFP antibodies. Input designates cell lysate not subjected to PD. All data shown in this figure are representative of three independent experiments. The position of migration of the molecular weight markers is shown on the left of the blots and gels. CaM, calmodulin.

Article Snippet: In turn, Ca 2+ /CaM may stabilize α‐helical re‐folding of KSR1‐CA3, as previously observed for other Ca 2+ /CaM binding partners (Gifford et al., ; Meador et al., ; Tidow & Nissen, ; Yamauchi et al., ).

Techniques: Binding Assay, Construct, Generated, Purification, Incubation, Negative Control, SDS Page, Staining, Western Blot, Transfection, Mutagenesis, Migration, Molecular Weight

Journal: Protein Science : A Publication of the Protein Society

Article Title: Disruption of Ca 2+ /calmodulin: KSR1 interaction lowers ERK activation

doi: 10.1002/pro.4982

Figure Lengend Snippet: The relaxed structures of extended Ca 2+ /CaM interacting with globular KSR1‐CA3. Snapshots showing the best representative conformations from the ensemble clusters for selected configurations (left panels). Violin plots represent the intermolecular salt bridge pair distance for the Ca 2+ /CaM:KSR1‐CA3 complex (right panels). In KSR1‐CA3, the transparent surface encompasses the Ca 2+ /CaM binding region of KSR1‐CA3 (residues 354–377). Key residues involved in binding are marked in green for KSR1 and purple for CaM. The dark and light blue spheres represent calcium and zinc ions, respectively. CaM, calmodulin.

Article Snippet: In turn, Ca 2+ /CaM may stabilize α‐helical re‐folding of KSR1‐CA3, as previously observed for other Ca 2+ /CaM binding partners (Gifford et al., ; Meador et al., ; Tidow & Nissen, ; Yamauchi et al., ).

Techniques: Binding Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Disruption of Ca 2+ /calmodulin: KSR1 interaction lowers ERK activation

doi: 10.1002/pro.4982

Figure Lengend Snippet: The relaxed structures of collapsed Ca 2+ /CaM binding to α‐helical peptides of KSR1‐CA3. (a) Violin plots representing the binding free energy (Δ G binding ) of the α‐helical peptides of KSR1‐CA3 interacting with collapsed Ca 2+ /CaM. (b) Correlation of the binding free energy with the van der Waals (vdW) interactions for the Ca 2+ /CaM:KSR1‐CA3 complex. (c and d) Snapshots showing the most favorable conformations from the ensemble clusters for the H8 (c) and H7 (d) simulations. The α‐helical structures of KSR1‐CA3 are colored gray. Hydrophobic, polar/glycine, positively charged, and negatively charged residues are colored white, green, blue, and red, respectively. For Ca 2+ /CaM, only the C‐lobe is shown. Key residues for interaction are marked in green for KSR1 and purple for CaM. CaM, calmodulin.

Article Snippet: In turn, Ca 2+ /CaM may stabilize α‐helical re‐folding of KSR1‐CA3, as previously observed for other Ca 2+ /CaM binding partners (Gifford et al., ; Meador et al., ; Tidow & Nissen, ; Yamauchi et al., ).

Techniques: Binding Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Disruption of Ca 2+ /calmodulin: KSR1 interaction lowers ERK activation

