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: 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

Journal: Acta Neuropathologica

Article Title: Galectin-3 shapes toxic alpha-synuclein strains in Parkinson’s disease

doi: 10.1007/s00401-023-02585-x

Figure Lengend Snippet: Antibodies used for immunostainings

Article Snippet: Goat anti-GAL3 , R&D Systems, Bio-techne , Minneapolis, MN, USA , AF-1197 , 1:1000.

Techniques:

Journal: Acta Neuropathologica

Article Title: Galectin-3 shapes toxic alpha-synuclein strains in Parkinson’s disease

doi: 10.1007/s00401-023-02585-x

Figure Lengend Snippet: Antibodies used for western blot

Article Snippet: Goat anti-GAL3 , R&D Systems, Bio-techne , Minneapolis, MN, USA , AF-1197 , 1:1000.

Techniques: Western Blot

Journal: Acta Neuropathologica

Article Title: Galectin-3 shapes toxic alpha-synuclein strains in Parkinson’s disease

doi: 10.1007/s00401-023-02585-x

Figure Lengend Snippet: Galectin-3 (GAL3) is associated with Lewy Bodies (LB) and Pale Bodies (PB) in PD patients. a Immunofluorescence analysis of GAL3 in association with distinct forms of human α-synuclein (hSYN) aggregation. Β-sheet structure marker Methoxy-X04 was used to discriminate between LB and PB. Multiple core LB and PB are shown. GAL3 is present in both types of aggregates independently of neuromelanin presence. Scale bar 10 µm. b GAL3 is present in a diverse subset of hSYN accumulations with a precise negative correlation (blue arrows). Scale bar 10 µm. c Proportion of hSYN aggregates that are associated with GAL3. Methoxy-X04 was used as a specific marker of LB. Single (sLB) and multiple core LB (mLB) were discriminated ( p < 0.05). d Protein levels of GAL3 measured by ELISA in the Cortex of Control and PD Patients (PD-Cx) ( p < 0.001), and in the Substantia nigra (PD-SN) of PD patients

Article Snippet: Goat anti-GAL3 , R&D Systems, Bio-techne , Minneapolis, MN, USA , AF-1197 , 1:1000.

Techniques: Immunofluorescence, Marker, Enzyme-linked Immunosorbent Assay

Journal: Acta Neuropathologica

Article Title: Galectin-3 shapes toxic alpha-synuclein strains in Parkinson’s disease

doi: 10.1007/s00401-023-02585-x

Figure Lengend Snippet: GAL3 variably associates with lysosomes in the outer layers LB in all the studied patients. a GAL3 surrounding LB was found in all 6 patients studied (P.1–6). Variable amount of GAL3 vesicles was found. Note lower hSYN staining in the presence of GAL3. Scale bar 10 µm. b High resolution microscopy showed a ring-like pattern for GAL3 without any hSYN inside. Scale bar 10 µm. c Immunofluorescence analysis revealed that GAL3 is associated with recruited lysosomes (LAMP1) in the vicinities of LB. Scale bar 10 µm. d Combination of GAL3 immunohistochemistry with immunofluorescence showed that GAL3 is associated with autofluorescent lipofuscin vesicles in PD patients. Scale bar 10 µm. e Immunofluorescence analysis revealed that GAL3 accumulates inside MAP2 + neurons in the viccinities of LB. Scale bar 10 µm

Article Snippet: Goat anti-GAL3 , R&D Systems, Bio-techne , Minneapolis, MN, USA , AF-1197 , 1:1000.

Techniques: Staining, Microscopy, Immunofluorescence, Immunohistochemistry

Journal: Acta Neuropathologica

Article Title: Galectin-3 shapes toxic alpha-synuclein strains in Parkinson’s disease

