Journal: Molecular cell

Article Title: Ubiquitin profiling of lysophagy identifies actin stabilizer CNN2 as a target of VCP/p97 and uncovers a link to HSPB1.

doi: 10.1016/j.molcel.2022.06.012

Figure Lengend Snippet: Figure 2. CNN2 translocates to damaged lysosomes and is ubiquitylated for timely dissociation (A) Dynamic association of endogenous CNN2 with damaged lysosomes in HeLa cells. Immunofluorescence of CNN2 and Gal3 as lysosomal damage marker after mock or LLOMe treatment for indicated time periods. Note CNN2 translocation and dissociation before Gal3 clearance. (B) Graph represents the percentage of CNN2 and Gal3-positive vesicles among all Gal3-positive vesicles per cell. More than 30 cells were quantified per condition in each experiment (n = 4 biologically independent experiments). One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test, ** p = 0.0036, *** p = 0.0002, and **** p < 0.0001. Error bars represent the mean ± SEM. (C) Schematic domain structure of CNN2 with positions of identified ubiquitylation sites indicated. CH domain, calponin homology domain; ABS1/2, actin- binding sites. (D) HeLa cells expressing CNN2-GFP wild type or harboring lysine-to-arginine substitutions in the CH domain (CH-KR) following mock or LLOMe treatment as indicated. Note that CNN2 wild type dissociates from LAMP1 vesicles within 3 h, but the ubiquitylation mutants persist. See Figure S3A for CNN2-GFP 5xKR covering the 5 ubiquitylation sites detected by MS. (E) Quantification of (D). Percentage of LAMP1 vesicles positive for CNN2. More than 20 cells were quantified. One-way ANOVA with Tukey’s multiple comparison test, *** p = 0.0003 and **** p < 0.0001; ns, not significant. Error bars represent the mean ± SD. (F) Live-cell imaging of HeLa cells expressing CNN2-GFP CH-KR and mCherry-Gal3. Lysosomes were loaded with photosensitizer AIPcS2a, irradiated in the indicated area, and chased over the course of 1 h. See Figure S3E for wild type and CNN2 5xKR imaging data. (A, D, and F) Scale bars, 10 mm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rat monoclonal anti-Gal3 (IF, 1:500) Santa Cruz Biotechnology Cat#sc-23938; RRID:AB_627658 Rabbit monoclonal anti-LAMP1 (IF, 1:500) Cell Signaling Technology Cat#9091; RRID:AB_2687579 Rabbit polyclonal anti-CNN2 (IF, 1:500; WB, 1:1000) Thermo Fischer Scientific Cat#PA5-61878; RRID:AB_2639249 Mouse monoclonal anti-CNN2 (WB, 1:1000) Origene Cat#TA503688; RRID:AB_11124845 Rabbit polyclonal anti-LC3 (IF, 1:500; WB, 1:1000) MBL Cat#PM036; RRID:AB_2274121 Rabbit polyclonal anti-p62 (IF, 1:500; WB, 1:1000) Sigma-Aldrich Cat#P0067; RRID:AB_1841064 Mouse monoclonal anti-p62 (WB, 1:1000) Abnova Cat#A00008878 Mouse monoclonal anti-HSP27 (IF, 1:500) Thermo Fischer Scientific Cat#MA3-015; RRID:AB_325463 Rabbit polyclonal anti-HSP27 (WB, 1:1000) Thermo Fischer Scientific Cat#PA5-78010; RRID:AB_2735924 Rabbit polyclonal anti-TAX1BP1 (WB, 1:1000) Sigma Cat#HPA024432; RRID:AB_1857783 Mouse monoclonal anti-p97 (WB, 1:2000) Santa Cruz Biotechnology Cat#sc-57492; RRID:AB_793927 Mouse monoclonal anti-P4D1 (WB, 1:1000) Cell Signaling Technology Cat#3936; RRID:AB_331292 Mouse monoclonal anti-Tubulin (WB, 1:5000) Sigma-Aldrich Cat#T-5168; RRID:AB_477579 Mouse monoclonal anti-GFP (WB, 1:10000) Roche Cat#11814460001; RRID:AB_390913 horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (WB, 1:10000) Biorad Cat#170-6515; RRID:AB_11125142 horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (WB, 1:10000) Biorad Cat#1706516; RRID:AB_11125547 Alexa Fluor-conjugated goat anti-rabbit, Alexa Fluor 568 (IF, 1:500) Invitrogen Cat#A11011; RRID:AB_143157 Alexa Fluor-conjugated goat anti-rabbit, Alexa Fluor 488 (IF, 1:500) Life technologies Cat#A11034; RRID:AB_2576217 Alexa Fluor-conjugated goat anti-rabbit, Alexa Fluor 633 (IF, 1:500) Thermo Fisher Scientific Cat#A21071; RRID:AB_2535732 Alexa Fluor-conjugated goat anti-mouse, Alexa Fluor 488 (IF, 1:500) Thermo Fisher Scientific Cat#10696113 Alexa Fluor-conjugated goat anti-mouse, Alexa Fluor 594 (IF, 1:500) Thermo Fisher Scientific Cat#A-11032; RRID:AB_2534091 Alexa Fluor-conjugated goat anti-mouse, Alexa Fluor 633 (IF, 1:500) Thermo Fisher Scientific Cat#10246252 Alexa Fluor-conjugated goat anti-rat, Alexa Fluor 488 (IF, 1:500) Thermo Fisher Scientific Cat#A-11006; RRID:AB_2534074 Alexa Fluor-conjugated goat anti-rat, Alexa Fluor 568 (IF, 1:500) Thermo Fisher Scientific Cat#A-11077; RRID:AB_2534121 (Continued on next page) e1 Molecular Cell 82, 2633–2649.e1–e7, July 21, 2022

Techniques: Marker, Translocation Assay, Comparison, Binding Assay, Expressing, Live Cell Imaging, Irradiation, Imaging

Journal: Molecular cell

Article Title: Ubiquitin profiling of lysophagy identifies actin stabilizer CNN2 as a target of VCP/p97 and uncovers a link to HSPB1.

