Journal: Frontiers in Molecular Neuroscience

Article Title: Galectin-3 Negatively Regulates Hippocampus-Dependent Memory Formation through Inhibition of Integrin Signaling and Galectin-3 Phosphorylation

doi: 10.3389/fnmol.2017.00217

Figure Lengend Snippet: Regional distribution of galectins in the rat brain and endogenous galectin-3 expression in the mouse CA1 area. (A) Western blot analysis showing the expression level of galectin-1, galectin-3 and galectin-7 in several regions in the rat brain. (B) Quantitative analyses of galectin-1, galectin-3 and galectin-7 expression level in different brain regions. N = 4 each group. Gal-1: galectin-1, Gal-3: galectin-3, Gal-7: galectin-7. OB: olfactory bulb, FC: frontal cortex, ST: striatum, Amy: amygdale, Hip: hippocampus. Data are mean ± SEM. (C) Immunohistochemistry showing the distribution of galectin-3, NeuN and their co-localization in rat hippocampal CA1 area. Scale bar equals 50 μm.

Article Snippet: The clarified lysate (0.5 mg) was immunoprecipitated with 3 μl of anti-galectin-3 antibody (Catalog No. MAB1197, R&D systems, Minneapolis, MN, USA) or anti-integrin α3 antibody (Millipore, Bedford, MA, USA) at 4°C for 2 h. Twenty microliter (10% slurry) of the Mag Sepharose Xtra beads (GE Healthcare, Pittsburgh, PA, USA) was added to the IP reaction product for overnight to catch the immune complex.

Techniques: Expressing, Western Blot, Immunohistochemistry

Journal: Frontiers in Molecular Neuroscience

Article Title: Galectin-3 Negatively Regulates Hippocampus-Dependent Memory Formation through Inhibition of Integrin Signaling and Galectin-3 Phosphorylation

doi: 10.3389/fnmol.2017.00217

Figure Lengend Snippet: Contextual fear-conditioning training and NMDA decrease galectin-3 expression in the rat hippocampus. (A) Western blot showing galectin-3 expression in the CA1 area of trained and non-trained rats. (B) Quantitative analysis of galectin-3 expression in CA1 area of trained and non-trained rats ( t (1,18) = 15.27, P < 0.001). (C) Western blot and quantitative analysis of galectin-3 expression in the amygdala of trained and non-trained rats ( t (1,18) = 0.02, P > 0.05). (D) Western blot and quantitative analysis of galectin-3 expression in the striatum of trained and non-trained rats ( t (1,18) = 0.38, P > 0.05). N = 10 each group. (E) Western blot showing the effect of NMDA injection (12.5 mM) on the expression level of galectin-3 and pERK in rat CA1 area. Quantitative analysis of the effect of NMDA on (F) Galectin-3 expression ( t (1,10) = 51.11, P < 0.001) and (G) pERK1/ERK1, pERK2/ERK2 levels ( t (1,10) = 6.69, P < 0.001 for pERK1 and t (1,10) = 15.12, P < 0.001 for pERK2) in the CA1 area. N = 6 each group. Data are mean ± SEM. # P < 0.001.

Article Snippet: The clarified lysate (0.5 mg) was immunoprecipitated with 3 μl of anti-galectin-3 antibody (Catalog No. MAB1197, R&D systems, Minneapolis, MN, USA) or anti-integrin α3 antibody (Millipore, Bedford, MA, USA) at 4°C for 2 h. Twenty microliter (10% slurry) of the Mag Sepharose Xtra beads (GE Healthcare, Pittsburgh, PA, USA) was added to the IP reaction product for overnight to catch the immune complex.

Techniques: Expressing, Western Blot, Injection

Journal: Frontiers in Molecular Neuroscience

Article Title: Galectin-3 Negatively Regulates Hippocampus-Dependent Memory Formation through Inhibition of Integrin Signaling and Galectin-3 Phosphorylation