doi: 10.1007/s00401-023-02585-x

Figure Lengend Snippet: Recombinant galectin-3 (Gal3) impairs synuclein aggregation in vitro. a Thioflavin-T (ThT) aggregation assay showed a rapid aggregation for recombinant human α-synuclein (αSyn) that was impaired in the presence of recombinant Gal3 (purple line). Notably, carbohydrate recognition domain (CRD) mutation (Gal3 R186S ) reverted this effect. b Proteinase K (PK) digestion at increasing concentration of resultant conditions from a) showed a lower stability in the presence of Gal3 (red line). c When Gal3 was added to αSyn pre-formed fibrils (PFF) after aggregation was completed, an increased signal was observed in the presence of ThT after 15 h. d PK digestion at increasing concentration of resultant fibrils from ( c ) showed similar stability of PFF in the presence of Gal3. e Electron microscopy images after uranyl negative staining of PFF after 24 h incubation with Gal3 (right panels). Note a marked disorganization of the fibrils network after Gal3 incubation with increased shortened species (upper right panel), and the change of morphology (lower right panel) with rounded structures attached to the fibrils. Scale bar 1 µm (upper panels) and 200 nm (lower panels). f Native PAGE Western Blot of the final results obtained in c ) Note that Gal3 promoted an increase in smaller soluble species released by αSyn fibrils. g Direct interaction of Gal3 with different αSyn species was investigated by ELISA. 2 µM Gal3 concentration were precoated in a 96 well plate and 2 µM αSyn species were incubated. 450 nm absorbance was measured to detect bounded protein. All types of species presented high affinity for Gal3 coated well compared with the control condition in absence of αSyn ( p < 0.001). No relevant absorbance was detected in the absence of precoated Gal3 (data not shown). h Addition of sonicated PFF pre-incubated with gal3 (PFFgal3) for 30 min to dopaminergic cell line N27 for 48 h led to a decreased number of cells compared with PFF alone (** p < 0.01; *** p < 0.001). i Graphical abstract representing the hypothesis proposed based on our in vitro studies about Gal3-αSyn interaction. Gal3 could impact αSyn elongation in de novo formation of fibrils while also affecting structured fibrils with little impact on the dense core but release of small species

Article Snippet: Goat anti-GAL3 , R&D Systems, Bio-techne , Minneapolis, MN, USA , AF-1197 , 1:1000.

Techniques: Recombinant, In Vitro, Mutagenesis, Concentration Assay, Electron Microscopy, Negative Staining, Incubation, Clear Native PAGE, Western Blot, Enzyme-linked Immunosorbent Assay, Sonication

Journal: Acta Neuropathologica

Article Title: Galectin-3 shapes toxic alpha-synuclein strains in Parkinson’s disease

doi: 10.1007/s00401-023-02585-x

Figure Lengend Snippet: GAL3 early overexpression leads to chronic activation and neuronal internalization. a Western Blot against GAL3 from brain homogenates from WT and Gal3KO mice revealed constitutive expression of GAL3 in WT mice. b Western Blot quantification of total GAL3 protein in WT mesencephalon samples. No difference was found between contralateral (Right hemisphere, RH) and ipsilateral (Left hemisphere, LH) hemispheres. Data are expressed as percentage fold to actin. c Double immunofluorescence 6 months after adenovirus injection showed clusters of CD11B + microglial cells highly reactive for GAL3. Internalized pSYN led to overexpression of GAL3 in WT microglia. pSYN was internalized by microglia independently of GAL3 genotype. Scale bar 20 µm. d TNFα quantification in SN and STR was performed on a MesoScale Discovery platform analysing brain extracts from AAV5-hSYN injected SN and STR ( p < 0.05). e Neuronal primary cell culture from WT mice showed efficient Gal3 internalization after incubation with 0.8 µM gal3 for 10 days. Note no difference in endogenous αSyn staining after addition of Gal3. f hSYN/GAL3 double immunofluorescence from injection area of mice WT brains 2 weeks after injection revealed no colocalization and significant upregulation of GAL3. Scale bar 50 µm. GAL3 lo /hSYN colocalization (white arrow) can be found near highly reactive GAL3 + cell indicating GAL3 release and neuronal GAL3 internalization. Scale bar 10 µm. g hSYN/GAL3 double immunofluorescence of mice WT brains 4 weeks after adenovirus injection revealed neuronal GAL3 staining. Scale bar 10 µm

Article Snippet: Goat anti-GAL3 , R&D Systems, Bio-techne , Minneapolis, MN, USA , AF-1197 , 1:1000.

Techniques: Over Expression, Activation Assay, Western Blot, Expressing, Immunofluorescence, Injection, Cell Culture, Incubation, Staining