doi: 10.1016/j.molcel.2022.06.012

Figure Lengend Snippet: Figure 7. p97 and HSPB1 cooperate in removing ubiquitylated CNN2 from lysosomes (A) HSPB1 is trapped on CNN2 in the absence of p97 activity. Proximity biotinylation analyzed by western blot in indicated conditions of lysosome damage (LLOMe) and p97 inhibition (NMS-873). Cells expressing CNN2-APEX2 were treated with LLOMe for 1 h and chased for 2 h after washout prior to the addition of H2O2 to trigger biotinylation. (B) Loss of p97 or HSPB1 function impairs CNN2 dissociation in a non-additive manner. The time course of CNN2 localization to damaged lysosomes upon p97 inhibition (NMS-873), HSPB1 depletion, or a combination of both as indicated. Scale bars, 10 mm. (C) Quantification of (B). The graph represents the percentage of CNN2 and Gal3-positive vesicles among all Gal3-positive vesicles per cell. More than 30 cells were quantified per condition in each experiment (n = 3 biologically independent experiments). Two-way ANOVA with Tukey’s multiple comparison test, **** p < 0.0001, *** p = 0.0004, * p = 0.0118. Error bars represent the mean ± SEM. (D) HSPB1 acts downstream of ubiquitylation together with p97. HeLa cells stably expressing CNN2-GFP were LLOMe treated for 1 h and chased for 2 h prior to denaturing lysis upon indicated treatments after p97 inhibition (NMS-873), HSPB1 siRNA, or a combination of both as indicated. Ubiquitylation of CNN2 was assessed by western blot after immunoprecipitation using GFP nanobodies. Note that loss of HSPB1, or p97 inhibition, leads to the increased accumulation of ubiquitylated CNN2 after LLOMe-induced damage, but that effects are not additive. (E) Model. After lysosome damage, various resident proteins become ubiquitylated to serve as an anchor point for autophagy-receptor-mediated recruitment of the LC3-decorated phagophore. CNN2 is recruited by associating with the p62 autophagy receptor and then stabilizes actin filaments that assist phagophore formation. CNN2 needs to be subsequently ubiquitylated and removed by p97 with the help of HSPB1 to allow efficient phagophore formation.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rat monoclonal anti-Gal3 (IF, 1:500) Santa Cruz Biotechnology Cat#sc-23938; RRID:AB_627658 Rabbit monoclonal anti-LAMP1 (IF, 1:500) Cell Signaling Technology Cat#9091; RRID:AB_2687579 Rabbit polyclonal anti-CNN2 (IF, 1:500; WB, 1:1000) Thermo Fischer Scientific Cat#PA5-61878; RRID:AB_2639249 Mouse monoclonal anti-CNN2 (WB, 1:1000) Origene Cat#TA503688; RRID:AB_11124845 Rabbit polyclonal anti-LC3 (IF, 1:500; WB, 1:1000) MBL Cat#PM036; RRID:AB_2274121 Rabbit polyclonal anti-p62 (IF, 1:500; WB, 1:1000) Sigma-Aldrich Cat#P0067; RRID:AB_1841064 Mouse monoclonal anti-p62 (WB, 1:1000) Abnova Cat#A00008878 Mouse monoclonal anti-HSP27 (IF, 1:500) Thermo Fischer Scientific Cat#MA3-015; RRID:AB_325463 Rabbit polyclonal anti-HSP27 (WB, 1:1000) Thermo Fischer Scientific Cat#PA5-78010; RRID:AB_2735924 Rabbit polyclonal anti-TAX1BP1 (WB, 1:1000) Sigma Cat#HPA024432; RRID:AB_1857783 Mouse monoclonal anti-p97 (WB, 1:2000) Santa Cruz Biotechnology Cat#sc-57492; RRID:AB_793927 Mouse monoclonal anti-P4D1 (WB, 1:1000) Cell Signaling Technology Cat#3936; RRID:AB_331292 Mouse monoclonal anti-Tubulin (WB, 1:5000) Sigma-Aldrich Cat#T-5168; RRID:AB_477579 Mouse monoclonal anti-GFP (WB, 1:10000) Roche Cat#11814460001; RRID:AB_390913 horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (WB, 1:10000) Biorad Cat#170-6515; RRID:AB_11125142 horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (WB, 1:10000) Biorad Cat#1706516; RRID:AB_11125547 Alexa Fluor-conjugated goat anti-rabbit, Alexa Fluor 568 (IF, 1:500) Invitrogen Cat#A11011; RRID:AB_143157 Alexa Fluor-conjugated goat anti-rabbit, Alexa Fluor 488 (IF, 1:500) Life technologies Cat#A11034; RRID:AB_2576217 Alexa Fluor-conjugated goat anti-rabbit, Alexa Fluor 633 (IF, 1:500) Thermo Fisher Scientific Cat#A21071; RRID:AB_2535732 Alexa Fluor-conjugated goat anti-mouse, Alexa Fluor 488 (IF, 1:500) Thermo Fisher Scientific Cat#10696113 Alexa Fluor-conjugated goat anti-mouse, Alexa Fluor 594 (IF, 1:500) Thermo Fisher Scientific Cat#A-11032; RRID:AB_2534091 Alexa Fluor-conjugated goat anti-mouse, Alexa Fluor 633 (IF, 1:500) Thermo Fisher Scientific Cat#10246252 Alexa Fluor-conjugated goat anti-rat, Alexa Fluor 488 (IF, 1:500) Thermo Fisher Scientific Cat#A-11006; RRID:AB_2534074 Alexa Fluor-conjugated goat anti-rat, Alexa Fluor 568 (IF, 1:500) Thermo Fisher Scientific Cat#A-11077; RRID:AB_2534121 (Continued on next page) e1 Molecular Cell 82, 2633–2649.e1–e7, July 21, 2022

Techniques: Activity Assay, Western Blot, Inhibition, Expressing, Comparison, Stable Transfection, Lysis, Immunoprecipitation

Journal: Cell Death and Differentiation

Article Title: Galectin-3 promotes Aβ oligomerization and Aβ toxicity in a mouse model of Alzheimer’s disease