doi: 10.1002/pro.4982

Figure Lengend Snippet: Binding of Ca 2+ /CaM to KSR1 is mediated by hydrophobic interactions. (a) The GST‐tagged KSR1‐M fragment (amino acids 319–433) was produced in Escherichia coli and purified on glutathione‐Sepharose. GST‐tagged KSR1‐M was then incubated with pure CaM in the presence or absence of 10 μM ZnCl 2 . All incubations contained 1 mM CaCl 2 . Pull‐down (PD) with GST‐coated Sepharose beads (GST) was the negative control. Proteins bound to the beads were eluted and analyzed by SDS‐PAGE. The gel was cut at ~23 kDa. The upper portion of the gel was stained with Coomassie blue; the lower portion was analyzed by Western blotting and probed for CaM. Input designates pure CaM not subjected to PD. # designates an empty lane. Data are representative of four independent experiments. (b) GFP‐tagged KSR1 was expressed in HEK293T cells. Equal amounts of protein from cell lysates were subjected to immunoprecipitation (IP) using GFP‐Trap beads in the presence of 1 mM CaCl 2 and in the presence or absence of 10 μM ZnCl 2 . IP with control (Ctrl) agarose beads was carried out in parallel. Samples were resolved by Western blotting and probed for GFP and calmodulin. Inputs are aliquots of cell lysates prior to IP. Data are representative of three independent repetitions. (c) Protein sequence from amino acid 354–377 of wild‐type (WT) KSR1, KSR1‐I 354 D;F 355 D, and KSR1‐K 372 A;E 373 A. Residues in bold were mutated. Red indicates the introduced mutations. (d) GFP‐tagged KSR1‐WT, KSR1‐I 354 D;F 355 D, and KSR1‐K 372 A;E 373 A were expressed in HEK293T cells. Equal amounts of protein cell lysates were then subjected to CaM‐Sepharose PDs in the presence of 1 mM CaCl 2 . PDs with protein A‐Sepharose (ProA) beads were carried out as negative controls. Samples were resolved by Western blotting and probed for GFP. Inputs are equal amounts of protein cell lysate not subjected to PD. Data are representative of three independent repetitions.

Article Snippet: In turn, Ca 2+ /CaM may stabilize α‐helical re‐folding of KSR1‐CA3, as previously observed for other Ca 2+ /CaM binding partners (Gifford et al., ; Meador et al., ; Tidow & Nissen, ; Yamauchi et al., ).

Techniques: Binding Assay, Produced, Purification, Incubation, Negative Control, SDS Page, Staining, Western Blot, Immunoprecipitation, Control, Sequencing

Journal: Protein Science : A Publication of the Protein Society

Article Title: Disruption of Ca 2+ /calmodulin: KSR1 interaction lowers ERK activation

doi: 10.1002/pro.4982

Figure Lengend Snippet: Replacement of KSR1‐Phe 355 with Asp significantly attenuates binding to Ca 2+ /CaM. (a) GFP‐tagged wild‐type (WT) KSR1, KSR1‐I 354 D, KSR1‐F 355 D, and KSR1‐L 364 D were separately expressed in HEK293T cells. Cell lysates were supplemented with 1 mM CaCl 2 and incubated with CaM‐Sepharose beads. Pull‐downs (PDs) with protein A beads (ProA) served as negative controls. Samples were resolved by Western blotting and probed for GFP. Inputs designate aliquots of cell lysate not subjected to PD. In each panel, images for WT and mutated KSR1 were from the same membrane (irrelevant lanes were omitted). (b) The GFP‐KSR1 signal observed after PD was quantified using LI‐COR Image Studio software (mean ± SD, n = 5). Binding to KSR1‐WT was set as 1. Data were analyzed by one‐way ANOVA followed by Dunnet's test (***, p ≤ 0.001; ns, not significant). (c) GFP‐tagged KSR1‐WT and KSR1‐F 355 D were expressed separately in HEK293T cells. KSR1 was immunoprecipitated (IP) using GFP‐Trap beads from cell lysates in the presence of 1 mM CaCl 2 . Precipitations with control agarose beads (Ctrl) were carried out in parallel. Inputs are cell lysates not subjected to IP. Samples were resolved by Western blotting and probed for GFP, CaM, MEK, and 14‐3‐3. # indicates an empty lane. (d) The CaM, MEK, and 14‐3‐3 bands observed after IP were quantified (mean ± SD, n = 3). Band intensities were divided by the values observed with cells transfected with KSR1‐WT to normalize this condition to 1. Data were analyzed by one‐sample t tests (**, p ≤ 0.01; ns, not significant). All blots shown in this figure are representative of at least three biological replicates. CaM, calmodulin.

