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: Cardiovascular Research

Article Title: Endothelial OCT4 is atheroprotective by preventing metabolic and phenotypic dysfunction

doi: 10.1093/cvr/cvac036

Figure Lengend Snippet: EC-specific conditional knockout of Oct4 exacerbates atherosclerosis in Apoe−/− mice. (A) EC-specific-lineage-tracing and conditional Oct4 knockout mouse model. Schematic of Cdh5-CreERT2; Oct4Flox/Flox; Rosa-Stop-eYFP+/+Apoe−/− mouse (designated as Oct4Flox/Flox). Mice received 10 injections of tamoxifen (TMX) between 6 and 8 weeks of age. In response to tamoxifen, Cre-recombinase under the control of the Cdh5 gene promoter excises LoxP sites (black triangles), removing Oct4 exon 1 (responsible for pluripotency) and STOP codon upstream of eYFP specifically in EC. Finally, it provides simultaneous EC-lineage-tracing and EC-specific Oct4 knockout. (B) Schematic of animal experiments. (C) Representative western blot showing protein levels of OCT4, Histone H3, and GAPDH in nuclear (Nuc) and cytoplasmic (Cyto) extracts from EC isolated from aortas of EC-specific OCT4 knockout (Oct4Δ/Δ) and wild-type (Oct4+/+) mice. L, ladder; PC, positive control, A404 precursor cells. (D) Densitometry quantification of Western blots. Values = mean ± S.E.M.. *P < 0.05 by unpaired t-test; n = 3 independent experiments. (E–H) Sudan IV en face staining and quantification of lesion area in aortas of Oct4Δ/Δ and Oct4+/+ male (E and F) and female (G and H) mice after 10 or 18 weeks of WD feeding. Values = mean ± S.E.M.. *P < 0.05, #P = 0.052 by unpaired t-test; Oct4+/+: n = 9 for 10 weeks and n = 12 for 18 weeks of WD; Oct4Δ/Δ: n = 11 (females) and n = 10 (males) for 10 weeks and n = 12 for 18 weeks of WD. (I) Representative immunostaining of LGALS3 in BCAs collected from Oct4Δ/Δ and Oct4+/+ male mice after 4 weeks of WD feeding. Scale bar = 100 µm. (J) The percentage of male mice with LGALS3-positive staining in Oct4+/+ (n = 4/11 positive BCAs) and Oct4Δ/Δ (n = 7/8 positive BCAs) BCAs. BCAs were analysed at 60 µm distance from aortic arch. *P < 0.05 by Fisher’s exact test.

Article Snippet: Immunohistochemistry was performed with antibodies for LGALS3 (Cedarlane; CL8942AP; 1:500) and TER-119 (rat anti-mouse, Santa-Cruz Biotechnology Inc.; 1:200).

Techniques: Knock-Out, Control, Western Blot, Isolation, Positive Control, Staining, Immunostaining

Journal: Cardiovascular Research

Article Title: Endothelial OCT4 is atheroprotective by preventing metabolic and phenotypic dysfunction

doi: 10.1093/cvr/cvac036

Figure Lengend Snippet: EC-specific conditional knockout of Oct4 destabilizes atherosclerotic plaque. (A–C) Assessment of collagen fibre hue variations based on PicroSirius Red staining followed by polarized microscopy. (A) PicroSirius Red staining of representative BCA cross-sections of Oct4Δ/Δ and Oct4+/+ male mice fed WD for 18 weeks. Scale bar = 100 µm. The ratio between the amount of immature (green), intermediate (yellow), and mature (red) collagen fibrils showed a significant decrease in mature fibrils within the lesion (B) and the 30 µm fibrous cap area (C) of Oct4Δ/Δ mice (n = 13) as compared with control mice (n = 14), ±S.E.M.. *P < 0.05 (for mature fibrils) by unpaired Student t-test. (D) Immunofluorescence staining of DAPI, YFP, ACTA2, and LGALS3 on representative BCA sections of Oct4+/+ and Oct4Δ/Δ mice fed WD for 18 weeks, showing a noticeable increase in LGALS3+ cells in the lesion. Scale bar = 100 µm. (E and F) Quantification of the percentages of LGALS3+ lesion area (E) and the ratio of ACTA2+ cells over LGALS3+ cells within the 30 µm protective fibrous cap area (F) of BCAs collected from Oct4Δ/Δ and Oct4+/+ male and female mice after 18 weeks of WD feeding. Values = mean ± S.E.M., *P < 0.05 determined by either unpaired t-test with Welch correction (E) or unpaired t-test (F) for Oct4+/+ (n = 12 males; 12 females) vs. Oct4Δ/Δ (n = 12 males; 11 females) mice. (G) Immunostaining for the red blood cell marker TER119. Scale bar = 100 µm. Inserts indicate magnified areas. (H) The percentage of BCAs exhibiting intraplaque haemorrhage based on TER119 staining in Oct4+/+ (n = 4/13 positive BCAs) vs. Oct4Δ/Δ (n = 10/13 positive BCAs) male mice. BCAs were analysed at 120, 420, and 720 µm distance from aortic arch. If any of these three locations had any TER119-positive staining, that animal was marked as positive for haemorrhage. *P < 0.05 by Fisher’s exact test.