doi: 10.1038/s41418-019-0348-z

Figure Lengend Snippet: Aβ oligomerization is reduced in Gal-3 KO mice injected with Aβ. a WT and Gal-3 KO mice (3 months old) were injected with 1% NH 4 OH or Aβ (5 μg) in the CA1 area. Animals were sacrificed 48 h after injection and dissected hippocampal tissues were subjected to Western blot analysis of Aβ oligomerization and Gal-3 expression. b Quantified results of HMW and LMW Aβ oligomerization [ n = 4 per group; F (3,12) = 149.28, P < 0.001 for HMW; q = 26.22, P < 0.001 for the WT+NH 4 OH group versus the WT+Aβ group and q = 13.78, P < 0.001 for the WT+Aβ group versus the Gal-3 KO+Aβ group; F (3,12) = 48.02 and P < 0.001 for LMW; q = 14.31, P < 0.001 for the WT+NH 4 OH group versus the WT+Aβ group and q = 6.74, P < 0.001 for the WT+Aβ group versus the Gal-3 KO+Aβ group]. c Quantified results for Gal-3 expression in the same mouse groups [ F (3,12) = 83.43, P < 0.001; q = 15.63, P < 0.001 for the WT+NH 4 OH group versus the WT+Aβ group]. d Mice (3 months old) received the following intra-hippocampal injections: NH 4 OH (1%) injection and Flag-vector plasmid (0.4 μg) transfection; Aβ (5 μg) injection and Flag-vector plasmid transfection; NH 4 OH injection and Flag-Gal-3 plasmid transfection; and Aβ injection and Flag-Gal-3 plasmid transfection. The two injections were given 2 h apart and animals were sacrificed 48 h after the injection of NH 4 OH or Aβ. Dissected hippocampal tissues were analyzed for Aβ oligomerization. Tissue lysates were also subjected to immunoprecipitation and immunoblotting with anti-Flag antibody to confirm transfection and expression of the plasmid. e Quantified results for HMW and LMW Aβ oligomerization [ n = 4 per group; F (3,12) = 52.71, P < 0.001 for HMW; q = 9.09, P < 0.001 for the NH 4 OH+Flag-vector group versus the Aβ+Flag-vector group and q = 5.8, P < 0.01 for the Aβ+Flag-vector group versus the Aβ+Flag-Gal-3 group; F (3,12) = 72.63, P < 0.001 for LMW; q = 5.65, P < 0.01 for the NH 4 OH+Flag-vector group versus the Aβ+Flag-vector group and q = 12.38, P < 0.001 for the Aβ+Flag-vector group versus the Aβ+Flag-Gal-3 group]. Data are expressed as mean ± SEM. ** P < 0.01, # P < 0.001

Article Snippet: Immunoblotting was carried out using the following antibodies: rat anti-Gal-3 (1:5000, R&D systems), goat anti-Gal-1 (1:5000, R&D systems), rabbit anit-PIAS1 (1:3000, Epitomics, Burlingame, CA), mouse anti-CD10/NEP (1:500, Santa Cruz Biotechnology, Dallas, TX), mouse anti-prealbumin/transthyretin (1:500, Santa Cruz Biotechnology), rabbit anti-insulin degrading enzyme (1:3000, Abcam, Cambridge, UK), rabbit anti-FAK (1:2000, Abcam), rabbit anti-pFAK (phospho-Y397, 1:2000, Abcam), rabbit anti-CREB (1:2000, Cell Signaling, Danvers, MA), rabbit anti-pCREB (phospho-S133, 1:2000, Cell Signaling), rat anti-TREM2 (1:3000, Millipore, Bedford, MA), mouse anti-TLR4 (1:500, Santa Cruz Biotechnology), rabbit anti-CD40 (1:2000, Abcam), mouse anti-actin (1:150000, Millipore), and mouse anti-Flag M2 (1:10000, Sigma-Aldrich) antibodies.

Techniques: Injection, Western Blot, Expressing, Plasmid Preparation, Transfection, Immunoprecipitation

Journal: Cell Death and Differentiation

Article Title: Galectin-3 promotes Aβ oligomerization and Aβ toxicity in a mouse model of Alzheimer’s disease

doi: 10.1038/s41418-019-0348-z

Figure Lengend Snippet: Aβ oligomerization and Gal-3 expression are age-dependently increased in APP/PS1 mice. a Wild-type (3 months old) and APP/PS1 mice of different ages (3, 5, 8, and 11 months) were sacrificed and dissected hippocampal tissues were subjected to Western blot analysis for endogenous Aβ oligomers. b Quantified results for HMW and LMW Aβ oligomerization [ n = 4 per group; F (4,15) = 39.3, P < 0.001 for HMW and F (4,15) = 74.07, P < 0.001 for LMW]. Statistical significances for various comparisons are shown in the figure. c Western blot analysis showing the expression levels of Gal-3 and PIAS1 in hippocampal samples from the same batch of WT mice and APP/PS1 mice of different ages ( n = 4 per group). d Quantified results for Gal-3 expression in WT and APP/PS1 mice [ F (4,15) = 14.59, P < 0.001]. e Quantified results for PIAS1 expression in WT and APP/PS1 mice [ F (4,15) = 5.86, P < 0.01] The letter “m” indicates month. Statistical significances of various comparisons are shown in the figure. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01, # P < 0.001

Article Snippet: Immunoblotting was carried out using the following antibodies: rat anti-Gal-3 (1:5000, R&D systems), goat anti-Gal-1 (1:5000, R&D systems), rabbit anit-PIAS1 (1:3000, Epitomics, Burlingame, CA), mouse anti-CD10/NEP (1:500, Santa Cruz Biotechnology, Dallas, TX), mouse anti-prealbumin/transthyretin (1:500, Santa Cruz Biotechnology), rabbit anti-insulin degrading enzyme (1:3000, Abcam, Cambridge, UK), rabbit anti-FAK (1:2000, Abcam), rabbit anti-pFAK (phospho-Y397, 1:2000, Abcam), rabbit anti-CREB (1:2000, Cell Signaling, Danvers, MA), rabbit anti-pCREB (phospho-S133, 1:2000, Cell Signaling), rat anti-TREM2 (1:3000, Millipore, Bedford, MA), mouse anti-TLR4 (1:500, Santa Cruz Biotechnology), rabbit anti-CD40 (1:2000, Abcam), mouse anti-actin (1:150000, Millipore), and mouse anti-Flag M2 (1:10000, Sigma-Aldrich) antibodies.

Techniques: Expressing, Western Blot

Journal: Cell Death and Differentiation

Article Title: Galectin-3 promotes Aβ oligomerization and Aβ toxicity in a mouse model of Alzheimer’s disease

doi: 10.1038/s41418-019-0348-z

Figure Lengend Snippet: Endogenous Aβ oligomerization, Gal-3 expression, Iba1 and GFAP distribution, and amyloid plaque are reduced in APP/PS1;Gal-3 +/− mice. a Endogenous Aβ oligomerization was examined by Western blot analysis of samples obtained from 3-month-old WT;WT, APP/PS1;WT, WT;Gal-3 −/− and APP/PS1;Gal-3 +/− mice. b Quantified results for HMW and LMW Aβ oligomerization [ n = 4 per group; F (3,12) = 110.36; P < 0.001 for HMW; q = 19.91, P < 0.001 for the WT;WT group versus the APP/PS1;WT group and q = 4.13, P = 0.01 for the APP/PS1;WT group versus the APP/PS1;Gal-3 +/− group; F (3,12) = 77.72, P < 0.001 for LMW; q = 15.87, P < 0.001 for the WT;WT group versus the APP/PS1;WT group and q = 3.32, P < 0.05 for the APP/PS1;WT group versus the APP/PS1;Gal-3 +/− group]. c Immunohistochemical analysis of Gal-3 and Iba1 and merged images for 7-month-old mice of the four genotypes. Scale bar, 25 μm. d Immunohistochemical analysis of Gal-3 and GFAP and merged image in 7-month-old mice of the four genotypes. Scale bar, 25 μm. e Immunohistochemical analysis showing the distribution of Gal-3 and ProteoStat in 11-month-old mice of the four genotypes. Scale bar, 200 μm. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01, # P < 0.001