Article Snippet: In turn, Ca 2+ /CaM may stabilize α‐helical re‐folding of KSR1‐CA3, as previously observed for other Ca 2+ /CaM binding partners (Gifford et al., ; Meador et al., ; Tidow & Nissen, ; Yamauchi et al., ).

Techniques: Binding Assay, Incubation, Western Blot, Membrane, Software, Immunoprecipitation, Control, Transfection

Journal: Protein Science : A Publication of the Protein Society

Article Title: Disruption of Ca 2+ /calmodulin: KSR1 interaction lowers ERK activation

doi: 10.1002/pro.4982

Figure Lengend Snippet: Model of Ca 2+ /CaM binding to the CA3 domain of KSR1. (1) Binding of four Ca 2+ ions (dark blue spheres) to apo‐CaM (red) induces a conformational shift in CaM to its extended Ca 2+ ‐loaded conformation. (2) Ca 2+ /CaM in its collapsed conformation then binds to KSR1 by wrapping around the CA3 domain (purple), which stabilizes it in an α‐helical conformation. (3) In cells, the Ca 2+ /CaM:KSR1 complex promotes EGF‐stimulated activation of ERK. Figure generated with BioRender. CaM, calmodulin; EGF, epidermal growth factor.

Article Snippet: In turn, Ca 2+ /CaM may stabilize α‐helical re‐folding of KSR1‐CA3, as previously observed for other Ca 2+ /CaM binding partners (Gifford et al., ; Meador et al., ; Tidow & Nissen, ; Yamauchi et al., ).

Techniques: Binding Assay, Activation Assay, Generated

Journal: PLOS ONE

Article Title: Sound waves alter the viability of tobacco cells via changes in cytosolic calcium, membrane integrity, and cell wall composition

doi: 10.1371/journal.pone.0299055

Figure Lengend Snippet: The total Ca 2+ concentration of untreated (control) and treated tobacco cells with sound pressures of 60, 75, and 90 dB for 15,30,45, and 60 min are shown (a) . The increase of cytosolic Ca 2+ was determined by the ratio of Ca 2+ -bound Fura-2 to Ca 2+ -free Fura-2 (340/380 nm) (b) . Different letters denoted on bars indicate significant differences (Duncan test, p ≤ 0.05). The overall coefficient of variation (OCV) was classified as low when it varied from 0.45 to 32.2%, medium from 32.2 to 63.95%, and high from 63.95 to 95.7%.

Article Snippet: The concentration of cytosolic free Ca 2+ was determined by loading cells with the acetoxymethyl ester of the Ca 2+ -binding dye Fura-2 (Fura-2-AM, Molecular Probes, Sigma-Aldrich, USA) in the dark at 28°C for 1 h. The cells were then washed three times with fresh medium and left for 15 min to have the Fura-2 crossed cell membranes and entered the cells where the cytosolic esterases cleave the acetoxymethyl hydrophobic side chains and produce the hydrophilic permeable fluorescent dye/ Ca 2+ complex.

Techniques: Concentration Assay

Journal: Computational and Structural Biotechnology Journal

Article Title: Calcineurin B inhibits calcium oxalate crystallization, growth and aggregation via its high calcium-affinity property

doi: 10.1016/j.csbj.2023.07.038

Figure Lengend Snippet: Ca 2+ -affinity assay. Equal amount of recombinant human CNB, TUPs or lysozyme (negative control) in coating buffer, or equal volume of coating buffer without any protein added (blank control) was immobilized in each well of a microplate and incubated with 0.1 mM CaCl 2 ·2 H 2 O at 25 °C for 60 min (A): The standard curve of [Ca 2+ ] measured by Ca 2+ -binding colorimetric assay using Arsenazo III reagent. (B): The starting (T 0 ) and remaining (T 60 ) [Ca 2+ ] in each well were then measured using Arsenazo III reagent and the standard curve. (C): Ca 2+ consumption, which determines Ca 2+ -affinity, was then calculated. The data were derived from three independent experiments using different samples and are reported as mean ± SD. * ** = P < 0.0001 vs. blank control.