doi: 10.3389/fnmol.2017.00217

Figure Lengend Snippet: Overexpressionof galectin-3 impairs fear memory but fear memory is enhanced in galectin-3 knockout (KO) mice. (A) Immediate freezing response ( t (1,14) = 0.74, P > 0.05) and retention performance ( t (1,14) = 2.95, P = 0.01) in rats transfected with Flag-galectin-3WT plasmid or Flag-vector plasmid. N = 8 each group. (B) Immunoprecipitation and immunoblotting against the Flag tag confirming the expression of galectin-3 in Flag-galectin-3 transfected animals (left). Immunohistochemistry with Cy3-conjugated secondary antibody against Flag confirming the expression of galectin-3 in rat CA1 area (right). Scale bar equals 250 μm. (C) Immediate freezing response ( t (1,18) = 1.1, P > 0.05) and retention performance ( t (1,18) = 2.14, P < 0.05) in wild-type (WT) and galectin-3 KO (Gal-3 −/− ) mice. N = 10 each group. (D) Western blot showing the expression level of galectin-3, IL-6 and TNFα in WT and Gal-3 −/− mice ( t (1,18) = 0.4 for IL-6 and t (1,18) = 0.54 for TNFα, both P > 0.05). N = 10 each group. (E) fEPSP slope in WT and galectin-3 KO mice subjected to TBS stimulation and LTP recording ( t (1,14) = 4.32, P < 0.001). N = 8 each group. TBS: theta burst stimulation. (F) The input/output (I/O) fEPSP slope ( F (1,32) = 0.045, P > 0.05), (G) the amplitude of fiber volley ( F (1,32) = 0.004, P > 0.05) and (H) the I/O vs. fiber volley curve ( F (1,32) = 0.36, P > 0.05) were generated in response to different intensities of presynaptic fiber stimulation of the Schaffer collateral pathway. N = 8 each group. Data are mean ± SEM. * P < 0.05, ** P < 0.01, # P < 0.001.

Article Snippet: The clarified lysate (0.5 mg) was immunoprecipitated with 3 μl of anti-galectin-3 antibody (Catalog No. MAB1197, R&D systems, Minneapolis, MN, USA) or anti-integrin α3 antibody (Millipore, Bedford, MA, USA) at 4°C for 2 h. Twenty microliter (10% slurry) of the Mag Sepharose Xtra beads (GE Healthcare, Pittsburgh, PA, USA) was added to the IP reaction product for overnight to catch the immune complex.

Techniques: Knock-Out, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, FLAG-tag, Expressing, Immunohistochemistry, Generated

Journal: Frontiers in Molecular Neuroscience

Article Title: Galectin-3 Negatively Regulates Hippocampus-Dependent Memory Formation through Inhibition of Integrin Signaling and Galectin-3 Phosphorylation

doi: 10.3389/fnmol.2017.00217

Figure Lengend Snippet: Water-maze training decreases galectin-3 expression in the rat hippocampus and spatial memory is enhanced in galectin-3 KO mice. (A) Western blot showing galectin-3 expression in CA1 area of rats subjected to water-maze training or swimming control. The quantified result is also shown ( t (1,10) = 23.75, P < 0.001). N = 6 each group. (B) Spatial acquisition of WT and galectin-3 KO mice ( F (1,14) = 5.34, q = 3.33, P < 0.05). (C) Representative swim pattern and probe trial performance of WT and galectin-3 KO mice (Chi square followed by t -test, t (1,14) = 1.61, P > 0.05). N = 8 each group. (D) Visible platform learning of WT and galectin-3 KO mice ( F (1,14) = 0.56, P > 0.05). (E) The swim speed of WT and galectin-3 KO mice ( t (1,14) = 2.06, P > 0.05). N = 8 each group. Data are mean ± SEM. # P < 0.001.

Article Snippet: The clarified lysate (0.5 mg) was immunoprecipitated with 3 μl of anti-galectin-3 antibody (Catalog No. MAB1197, R&D systems, Minneapolis, MN, USA) or anti-integrin α3 antibody (Millipore, Bedford, MA, USA) at 4°C for 2 h. Twenty microliter (10% slurry) of the Mag Sepharose Xtra beads (GE Healthcare, Pittsburgh, PA, USA) was added to the IP reaction product for overnight to catch the immune complex.

Techniques: Expressing, Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: Galectin-3 Negatively Regulates Hippocampus-Dependent Memory Formation through Inhibition of Integrin Signaling and Galectin-3 Phosphorylation