Article Snippet: Immunoblotting was carried out using the following antibodies: rat anti-Gal-3 (1:5000, R&D systems), goat anti-Gal-1 (1:5000, R&D systems), rabbit anit-PIAS1 (1:3000, Epitomics, Burlingame, CA), mouse anti-CD10/NEP (1:500, Santa Cruz Biotechnology, Dallas, TX), mouse anti-prealbumin/transthyretin (1:500, Santa Cruz Biotechnology), rabbit anti-insulin degrading enzyme (1:3000, Abcam, Cambridge, UK), rabbit anti-FAK (1:2000, Abcam), rabbit anti-pFAK (phospho-Y397, 1:2000, Abcam), rabbit anti-CREB (1:2000, Cell Signaling, Danvers, MA), rabbit anti-pCREB (phospho-S133, 1:2000, Cell Signaling), rat anti-TREM2 (1:3000, Millipore, Bedford, MA), mouse anti-TLR4 (1:500, Santa Cruz Biotechnology), rabbit anti-CD40 (1:2000, Abcam), mouse anti-actin (1:150000, Millipore), and mouse anti-Flag M2 (1:10000, Sigma-Aldrich) antibodies.

Techniques: Expressing, Western Blot, Immunohistochemical staining

Journal: Cell Death and Differentiation

Article Title: Galectin-3 promotes Aβ oligomerization and Aβ toxicity in a mouse model of Alzheimer’s disease

doi: 10.1038/s41418-019-0348-z

Figure Lengend Snippet: Endogenous Gal-3 expression is decreased but memory performance is improved in APP/PS1;Gal-3 +/− mice. a Endogenous expression levels of Gal-3 and NEP were examined by Western blot analysis in 3-month-old WT;WT, APP/PS1;WT, WT;Gal-3 −/− and APP/PS1;Gal-3 +/− mice. b Quantified results for Gal-3 and NEP expression [ n = 4 per group; for Gal-3, F (3,12) = 53.47, P < 0.001; q = 10.43, P < 0.001 for the WT;WT group versus the APP/PS1;WT group; q = 6.46, P < 0.001 for the APP/PS1;WT group versus the APP/PS1;Gal-3 +/− group and q = 3.98, P < 0.05 for the WT;WT group versus the APP/PS1;Gal-3 +/− group; for NEP, F (3,12) = 27.76, P < 0.001; q = 12.04, P < 0.001 for the WT;WT group versus the APP/PS1;WT group; q = 9.72, P < 0.001 for the WT;WT group versus the WT;Gal-3 −/− group and q = 6.29, P < 0.01 for the WT;WT group versus the APP/PS1;Gal-3 +/− group]. c Acquisition performance obtained by assessing the water maze learning of 8-month-old mice of the four genotypes [ n = 7 per group; F (3,24) = 28.72, P < 0.001; q = 9.33, P < 0.001 for the APP/PS1;WT group versus the WT;WT group and q = 3.59, P < 0.05 for the APP/PS1;Gal-3 +/− group versus the APP/PS1;WT group]. d Retention performance of the same mice described in ( c ) [ n = 7 per group; F (3,24) = 4.76, P < 0.01; q = 3.94, P < 0.05 for the APP/PS1;WT group versus the WT;WT group; q = 3.25, P < 0.05 for the APP/PS1;Gal-3 +/− group versus the APP/PS1;WT group for the target quadrant]. Representative swim patterns from each group are also shown (upper panel). Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01, # P < 0.001

Article Snippet: Immunoblotting was carried out using the following antibodies: rat anti-Gal-3 (1:5000, R&D systems), goat anti-Gal-1 (1:5000, R&D systems), rabbit anit-PIAS1 (1:3000, Epitomics, Burlingame, CA), mouse anti-CD10/NEP (1:500, Santa Cruz Biotechnology, Dallas, TX), mouse anti-prealbumin/transthyretin (1:500, Santa Cruz Biotechnology), rabbit anti-insulin degrading enzyme (1:3000, Abcam, Cambridge, UK), rabbit anti-FAK (1:2000, Abcam), rabbit anti-pFAK (phospho-Y397, 1:2000, Abcam), rabbit anti-CREB (1:2000, Cell Signaling, Danvers, MA), rabbit anti-pCREB (phospho-S133, 1:2000, Cell Signaling), rat anti-TREM2 (1:3000, Millipore, Bedford, MA), mouse anti-TLR4 (1:500, Santa Cruz Biotechnology), rabbit anti-CD40 (1:2000, Abcam), mouse anti-actin (1:150000, Millipore), and mouse anti-Flag M2 (1:10000, Sigma-Aldrich) antibodies.

Techniques: Expressing, Western Blot

Journal: Cell Death and Differentiation

Article Title: Galectin-3 promotes Aβ oligomerization and Aβ toxicity in a mouse model of Alzheimer’s disease

doi: 10.1038/s41418-019-0348-z

Figure Lengend Snippet: Galectin-3 associates with Aβ and interacts with Aβ. a Immunohistochemical analysis showing the distributions of endogenous Gal-3 and Aβ in hippocampal samples of WT (left panel) and APP/PS1 (right panel) mice at 11 months of age. Scale bar, 200 μm. b Co-IP experiment showing preferential association of Gal-3 with endogenous Aβ monomers in 8-month-old APP/PS1 mice. Experiments were performed in duplicate. c Different amounts of recombinant human Gal-3 protein (0.25, 0.5, 1, and 2 μg) were added to a solution containing Aβ42 peptide (15 μg), the mixture was subjected to a thioflavin-T assay, and fluorescence was measured hourly at different time points. The fluorescence plateaued at the 7-h time point. Experiments were performed in triplicate. The abbreviation “rh” indicates “recombinant human”. Data are expressed as mean ± SEM

Article Snippet: Immunoblotting was carried out using the following antibodies: rat anti-Gal-3 (1:5000, R&D systems), goat anti-Gal-1 (1:5000, R&D systems), rabbit anit-PIAS1 (1:3000, Epitomics, Burlingame, CA), mouse anti-CD10/NEP (1:500, Santa Cruz Biotechnology, Dallas, TX), mouse anti-prealbumin/transthyretin (1:500, Santa Cruz Biotechnology), rabbit anti-insulin degrading enzyme (1:3000, Abcam, Cambridge, UK), rabbit anti-FAK (1:2000, Abcam), rabbit anti-pFAK (phospho-Y397, 1:2000, Abcam), rabbit anti-CREB (1:2000, Cell Signaling, Danvers, MA), rabbit anti-pCREB (phospho-S133, 1:2000, Cell Signaling), rat anti-TREM2 (1:3000, Millipore, Bedford, MA), mouse anti-TLR4 (1:500, Santa Cruz Biotechnology), rabbit anti-CD40 (1:2000, Abcam), mouse anti-actin (1:150000, Millipore), and mouse anti-Flag M2 (1:10000, Sigma-Aldrich) antibodies.