Article Snippet: The starting (T 0 ) and remaining (T 60 ) [Ca 2+ ] in each well were measured by a Ca 2+ -binding colorimetric assay using Arsenazo III reagent (BioSystems S.A.; Barcelona, Spain) , .

Techniques: Recombinant, Negative Control, Incubation, Binding Assay, Colorimetric Assay, Derivative Assay

Journal: The Journal of Physiology

Article Title: Long QT syndrome‐associated calmodulin variants disrupt the activity of the slowly activating delayed rectifier potassium channel

doi: 10.1113/JP284994

Figure Lengend Snippet: Left shows the location of the mutations in the crystal structure of Ca 2+ /CaM (PDB: 1CLL). Right illustrates their location within the C‐lobe, all of which occur within residues which directly coordinate Ca 2+ , as depicted by black lines. Mutants D95V and N97I are located in the third Ca 2+ ‐binding site of CaM (EF‐hand III), whereas D131H is found in the fourth EF‐hand (EF‐hand IV).

Article Snippet: In the case of the variants studied (D95V, N97I and D131H), substitutions occur at residues which directly coordinate Ca 2+ at the C‐lobe of the protein, resulting in reduced Ca 2+ ‐binding (Crotti et al., ; Makita et al., ; Pipilas et al., ; Sondergaard et al., ; Vassilakopoulou et al., ) and reduced conformational plasticity in response to increases in [Ca 2+ ].

Techniques: Binding Assay

Journal: The Journal of Physiology

Article Title: Long QT syndrome‐associated calmodulin variants disrupt the activity of the slowly activating delayed rectifier potassium channel

doi: 10.1113/JP284994

Figure Lengend Snippet: Summary of IKs densities and voltage sensitivity at resting (100 n m ) and high (1 μ m ) intracellular Ca 2+ levels . Data are means ± s.e.m. Statistical significance was determined using two‐way ANOVA with Dunnett's multiple comparisons tests (for current densities) and one‐way ANOVA with Dunnett's multiple comparisons tests (for V 1/2 activation)

Article Snippet: In the case of the variants studied (D95V, N97I and D131H), substitutions occur at residues which directly coordinate Ca 2+ at the C‐lobe of the protein, resulting in reduced Ca 2+ ‐binding (Crotti et al., ; Makita et al., ; Pipilas et al., ; Sondergaard et al., ; Vassilakopoulou et al., ) and reduced conformational plasticity in response to increases in [Ca 2+ ].

Techniques: Activation Assay

Journal: The Journal of Physiology

Article Title: Long QT syndrome‐associated calmodulin variants disrupt the activity of the slowly activating delayed rectifier potassium channel

doi: 10.1113/JP284994

Figure Lengend Snippet: A , two dimensional 1 H, 15 N HSQC NMR spectra of Ca 2+ /CaM variants. Overlay of spectra collected from uniformly labelled 15 N CaM proteins in the presence of 1 m m CaCl 2 . Spectra were collected at 30°C on 700/800 MHz NMR spectrometers (Bruker). B , chemical shift perturbation of Ca 2+ ‐saturated, LQTS‐associated CaM mutants compared with CaM‐WT. B , top panel, schematic of the distribution of key structural features of CaM (N‐lobe: 1−72, linker region: 73−87, C‐lobe: 88−148). The regions containing the Ca 2+ ‐binding EF‐hands are outlined (EF‐hand I: 21−32, EF‐hand II: 57−68, EF‐hand III: 94−105, EF‐hand IV: 130−141). B , bottom panels, chemical shift differences ( 15 N and 1 H) between the residues of Ca 2+ ‐saturated CaM‐WT and LQTS‐associated variants in the presence of 1 m m CaCl 2 . Residues for which chemical shift differences could not be calculated are shown with an arbitrary value of −0.1 ppm. Chemical shift differences were expressed in ppm as Δδ = [(ΔH) 2 +(0.15ΔN) 2 ] 1/2 .

