recombinant protein galanin  (USV Limited)

Journal: eLife

Article Title: Co-aggregation and secondary nucleation in the life cycle of human prolactin/galanin functional amyloids

doi: 10.7554/eLife.73835

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , Galanin , USV Limited (Mumbai, India) , Custom synthesis , .

Techniques: Control, Recombinant, Software

Journal: Glia

Article Title: Galanin is an autocrine myelin and oligodendrocyte trophic signal induced by leukemia inhibitory factor.

doi: 10.1002/glia.22798

Figure Lengend Snippet: FIGURE 2: LIF-induced protein changes in oligodendrocytes and conditioned medium. (a) Concentrated media of rat oligodendrocyte (OC, upper panel) and CG4 cell cultures (middle panel), cultured for 48 h, were subject to Western blotting using anti-galanin antibodies. Cultures exposed to LIF at 100 ng/mL showed marked up-regulation of a 14 kD band consistent with secretion of preprogalanin in com- parison with cells exposed only to insulin (Nil). Rat astrocytes (lower panel) did not exhibit the same LIF-induced response (100 ng/mL, 48 h). Independent replicate experiments are shown in each blot, and major protein bands visualized by Amido Black stains of the same membranes are shown as loading controls. (b) Western blot of concentrated media of OC cultures exposed to either LIF or insulin showed evidence of a band at 7 kD, consistent with IGF2 secretion. Unlike preprogalanin, IGF2 levels were not increased by LIF at the protein level.

Article Snippet: Cells were maintained in SATO media for 72 h in the presence of: (1) no additional factors, (2) recombinant rat galanin (1 nM), recombinant rat TNF-a (100 ng/mL R&D systems) and both galanin (1 nM) and TNF-a (100 ng/mL).

Techniques: Cell Culture, Western Blot

Journal: Glia

Article Title: Galanin is an autocrine myelin and oligodendrocyte trophic signal induced by leukemia inhibitory factor.

doi: 10.1002/glia.22798

Figure Lengend Snippet: FIGURE 3: Galanin-containing secretory granules in oligodendrocytes are induced by LIF in vitro and in vivo. CNPase-positive oligoden- drocytes (OC) (red) in culture containing insulin (a) lacked galanin expression (green). After exposure to LIF (100 ng/mL, 48 h), CNPase positive OC (b) showed punctate cytoplasmic galanin staining (arrow). CNPase positive OC (c, red) showed some granular cytoplasmic staining for secretogranin-2 (green), but this was markedly up-regulated after 48 h of LIF exposure (d, arrow). CNPase (red) positive OC were visualized in linear arrays in the corpus callosum (cc, panels e and f). After in vivo exposure to cuprizone for 18 days, 75% of OC in the cc of wild-type mice exhibited cytoplasmic galanin immunoreactivity (green, e, arrows), whereas OC in the cc of LIF knockout mice exhibit no galanin positivity (f). Images in g–i are from PLP-dsRED mice expressing the dsRED fluorophore, stained with anti-galanin anti- body (green). After 21 days of systemic cuprizone exposure, in the paraventricular thalamic nucleus (g), galanin-positive neuronal cell bodies and fibers were seen next to galanin-negative OC (red). In contrast, in the demyelinating cc around 75% of PLP-ds red positive OC expressed cytoplasmic galanin (h, arrows). Occasional dsRED negative cells also expressed galanin (arrowhead). (i) High power image from the cc illustrating granular cytoplasmic galanin staining in a dsRED positive cell. Scale bars for panels a–f, i 5 10 lm; for panels g and h 5 50 lm.

Article Snippet: Cells were maintained in SATO media for 72 h in the presence of: (1) no additional factors, (2) recombinant rat galanin (1 nM), recombinant rat TNF-a (100 ng/mL R&D systems) and both galanin (1 nM) and TNF-a (100 ng/mL).

Techniques: In Vitro, In Vivo, Expressing, Staining, Knock-Out

Journal: Glia

Article Title: Galanin is an autocrine myelin and oligodendrocyte trophic signal induced by leukemia inhibitory factor.