Techniques: Immunohistochemical staining, Co-Immunoprecipitation Assay, Recombinant, ThT Assay, Fluorescence

Journal: Cell Death and Differentiation

Article Title: Galectin-3 promotes Aβ oligomerization and Aβ toxicity in a mouse model of Alzheimer’s disease

doi: 10.1038/s41418-019-0348-z

Figure Lengend Snippet: Neprilysin (NEP) expression is increased and integrin signaling is enhanced in Gal-3 KO mice. a Dissected hippocampi of naïve WT and Gal-3 KO mice (3-month-old) were subjected to various Western blot assays. Representative gel patterns obtained for NEP, IDE, TTR, and Gal-3 are shown. b Quantified results for NEP expression [ t (1,8) = 8.61, P < 0.001; left panel], IDE expression [ t (1,8) = 0.72, P > 0.05; middle panel] and TTR expression [ t (1,8) = 1.32, P > 0.05; right panel]. c Representative gel patterns obtained for pFAK, FAK, pCREB, and CREB, and their quantified results [ t (1,8) = 11.14, P < 0.001 for pFAK/FAK; t (1,8) = 0.57, P > 0.05 for FAK/Actin; t (1,8) = 6.72, P < 0.001 for pCREB/CREB and t (1,8) = 0.67, P > 0.05 for CREB/Actin]. d ChIP assay examining the binding of endogenous CREB to the NEP promoter in WT and Gal-3 KO mice, and the quantified results [ t (1,8) = 9.06, P < 0.001]. Data are expressed as mean ± SEM. # P < 0.001

Article Snippet: Immunoblotting was carried out using the following antibodies: rat anti-Gal-3 (1:5000, R&D systems), goat anti-Gal-1 (1:5000, R&D systems), rabbit anit-PIAS1 (1:3000, Epitomics, Burlingame, CA), mouse anti-CD10/NEP (1:500, Santa Cruz Biotechnology, Dallas, TX), mouse anti-prealbumin/transthyretin (1:500, Santa Cruz Biotechnology), rabbit anti-insulin degrading enzyme (1:3000, Abcam, Cambridge, UK), rabbit anti-FAK (1:2000, Abcam), rabbit anti-pFAK (phospho-Y397, 1:2000, Abcam), rabbit anti-CREB (1:2000, Cell Signaling, Danvers, MA), rabbit anti-pCREB (phospho-S133, 1:2000, Cell Signaling), rat anti-TREM2 (1:3000, Millipore, Bedford, MA), mouse anti-TLR4 (1:500, Santa Cruz Biotechnology), rabbit anti-CD40 (1:2000, Abcam), mouse anti-actin (1:150000, Millipore), and mouse anti-Flag M2 (1:10000, Sigma-Aldrich) antibodies.

Techniques: Expressing, Western Blot, Binding Assay

Journal: Cell Death and Differentiation

Article Title: Galectin-3 promotes Aβ oligomerization and Aβ toxicity in a mouse model of Alzheimer’s disease

doi: 10.1038/s41418-019-0348-z

Figure Lengend Snippet: Aβ oligomerization and Gal-3 expression are increased in AD patients. The frontal lobe lysates of normal subjects and AD patients were subjected to Western blot analysis for Aβ oligomerization and the expression of Gal-3 and Gal-1 ( n = 4 per group). b – d Quantified results for ( b ) HMW [ t (1,6) = 9.72, P < 0.001] and LMW [ t (1,6) = 7.21, P < 0.001] Aβ oligomerization, c Gal-3 expression [ t (1,6) = 4.92, P < 0.01] and d Gal-1 expression [ t (1,6) = 0.43, P > 0.05]. e Immunohistochemical analysis showing the distributions of Gal-3 and ProteoStat staining in normal subjects and AD patients ( n = 3 per group). Scale bar, 25 μm. DAPI was used to stain nuclei. Data are expressed as mean ± SEM. ** P < 0.01, # P < 0.001

Article Snippet: Immunoblotting was carried out using the following antibodies: rat anti-Gal-3 (1:5000, R&D systems), goat anti-Gal-1 (1:5000, R&D systems), rabbit anit-PIAS1 (1:3000, Epitomics, Burlingame, CA), mouse anti-CD10/NEP (1:500, Santa Cruz Biotechnology, Dallas, TX), mouse anti-prealbumin/transthyretin (1:500, Santa Cruz Biotechnology), rabbit anti-insulin degrading enzyme (1:3000, Abcam, Cambridge, UK), rabbit anti-FAK (1:2000, Abcam), rabbit anti-pFAK (phospho-Y397, 1:2000, Abcam), rabbit anti-CREB (1:2000, Cell Signaling, Danvers, MA), rabbit anti-pCREB (phospho-S133, 1:2000, Cell Signaling), rat anti-TREM2 (1:3000, Millipore, Bedford, MA), mouse anti-TLR4 (1:500, Santa Cruz Biotechnology), rabbit anti-CD40 (1:2000, Abcam), mouse anti-actin (1:150000, Millipore), and mouse anti-Flag M2 (1:10000, Sigma-Aldrich) antibodies.

Techniques: Expressing, Western Blot, Immunohistochemical staining, Staining

Journal: American Journal of Translational Research

Article Title: The combination of circRNA-UMAD1 and Galectin-3 in peripheral circulation is a co-biomarker for predicting lymph node metastasis of thyroid carcinoma

doi:

Figure Lengend Snippet: Circulating Gal3 is a potential target for the diagnosis of LNM. TCGA database analysis of the expression level of Gal3 in serval solid cancer indicated that Gal3 is 100% positive in thyroid cancer, which is more than the rate in other cancers (A). The protein level of Gal3 in the serum of PTC patients was detected by ELISA analysis, showing that LNM patients express more Gal3 (B). Gal3 expression is significantly related to LNM based on univariant analysis in 50 PTC patients (C). Sequencing TCGA data downloaded from the LinkedOmics website demonstrated that the RNA level of Gal3 is upregulated in tumor tissues (D), and is associated with the stage of PTC (E) and LNM (F). Unsupervised hierarchical clustering heat map shows the expression of different genes between non-LNM and LNM PTC samples (G). Unpaired t-test and One-way ANOVA multiple comparisons test were performed to obtain P values in comparisons of two or more groups. * indicates P<0.05.