Article Snippet: In the case of the variants studied (D95V, N97I and D131H), substitutions occur at residues which directly coordinate Ca 2+ at the C‐lobe of the protein, resulting in reduced Ca 2+ ‐binding (Crotti et al., ; Makita et al., ; Pipilas et al., ; Sondergaard et al., ; Vassilakopoulou et al., ) and reduced conformational plasticity in response to increases in [Ca 2+ ].

Techniques: Binding Assay

Journal: Acta Neuropathologica Communications

Article Title: T cell-activation in neuromyelitis optica lesions plays a role in their formation

doi: 10.1186/2051-5960-1-85

Figure Lengend Snippet: T cells with different CNS antigen-specificity are activated to different extent in the CNS. Analysis of surface markers by flow cytometry. Histograms are shown. GFP-labeled MBP-, S100β-, and MOG-specific T cells isolated from the spleen (blue) or spinal cord (green) of recipient rats were isolated at the acute phase of clinical symptoms and analyzed for the expression of T cell receptors (TCR), interleukin-2 receptors (IL-2R) or the Ox40 antigen, using specific antibodies for these molecules and an isotype control (IgG). MBP-specific T cells were strongly activated, as evidenced by a down-regulation of TCR, and an up-regulation of IL-2R and the Ox40 antigen. S100β-specific T cells showed an intermediated degree of activation (i.e. no downregulation of TCR, weak up-regulation of IL-2R and Ox40 antigen), and MOG-specific T cells were not noticeably activated, as revealed by the lack of upregulation of IL-2R and the Ox40 antigen).

Article Snippet: T cell lines against myelin basic protein (MBP, from guinea pig, Sigma), myelin oligodendrocyte glycoprotein (MOG, recombinant N-terminal peptide 1–125, rat, own production) and the astrocytic Ca 2+ binding protein S100β (bovine, Sigma) were intraperitoneally injected to induce EAE.

Techniques: Flow Cytometry, Labeling, Isolation, Expressing, Activation Assay

Journal: Acta Neuropathologica Communications

Article Title: T cell-activation in neuromyelitis optica lesions plays a role in their formation

doi: 10.1186/2051-5960-1-85

Figure Lengend Snippet: T cells infiltration of the spinal cord following the initiation of NMO-like lesions in NMO-IgG seropositive animals by T cells with different CNS antigen-specificities. (A-F) T cells specific for MBP (A,B) , S100β (C,D) and MOG (E,F) were used to induce CNS inflammation, followed by transfer of NMO-IgG 4 days later. The animals were sacrificed 5 days after T cell transfer. For histological evaluation, their spinal cords were reacted with anti-CD3 antibodies (brown reaction product) and counterstained with hematoxylin to reveal nuclei (blue). bars = 500 μm (A,C,E) and 100 μm (B,D,F) . (G) The average number of T cells per mm 2 of lesions was determined by evaluating 5 representative spinal cord cross sections (1 cervical, 2 thoracal, 2 lumbar cross sections) per animal, using 5 animals (MBP- and MOG-specific T cells) or 4 animals (S100β-specific T cells) per group. Asterisks indicate statistically significant (p < 0,05) differences between individual CNS antigen specificities of the T cells used to induce CNS inflammation (Kruskal-Wallis followed by Mann–Whitney U test and Bonferroni-Holm correction; p = 0,0476 for MBP/S100β and S100β/MOG, p = 0.858 for MBP/MOG). (H) Numbers of ED1 + cells (activated microglia/macrophages) in spinal cord cross sections. The cell numbers were determined by evaluating one complete spinal cord cross section per animal, using 5 animals (MBP- and MOG-specific T cells) or 4 animals (S100β-specific T cells) per group. Asterisks indicate statistically significant (p < 0,05) differences between individual CNS antigen specificities of the T cells used to induce CNS inflammation (Kruskal-Wallis followed by Mann–Whitney U test and Bonferroni-Holm correction; p = 0,048 for MBP/S100β, p = 0,024 for MBP/MOG, and p = 0,189 for S100β/MOG).