doi: 10.3389/fnmol.2017.00217

Figure Lengend Snippet: Galectin-3 negatively regulates fear memory through inhibition of integrin α3-mediated signaling. (A) Left and middle panel, Co-IP experiment showing galectin-3 association with integrin α3 and vice versa in rat CA1 area. This association is reduced after training (upper panels). Western blot showing the expression of integrin α3 and galectin-3 in trained and non-trained animals (lower-left panel). Right panel, Co-IP experiment showing that galectin-3 is not associated with NR1 in rat hippocampus, and fear-conditioning training does not apparently alter this relationship. Western blot showing that NR1 expression is increased, but galectin-3 expression is decreased in hippocampal cell lysate of trained animals (lower panel). Experiments are in two repeats. (B) Western blot showing the expression level of integrin α3 ( t (1,10) = 5.18, P < 0.001), phospho-focal adhesion kinase (FAK; t (1,10) = 36, P < 0.001) and FAK in trained and non-trained animals. (C) Quantitative analysis of integrin α3 and phospho-FAK expression in trained and non-trained animals. N = 6 each group. (D) Immediate freezing response ( F (2,24) = 0.33, P > 0.05) and retention performance in galectin-3 siRNA + Flag-vector and galectin-3 siRNA + Flag-FAKY397F co-transfected animals ( F (2,24) = 7.0, P < 0.001; q = 3.81, P = 0.01 comparing the galectin-3 siRNA + Flag-vector group with control siRNA + Flag-vector group; q = 5.08, P < 0.01 comparing the galectin-3 siRNA + Flag-FAKY397F group with galectin-3 siRNA + Flag-vector group). N = 9 each group. (E) Western blot showing the expression level of galectin-3 ( F (2,24) = 476.38, P < 0.001; q = 36.42 and q = 39.05, respectively; both P < 0.001), phospho-FAK ( F (2,24) = 87.36, P < 0.001; q = 18.63, P < 0.001 comparing the galectin-3 siRNA + Flag-vector group with control siRNA + Flag-vector group; q = 10.63, P < 0.001 comparing the galectin-3 siRNA + Flag-FAKY397F group with galectin-3 siRNA + Flag-vector group) and FAK in galectin-3 siRNA + Flag-vector and galectin-3 siRNA + Flag-FAKY397F co-transfected animals. N = 9 each group. Immunoprecipitation and immunoblotting against Flag confirming the expression of FAKY397F in Flag-FAKY397F-transfected animals (lower panel). (F) Quantitative analysis of galectin-3, phospho-FAK and FAK expression in the same animals. N = 9 each group. Data are mean ± SEM. ** P < 0.01, # P < 0.001.

Article Snippet: The clarified lysate (0.5 mg) was immunoprecipitated with 3 μl of anti-galectin-3 antibody (Catalog No. MAB1197, R&D systems, Minneapolis, MN, USA) or anti-integrin α3 antibody (Millipore, Bedford, MA, USA) at 4°C for 2 h. Twenty microliter (10% slurry) of the Mag Sepharose Xtra beads (GE Healthcare, Pittsburgh, PA, USA) was added to the IP reaction product for overnight to catch the immune complex.

Techniques: Inhibition, Co-Immunoprecipitation Assay, Western Blot, Expressing, Plasmid Preparation, Transfection, Immunoprecipitation

Journal: Frontiers in Molecular Neuroscience

Article Title: Galectin-3 Negatively Regulates Hippocampus-Dependent Memory Formation through Inhibition of Integrin Signaling and Galectin-3 Phosphorylation

doi: 10.3389/fnmol.2017.00217

Figure Lengend Snippet: Galectin-3 negatively regulates fear memory through phosphorylation of galectin-3 at Ser-6. (A) Co-IP experiment (immunoprecipitation with anti-galectin-3 antibody and immunoblotting with anti-p-Ser antibody) showing the candidate p-galectin-3 band in non-trained animals in rat CA1 area. This association is dramatically reduced after training (upper panel). Western blot showing the expression level of galectin-3 in trained and non-trained animals (lower panel). Experiments are in two repeats. (B) Immediate freezing response ( F (2,21) = 0.88, P > 0.05) and retention performance in galectin-3WT and galectin-3S6A-transfected animals ( F (2,21) = 8.89, P < 0.001; q = 3.49, P < 0.05 comparing the galectin-3WT with Flag-vector group; q = 5.93, P = 0.01 comparing the Flag-galectin-3S6A group with Flag-galectin-3WT group; q = 2.44, P > 0.05 comparing the Flag-galectin-3S6A group with Flag-vector group). N = 8 each group. Immunoprecipitation and immunoblotting against the Flag tag confirming the expression of galectin-3 and galectin-3S6A in Flag-galectin-3WT and Flag-galectin-3S6A-transfected animals, respectively (lower panel). (C) Immediate freezing response ( F (2,21) = 0.01, P > 0.05) and retention performance in galectin-3WT and galectin-3S6D-transfected animals ( F (2,21) = 11.86, P < 0.001; q = 3.42, P < 0.05 comparing the galectin-3WT with Flag-vector group; q = 3.47, P < 0.05 comparing the Flag-galectin-3S6D group with Flag-galectin-3WT group; q = 6.89, P < 0.001 comparing the Flag-galectin-3S6D group with Flag-vector group). N = 8 each group. Immunoprecipitation and immunoblotting against the Flag tag confirming the expression of galectin-3 and galectin-3S6D in Flag-galectin-3WT and Flag-galectin-3S6D-transfected animals, respectively (lower panel). Data are mean ± SEM. * P < 0.05, ** P < 0.01.

Article Snippet: The clarified lysate (0.5 mg) was immunoprecipitated with 3 μl of anti-galectin-3 antibody (Catalog No. MAB1197, R&D systems, Minneapolis, MN, USA) or anti-integrin α3 antibody (Millipore, Bedford, MA, USA) at 4°C for 2 h. Twenty microliter (10% slurry) of the Mag Sepharose Xtra beads (GE Healthcare, Pittsburgh, PA, USA) was added to the IP reaction product for overnight to catch the immune complex.

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Expressing, Transfection, Plasmid Preparation, FLAG-tag