Article Snippet: The membranes were incubated with primary antibodies specific to Gal3 (Cell Signaling, #87985), NOTCH1 (Cell Signaling, #3608), Hes1 (Cell Signaling, #11988), TGF-β1 (Abcam, ab92486), ErbB2 (Abcam, ab237715), ErbB4 (Abcam, ab19391), NF-κB (p65) (Abcam, ab7970) and GAPDH (Bioworld BS606030), also corresponding secondary HRP-conjugated goat anti-rabbit (Jackson.US) were immunoreacted.

Techniques: Biomarker Discovery, Expressing, Enzyme-linked Immunosorbent Assay, Sequencing

Journal: American Journal of Translational Research

Article Title: The combination of circRNA-UMAD1 and Galectin-3 in peripheral circulation is a co-biomarker for predicting lymph node metastasis of thyroid carcinoma

doi:

Figure Lengend Snippet: Relationship between Gal3 expression and pathological features in PTC patients with or without LNM

Article Snippet: The membranes were incubated with primary antibodies specific to Gal3 (Cell Signaling, #87985), NOTCH1 (Cell Signaling, #3608), Hes1 (Cell Signaling, #11988), TGF-β1 (Abcam, ab92486), ErbB2 (Abcam, ab237715), ErbB4 (Abcam, ab19391), NF-κB (p65) (Abcam, ab7970) and GAPDH (Bioworld BS606030), also corresponding secondary HRP-conjugated goat anti-rabbit (Jackson.US) were immunoreacted.

Techniques: Expressing

Journal: American Journal of Translational Research

Article Title: The combination of circRNA-UMAD1 and Galectin-3 in peripheral circulation is a co-biomarker for predicting lymph node metastasis of thyroid carcinoma

doi:

Figure Lengend Snippet: Enrichment of miRNA and circRNA sequencing profiles. Flow chart of the circRNA/miRNA/mRNA screen to identify regulators upstream of Gal3 (A). Pathological morphology was validated by HE staining of two paired primary and LNM tumor samples that were prepared for RNA sequencing (B). Unsupervised hierarchical clustering shows distinct miRNA (C) and circRNA (D) expression with green representing decreased expression. Significantly enriched pathways in genes regulated by differentially expressed cancer-related miRNAs (E) and circRNAs (F) were identified by KEGG pathway enrichment analysis. Western blot analysis for ErbB, NOTCH, NF-κB and TGF-β pathways (G). IF staining for Gal3 and ErbB4 in original tumor and Lymph node metastasis tissues (H). bar = 20 μm.

Article Snippet: The membranes were incubated with primary antibodies specific to Gal3 (Cell Signaling, #87985), NOTCH1 (Cell Signaling, #3608), Hes1 (Cell Signaling, #11988), TGF-β1 (Abcam, ab92486), ErbB2 (Abcam, ab237715), ErbB4 (Abcam, ab19391), NF-κB (p65) (Abcam, ab7970) and GAPDH (Bioworld BS606030), also corresponding secondary HRP-conjugated goat anti-rabbit (Jackson.US) were immunoreacted.

Techniques: Sequencing, Staining, RNA Sequencing, Expressing, Western Blot

Journal: American Journal of Translational Research

Article Title: The combination of circRNA-UMAD1 and Galectin-3 in peripheral circulation is a co-biomarker for predicting lymph node metastasis of thyroid carcinoma

doi:

Figure Lengend Snippet: miR-873 was identified to act as an upstream regulator of Gal3. Unsupervised hierarchical clustering heat map of miRNA expression fold-change from TCGA data shows the top 50 miRNAs with decreased expression in the LNM group. Nine miRNAs were consistent with our sequencing data, as shown by the black arrow (A). A Venn diagram showing that miR-873 is a potential regulator of Gal3 and is related to LNM in PTC (B). The putative binding site of miR-873-5p on Gal3 is shown, including the wild-type and mutated Gal3 3’-UTR sequences which were used to create the wild-type and mutant luciferase reporter constructs. The similarity between 12 putative complementary binding sites (red letters) of the Gal3 3’-UTRs from different species (C). A luciferase reporter assay demonstrated that miR-873 inhibited the transcription of the wild-type but not the mutant 3’-UTRs of Gal3 (D). The expression of endogenous Gal3 RNA was inhibited in miR-873-mimic-infected HEK293 cells compared to the control, as detected by qRT-PCR and normalized to GAPDH mRNA (E), while it was increased in the miR-873 inhibitor group (F). The RNA level of miR-873 was validated in LNM-positive tumor tissues compared to the negative control by analyzing the TCGA profile. N0: non-LNM; N1: LNM (G). miR-873 was validated again in a different metastatic lymph node group, N0: non-LNM; N1<4; 410 (H). Relative expression of miR-873 in circulation was detected by qRT-PCR between non-LNM and LNM patients (I). Unpaired t-test and one-way ANOVA multiple comparisons test were performed to obtain P values in comparisons of two or more groups. * indicates P<0.05.

Article Snippet: The membranes were incubated with primary antibodies specific to Gal3 (Cell Signaling, #87985), NOTCH1 (Cell Signaling, #3608), Hes1 (Cell Signaling, #11988), TGF-β1 (Abcam, ab92486), ErbB2 (Abcam, ab237715), ErbB4 (Abcam, ab19391), NF-κB (p65) (Abcam, ab7970) and GAPDH (Bioworld BS606030), also corresponding secondary HRP-conjugated goat anti-rabbit (Jackson.US) were immunoreacted.

Techniques: Expressing, Sequencing, Binding Assay, Mutagenesis, Luciferase, Construct, Reporter Assay, Infection, Control, Quantitative RT-PCR, Negative Control

Journal: American Journal of Translational Research

Article Title: The combination of circRNA-UMAD1 and Galectin-3 in peripheral circulation is a co-biomarker for predicting lymph node metastasis of thyroid carcinoma

doi:

Figure Lengend Snippet: CircRNA UMAD1 is associated with LNM and Gal3 expression. Expression of hsa-circ-UMAD1 quantified by at least three independent qRT-PCR experiments in the cohort comparing non-LNM tumors (absent, n = 23) and LNM tumors (present, n = 27). Unpaired t-tests were performed to identify significant differences. The relative expression level of circRNA UMAD1 was normalized to the level of β-actin (A). CircRNA UMAD1 expression was significantly related to LNM and primary side location based on univariant analysis of 50 PTC patients, which is described in Table 1 (B). The correlation between the expression of circRNA UMAD1 and Gal3 was evaluated by Person’s correlation test (r = 0.35, P = 0.00138) (C). All data are expressed as the median ± range with three independent experiments.

Article Snippet: The membranes were incubated with primary antibodies specific to Gal3 (Cell Signaling, #87985), NOTCH1 (Cell Signaling, #3608), Hes1 (Cell Signaling, #11988), TGF-β1 (Abcam, ab92486), ErbB2 (Abcam, ab237715), ErbB4 (Abcam, ab19391), NF-κB (p65) (Abcam, ab7970) and GAPDH (Bioworld BS606030), also corresponding secondary HRP-conjugated goat anti-rabbit (Jackson.US) were immunoreacted.