Article Snippet: T cell lines against myelin basic protein (MBP, from guinea pig, Sigma), myelin oligodendrocyte glycoprotein (MOG, recombinant N-terminal peptide 1–125, rat, own production) and the astrocytic Ca 2+ binding protein S100β (bovine, Sigma) were intraperitoneally injected to induce EAE.

Techniques: MANN-WHITNEY

Journal: Acta Neuropathologica Communications

Article Title: T cell-activation in neuromyelitis optica lesions plays a role in their formation

doi: 10.1186/2051-5960-1-85

Figure Lengend Snippet: Entry of human immunglobulins to lesions provoked by different CNS antigen-specific T cells in NMO-IgG seropositive animals. (A-F) T cells specific for MBP (A,B) , S100β (C,D) and MOG (E,F) were used to induce CNS inflammation, followed by transfer of NMO-IgG 4 days after T cell transfer. The animals were sacrificed 5 days after T cell transfer. For histological evaluation, their spinal cords were reacted with anti-human IgG (brown reaction product) and counterstained with hematoxylin to reveal nuclei (blue). Overviews (A,C,E) and details (B,D,F) of representative spinal cord sections are shown. Bars = 100 μm.

Article Snippet: T cell lines against myelin basic protein (MBP, from guinea pig, Sigma), myelin oligodendrocyte glycoprotein (MOG, recombinant N-terminal peptide 1–125, rat, own production) and the astrocytic Ca 2+ binding protein S100β (bovine, Sigma) were intraperitoneally injected to induce EAE.

Techniques:

Journal: Acta Neuropathologica Communications

Article Title: T cell-activation in neuromyelitis optica lesions plays a role in their formation

doi: 10.1186/2051-5960-1-85

Figure Lengend Snippet: Loss of AQP4 reactivity in NMO-like lesions initiated by T cells with different CNS antigen-specificities. (A-F) T cells specific for MBP (A,B) , S100β (C,D) and MOG (E,F) were used to induce CNS inflammation, followed by transfer of NMO-IgG 4 days later. The animals were sacrificed 5 days after T cell transfer. For histological evaluation, their spinal cords were reacted with anti-AQP4 antibodies (brown reaction product) and counterstained with hematoxylin to reveal nuclei (blue). bars = 500 μm (A,C,E) and 100 μm (B,D,F) . (G) The average number of lesions with AQP4 loss per spinal cord cross section, as determined by evaluating 5 representative spinal cord cross sections (1 cervical, 2 thoracal, and 2 lumbar cross sections) per animal, using 5 animals (MBP, MOG) and 4 animals (S100β) per group. Asterisks indicate statistically significant differences between individual CNS antigen specificities of the T cells used to induce CNS inflammation (ANOVA-Holm Sidak; p < 0,001 for MBP-specific T cells compared to MOG-specific T cells; p = 0,008 for MBP-specific T cells compared to S100β-specific T cells; and p = 0,005 for S100β-specific T cells compared to MOG-specific T cells). (H) The largest lesion with AQP4 loss per animal, using 5 animals (MBP, MOG) and 4 animals (S100β) per group. Asterisks indicate statistically significant differences between individual CNS antigen specificities of the T cells used to induce CNS inflammation (Mann–Whitney U test with Bonferroni-Holm correction; p = 0,732 for MBP/S100β, p = 0,024 for MBP/MOG, p = 0,048 for S100β/MOG).