doi: 10.1002/glia.22798

Figure Lengend Snippet: FIGURE 4: Galanin is a survival factor for oligodendrocytes. (a) and (b) Oligodendrocyte (OC) survival 48 or 72 h after addition of either recombinant rat galanin (3.2 ng/mL 5 1 nM), LIF (100 ng/mL), NT3 (5 ng/mL), or insulin (5 mg/mL), compared with basal media only. Post- mitotic OC were plated at 100 cells/well (20 wells per condition), and the data are presented as live cell counts (mean 6 SD). Each growth factor condition significantly increased survival above basal (*P < 0.05, ***P < 0.001, ANOVA). (c) To assess any proliferative effect of galanin, oligodendrocyte progenitor cells (OPCs) were exposed to galanin (1 nM), PDGF (5 ng/mL), PDGF (2.5 ng/mL) and the combination of PDGF (2.5 ng/mL) and galanin (1 nM). Maximal-dose PDGF (5 ng/mL) induced around 40% BRDU-positivity, whereas gal- anin induced none. The combination of half-maximal dose PDGF and galanin did not promote OPC proliferation above that induced by half-maximal dose PDGF alone (*** denotes significant difference to basal proliferation rate, P < 0.001, ANOVA, data shown are mean % 6 SD). (d) Relative mRNA expression of the three known galanin receptor species in postmitotic OC assessed by real-time PCR. GalR2 mRNA is around 900-fold more abundantly expressed than the other receptor species (mean of three separate experiments shown, low- est GalR1 expression was standardized to 1, error bars 5 SD, *** denotes significant difference compared with GalR1 expression, P < 0.001, ANOVA). (e) Time-course of phosphorylation of key signaling intermediaries after galanin (1mg/mL) exposure in vitro. ERK 1/2 is rapidly phosphorylated, whereas AKT and S6 phosphorylation levels are unaltered. Representative images of duplicate experiments shown.

Article Snippet: Cells were maintained in SATO media for 72 h in the presence of: (1) no additional factors, (2) recombinant rat galanin (1 nM), recombinant rat TNF-a (100 ng/mL R&D systems) and both galanin (1 nM) and TNF-a (100 ng/mL).

Techniques: Recombinant, Expressing, Real-time Polymerase Chain Reaction, Phospho-proteomics, In Vitro

Journal: Glia

Article Title: Galanin is an autocrine myelin and oligodendrocyte trophic signal induced by leukemia inhibitory factor.

doi: 10.1002/glia.22798

Figure Lengend Snippet: FIGURE 5: LIF-mediated oligodendrocyte survival in vitro is galanin-dependent. Mouse oligodendrocyte progenitor cells from wild-type (WT) and galanin knockout (Gal KO) mice were expanded in vitro and then plated under differentiating condi- tions at high density in Sato (Nil) or Sato plus LIF (100 ng/mL). Live cells were counted at 48, 72, and 96 h under bright field microscopy (average of 10 fields in each of two plates/condi- tion). Results were combined from independent duplicate experi- ments, and survival is expressed as mean (6SD) live cell count/ field. Addition of LIF to WT oligodendrocyte (OC) cultures increased survival at 48, 72, and 96 h. Addition of LIF to Gal KO OC cultures did not produce a survival effect at any time-point (***P < 0.001; *P < 0.05, ANOVA).

Article Snippet: Cells were maintained in SATO media for 72 h in the presence of: (1) no additional factors, (2) recombinant rat galanin (1 nM), recombinant rat TNF-a (100 ng/mL R&D systems) and both galanin (1 nM) and TNF-a (100 ng/mL).

Techniques: In Vitro, Knock-Out, Microscopy, Cell Counting

Journal: Glia

Article Title: Galanin is an autocrine myelin and oligodendrocyte trophic signal induced by leukemia inhibitory factor.

doi: 10.1002/glia.22798

Figure Lengend Snippet: FIGURE 6: Galanin knockout mice exhibit more severe cuprizone demyelination. Galanin knockout (KO) mice (n 5 11) and wild-type mice (n 5 11) were fed cuprizone 0.2% in milled chow for 4 weeks. Myelinated axon density was quantitated in the severely demyelinated caudal portion of the corpus callosum (a, black square) in two fields/mouse from methylene-blue stained thin sections (b and c). (b) Illus- trates severe loss of myelinated axons in a galanin KO mouse, (c) shows less severe loss from a wild-type mouse, arrowheads indicate myelinated axons, scale bars 5 40 mm, unit area 5 17,400 mm2. Galanin KO mice had less residual myelinated axons than wild-type mice (d, average myelinated axon densities from individual mice shown in scatterplot, lines indicate means, ***P < 0.001, t-test).

Article Snippet: Cells were maintained in SATO media for 72 h in the presence of: (1) no additional factors, (2) recombinant rat galanin (1 nM), recombinant rat TNF-a (100 ng/mL R&D systems) and both galanin (1 nM) and TNF-a (100 ng/mL).

Techniques: Knock-Out, Staining