Techniques: Expressing, Quantitative RT-PCR

Journal: American Journal of Translational Research

Article Title: The combination of circRNA-UMAD1 and Galectin-3 in peripheral circulation is a co-biomarker for predicting lymph node metastasis of thyroid carcinoma

doi:

Figure Lengend Snippet: ROC curves for individual biomarkers circRNA UMAD1 and Gal3 or combination. ROC curve analysis of circRNA UMAD1 calculates the best cutoff point to discriminate between the LNM and primary tumor groups. The area under the curve (AUC) (SE) = 0.718 (0.072); 95% confidence interval was 0.576 to 0.86; P = 0.0084 (A). ROC curve analysis of Gal3 between these two groups. AUC (SE) = 0.825 (0.061); 95% confidence interval was 0.704 to 0.945; P<0.001 (B). ROC curve analysis of circRNA UMAD1 combined with Gal3 for distinguishing patients in the LNM vs. primary tumor groups. AUC (SE) = 0.87 (0.051); 95% confidence interval was 0.766 to 0.97; P<0.001 (C). Likelihood ratio = 3.75. P<0.01, ROC, receiver operating characteristic; SE, standard error.

Article Snippet: The membranes were incubated with primary antibodies specific to Gal3 (Cell Signaling, #87985), NOTCH1 (Cell Signaling, #3608), Hes1 (Cell Signaling, #11988), TGF-β1 (Abcam, ab92486), ErbB2 (Abcam, ab237715), ErbB4 (Abcam, ab19391), NF-κB (p65) (Abcam, ab7970) and GAPDH (Bioworld BS606030), also corresponding secondary HRP-conjugated goat anti-rabbit (Jackson.US) were immunoreacted.

Techniques:

Journal: Acta Neuropathologica

Article Title: Galectin-3, a novel endogenous TREM2 ligand, detrimentally regulates inflammatory response in Alzheimer’s disease

doi: 10.1007/s00401-019-02013-z

Figure Lengend Snippet: Galectin-3 is increased in human and mouse AD brains and marks a microglial phenotype associated with Aβ plaques. a Western blot analyses of cortex from human AD cases ( n = 6) and age-matched healthy controls ( n = 5). b Galectin-3 (gal3) staining mainly coincided with Iba1 + microglia found around Aβ plaques in human AD brains. c Immunohistochemistry showed high levels of gal3 in microglia in AD brains, as compared to Iba1-staining (low levels of gal3 was detected in association to blood vessels). d Gal3 protein was significantly upregulated in the cortex of 5xFAD mice in a time-dependent fashion (WT, 6 months old). e Gal3 expression was found in Iba1 + cells around Αβ plaques. Statistical significance was calculated by one-way ANOVA with Tukey’s correction ( d ) or Student’s t test ( a ). *p < 0.05; **p < 0.01. Data are shown as mean ± SD. The human AD cases are described in suppl. Tables S1 and S2 (online resources 8 and 9)

Article Snippet: Antibodies used for this study: anti-rabbit iNOS primary antibody (1:5000, Santa Cruz), anti-rat gal3 antibody (1:3000, M38 clone from Hakon Leffler’s lab, in-house antibody), anti-goat gal3 antibody (1:1000, R&D Systems), anti-mouse actin antibody 1:10,000 (Sigma-Aldrich), anti-human Aβ antibody (1:5000, Covance), anti-rabbit Iba-1 antibody (1:500, Wako), anti-mouse TLR4 antibody (1:1000, Santa Cruz), anti-mouse NLRP3 antibody (1:5000, Adipogen), anti-rabbit C83 antibody (369) (1:1000, Gunnar Gouras Laboratory, BMC, Lund, Sweden), TREM2 antibody 1:500 (AF1729, anti-sheep) anti-rabbit IDE-1 antibody (1:1000, Calbiochem), anti-Rabbit p-Tau (pTau181, 1:1000, Santa Cruz), anti-mouse Aβ (1:1000, Sigma-Aldrich), anti-CD45 rat monoclonal (clone IBL-3/16, 1:500, AbD Serotec), anti-CD68 (1:500, eBiosciences) and anti-Clec7a rabbit monoclonal (1:500, abcam).

Techniques: Western Blot, Staining, Immunohistochemistry, Expressing

Journal: Acta Neuropathologica

Article Title: Galectin-3, a novel endogenous TREM2 ligand, detrimentally regulates inflammatory response in Alzheimer’s disease

doi: 10.1007/s00401-019-02013-z

Figure Lengend Snippet: Galectin-3 deficiency/inhibition reduces the microglial inflammatory response in vitro. a Reduced cytokine levels in culture medium from Gal3KO primary microglial cultures compared to WT after fΑβ treatment for 12 h. b WT primary microglial cultures increase the release of gal3 upon stimulation with fAβ. In vitro experiments represent a minimum of three independent experiments. Statistical significance was calculated by Student’s t-test ( b ), or one-way ANOVA with Tukey’s correction ( a ) *p < 0.05; **p < 0.01; ***p < 0.001. Data are shown as mean ± SD

Article Snippet: Antibodies used for this study: anti-rabbit iNOS primary antibody (1:5000, Santa Cruz), anti-rat gal3 antibody (1:3000, M38 clone from Hakon Leffler’s lab, in-house antibody), anti-goat gal3 antibody (1:1000, R&D Systems), anti-mouse actin antibody 1:10,000 (Sigma-Aldrich), anti-human Aβ antibody (1:5000, Covance), anti-rabbit Iba-1 antibody (1:500, Wako), anti-mouse TLR4 antibody (1:1000, Santa Cruz), anti-mouse NLRP3 antibody (1:5000, Adipogen), anti-rabbit C83 antibody (369) (1:1000, Gunnar Gouras Laboratory, BMC, Lund, Sweden), TREM2 antibody 1:500 (AF1729, anti-sheep) anti-rabbit IDE-1 antibody (1:1000, Calbiochem), anti-Rabbit p-Tau (pTau181, 1:1000, Santa Cruz), anti-mouse Aβ (1:1000, Sigma-Aldrich), anti-CD45 rat monoclonal (clone IBL-3/16, 1:500, AbD Serotec), anti-CD68 (1:500, eBiosciences) and anti-Clec7a rabbit monoclonal (1:500, abcam).