Article Snippet: T cell lines against myelin basic protein (MBP, from guinea pig, Sigma), myelin oligodendrocyte glycoprotein (MOG, recombinant N-terminal peptide 1–125, rat, own production) and the astrocytic Ca 2+ binding protein S100β (bovine, Sigma) were intraperitoneally injected to induce EAE.

Techniques: MANN-WHITNEY

Journal: Acta Neuropathologica Communications

Article Title: T cell-activation in neuromyelitis optica lesions plays a role in their formation

doi: 10.1186/2051-5960-1-85

Figure Lengend Snippet: Differences in T cell activation translate into differences in IFN-γ production, which affects the microglial expression of complement factors and complement inhibitors. (A) Normalized relative expression of IFN-γ mRNA in relation to the house-keeping gene beta actin (calculated using the following equation: 2 -ΔCt = 2 -[Ct(GOI)-Ct(HKG)] ( GOI – Gene of interest; HKG – house-keeping gene; ) are shown. Statistically significant differences (*, as determined by one-way ANOVA followed by Bonferroni’s post-hoc testing) were observed between MBP-specific T cells and their MOG- or S100β-specific counterparts. (B) Pathways contributing to the complement cascade and alterations in gene expression (encircled in red: upregulation; encircled in green: downregulation) of complement factors and inhibitors by IFN-γ treated microglia. (C-D) Changes in gene expression of complement factors and inhibitors (C) and of Fcgr3 (D) in IFN-γ treated microglia. These cells were treated for 48 hrs with 100 ng/ml IFN-γ. Subsequently, the mRNA of these cells was harvested and subjected to gene expression profiling. Log2-fold changes in gene expression and differences in the normalized signal intensities (nSI) of complement components/factors and Fcgr3 between IFN-γ and vehicle control-treated microglial cultures are shown (2 different, independent samples per treatment group). Genes with elevated expression in the IFN-γ treated group are labeled red, genes with lower expression levels are labeled green.

Article Snippet: T cell lines against myelin basic protein (MBP, from guinea pig, Sigma), myelin oligodendrocyte glycoprotein (MOG, recombinant N-terminal peptide 1–125, rat, own production) and the astrocytic Ca 2+ binding protein S100β (bovine, Sigma) were intraperitoneally injected to induce EAE.

Techniques: Activation Assay, Expressing, Labeling

Journal: PLoS ONE

Article Title: Nerve Terminal Degeneration Is Independent of Muscle Fiber Genotype in SOD1 G93A Mice

doi: 10.1371/journal.pone.0009802

Figure Lengend Snippet: A. Low magnification view of a wild-type MG muscle transplanted 2 months earlier into a transgenic mouse that expresses EGFP (green) under the direction of the S100 promoter. Endplate ACHRs are stained with α−Btx (red). This panel illustrates the general finding that all endplate staining colocalized with EGFP. B. EGFP fluorescence was absent at motor endplates in an MG muscle from an EGFP-expressing transgenic transplanted into a wild-type host (B1) but the same endplates stained positively for S100 (green, B2), demonstrating that terminal Schwann cells were derived from the host. C. To exclude the possible survival of wild-type TSCs after transplantation of wild-type muscle into the ECFP transgenic (C1), muscle sections were first labeled for S100 (C2) but all labeling was found to colocalize with EGFP fluorescence (C3) providing further evidence that wild-type TSCs did not survive transplantation.

Article Snippet: Synaptic vesicles were labeled using a rabbit polyclonal antibody directed at synaptophysin (1∶100, Santa Cruz Biotechnology), and terminal Schwann cells (TSC) were labeled with a rabbit polyclonal antibody against S100 Ca 2+ -binding protein (Dako).

Techniques: Transgenic Assay, Staining, Fluorescence, Expressing, Derivative Assay, Transplantation Assay, Labeling