Techniques: Inhibition, In Vitro

Journal: Acta Neuropathologica

Article Title: Galectin-3, a novel endogenous TREM2 ligand, detrimentally regulates inflammatory response in Alzheimer’s disease

doi: 10.1007/s00401-019-02013-z

Figure Lengend Snippet: Galectin-3 colocalizes with TREM2. a , b Iba1 + cells expressing galectin-3 (gal3) around Aβ plaque in 5xFAD mice. c Reduced number of Iba1 + microglial cells around Αβ plaques in 5xFAD/Gal3KO mice compared to 5xFAD mice (% of Αβ area). d Number of Iba1 + cells expressing gal3 in 5xFAD (% of Αβ area). e , f Gal3 and TREM2 in plaque-associated microglia in the brain of 5xFAD mice reveals colocalization of gal3 and TREM2. g Gal3 and TREM2 colocalization in 5xFAD mouse brain using STORM microscopy. Statistical significance was calculated by Student’s t test. *p < 0.05. Data are shown as mean ± SEM. All images were taken in 5xFAD mice at 18 months

Article Snippet: Antibodies used for this study: anti-rabbit iNOS primary antibody (1:5000, Santa Cruz), anti-rat gal3 antibody (1:3000, M38 clone from Hakon Leffler’s lab, in-house antibody), anti-goat gal3 antibody (1:1000, R&D Systems), anti-mouse actin antibody 1:10,000 (Sigma-Aldrich), anti-human Aβ antibody (1:5000, Covance), anti-rabbit Iba-1 antibody (1:500, Wako), anti-mouse TLR4 antibody (1:1000, Santa Cruz), anti-mouse NLRP3 antibody (1:5000, Adipogen), anti-rabbit C83 antibody (369) (1:1000, Gunnar Gouras Laboratory, BMC, Lund, Sweden), TREM2 antibody 1:500 (AF1729, anti-sheep) anti-rabbit IDE-1 antibody (1:1000, Calbiochem), anti-Rabbit p-Tau (pTau181, 1:1000, Santa Cruz), anti-mouse Aβ (1:1000, Sigma-Aldrich), anti-CD45 rat monoclonal (clone IBL-3/16, 1:500, AbD Serotec), anti-CD68 (1:500, eBiosciences) and anti-Clec7a rabbit monoclonal (1:500, abcam).

Techniques: Expressing, Microscopy

Journal: Acta Neuropathologica

Article Title: Galectin-3, a novel endogenous TREM2 ligand, detrimentally regulates inflammatory response in Alzheimer’s disease

doi: 10.1007/s00401-019-02013-z

Figure Lengend Snippet: Galectin-3 interacts with TREM2 through its carbohydrate-binding domain. a Fluorescent anisotropy assay for galectin-3 (gal3)/TREM2 interaction. Data are presented as % of TREM2–gal3 binding (gal3, WT and mutant gal3 with deficient carbohydrate-binding domain, R186S) and fluorescent probe interaction, by increasing concentrations of TREM2, together with the calculated K d values for the gal3/TREM2 interaction ( n = 2). b Control and DAP12 reporter cell lines were stimulated with increasing concentrations of gal3 (250 nM–2.5 µM), ionomycin and phosphatidylserine (PS). Statistical significance was calculated by Student’s t test or one-way ANOVA with Bonferroni’s post hoc test. *p < 0.05; **p < 0.01. Data are shown as mean ± SD

Article Snippet: Antibodies used for this study: anti-rabbit iNOS primary antibody (1:5000, Santa Cruz), anti-rat gal3 antibody (1:3000, M38 clone from Hakon Leffler’s lab, in-house antibody), anti-goat gal3 antibody (1:1000, R&D Systems), anti-mouse actin antibody 1:10,000 (Sigma-Aldrich), anti-human Aβ antibody (1:5000, Covance), anti-rabbit Iba-1 antibody (1:500, Wako), anti-mouse TLR4 antibody (1:1000, Santa Cruz), anti-mouse NLRP3 antibody (1:5000, Adipogen), anti-rabbit C83 antibody (369) (1:1000, Gunnar Gouras Laboratory, BMC, Lund, Sweden), TREM2 antibody 1:500 (AF1729, anti-sheep) anti-rabbit IDE-1 antibody (1:1000, Calbiochem), anti-Rabbit p-Tau (pTau181, 1:1000, Santa Cruz), anti-mouse Aβ (1:1000, Sigma-Aldrich), anti-CD45 rat monoclonal (clone IBL-3/16, 1:500, AbD Serotec), anti-CD68 (1:500, eBiosciences) and anti-Clec7a rabbit monoclonal (1:500, abcam).

Techniques: Binding Assay, Mutagenesis, Control

Journal: Acta Neuropathologica

Article Title: Galectin-3, a novel endogenous TREM2 ligand, detrimentally regulates inflammatory response in Alzheimer’s disease

doi: 10.1007/s00401-019-02013-z

Figure Lengend Snippet: Galectin-3 induces the formation of insoluble Αβ aggregates following injections of Αβ monomers in the hippocampi of WT mice. Αβ monomers were injected with galectin-3 (Gal3) (Aβ + Gal3) or without (Aβ) after 1 h Aβ monomers incubation w/o gal3 into the right or left hippocampi of WT mice, respectively. a Staining for Αβ and gal3 in the left hippocampus (only Aβ monomers injected). b , c Staining for Αβ and gal3 in the right hippocampus (Aβ + gal3 injected). Dashed frames in b are magnified and shown in c . d Thioflavin-S and Αβ staining of the left hippocampus (only Aβ injected). e Thioflavin-S and Αβ staining of the right hippocampus (Aβ + gal3 injected). White arrows point to gal3 and thioflavin-S + aggregates. f , g Iba1 and GFAP immunoreactivity in the right hippocampus (Αβ + gal3 were injected)

Article Snippet: Antibodies used for this study: anti-rabbit iNOS primary antibody (1:5000, Santa Cruz), anti-rat gal3 antibody (1:3000, M38 clone from Hakon Leffler’s lab, in-house antibody), anti-goat gal3 antibody (1:1000, R&D Systems), anti-mouse actin antibody 1:10,000 (Sigma-Aldrich), anti-human Aβ antibody (1:5000, Covance), anti-rabbit Iba-1 antibody (1:500, Wako), anti-mouse TLR4 antibody (1:1000, Santa Cruz), anti-mouse NLRP3 antibody (1:5000, Adipogen), anti-rabbit C83 antibody (369) (1:1000, Gunnar Gouras Laboratory, BMC, Lund, Sweden), TREM2 antibody 1:500 (AF1729, anti-sheep) anti-rabbit IDE-1 antibody (1:1000, Calbiochem), anti-Rabbit p-Tau (pTau181, 1:1000, Santa Cruz), anti-mouse Aβ (1:1000, Sigma-Aldrich), anti-CD45 rat monoclonal (clone IBL-3/16, 1:500, AbD Serotec), anti-CD68 (1:500, eBiosciences) and anti-Clec7a rabbit monoclonal (1:500, abcam).

Techniques: Injection, Incubation, Staining