Article Snippet: Immunohistochemistry was performed with antibodies for LGALS3 (Cedarlane; CL8942AP; 1:500) and TER-119 (rat anti-mouse, Santa-Cruz Biotechnology Inc.; 1:200).

Techniques: Knock-Out, Staining, Microscopy, Control, Immunofluorescence, Immunostaining, Marker

Journal: Frontiers in Cardiovascular Medicine

Article Title: Deficiency of Myeloid Pfkfb3 Protects Mice From Lung Edema and Cardiac Dysfunction in LPS-Induced Endotoxemia

doi: 10.3389/fcvm.2021.745810

Figure Lengend Snippet: Myeloid-specific Pfkfb3 deficiency attenuates LPS-induced inflammatory responses. (A) Representative images (left) and quantification (right) for immunohistochemical staining of the neutrophil marker Ly6G in lung sections of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 5). (B) Representative images (left) and quantification (right) for immunohistochemical staining of the macrophage marker Mac2 in lung sections of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 5). (C–E) qPCR analysis of the mRNA levels of Il1b (C) , Il6 (D) and Nos2 (E) in the lung of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 6). (F,G) ELISA analysis of Il1b (F) and Il6 (G) in serum of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 4). (H) NO levels in serum of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 3–4). All data are represented as mean ± SEM, ** P < 0.01 and *** P < 0.001 for Pfkfb3 WT vs. Pfkfb3 ΔMϕ (unpaired two-tailed Student's t test).

Article Snippet: After antigen retrieval with Antigen Unmasking Solution (H-3301, Vector Laboratories, Burlingame, CA, USA) at 98°C for 10 min, sections were blocked with avidin solution with 10% normal rabbit serum for 1 h at room temperature, and incubated in biotin blocking solution with primary antibodies against Mac2 (3 μg/mL, CL8942F, Cedarlane, Burlington, NC, USA), or Ly6G (3 μg/mL, 551459, BD biosciences, San Jose, CA, USA) at 4°C overnight.

Techniques: Immunohistochemical staining, Staining, Marker, Injection, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Neurobiology of disease

Article Title: Novel human neuronal tau model exhibiting neurofibrillary tangles and transcellular propagation

doi: 10.1016/j.nbd.2017.06.005

Figure Lengend Snippet: Neurofibrillary tangle formation in neurons derived from hiPSC. (A) Schematic of tau-RD constructs. Tau-RD-WT-YFP contains the wild type repeat domain. Tau-RD-PP-YFP contains the repeat domain with two substitutions of isoleucine-to-proline at positions 277 and 308. Tau-RD-LM-YFP contains the repeat domain with two mutations, both cause familial frontotemporal dementia with parkinsonism (FTLD-17). The tau-RD is tagged with YFP for visualization. (B) Neuronal cultures derived from NDC hiPSC, which were transduced with tau-RD lentivirus, show NFTs, which are thioflavin positive, only in cultures transduced with tau-RD-LM (arrows). Scale bar is 10μm

Article Snippet: To investigate this, we applied a novel human induced pluripotent stem cell (hiPSC) system we have developed to serve as a human neuronal model. We introduced the tau repeat domain (tau-RD) with P301L and V337M (tau-RD-LM) mutations into hiPSC-derived neurons and observed expression of tau-RD at levels similar to total tau in postmortem AD brains.

Techniques: Derivative Assay, Construct, Transduction

Journal: Neurobiology of disease

Article Title: Novel human neuronal tau model exhibiting neurofibrillary tangles and transcellular propagation

doi: 10.1016/j.nbd.2017.06.005

Figure Lengend Snippet: Exosomes derived from tau-RD-LM conditioned media injection in normal mouse brain. Representative NTA plot of averaged size/concentration for exosomes derived from (A) neuronal cell culture media (NiPSCE) and (B) tau-RD-LM conditioned media from induced pluripotent stem cells (NiPSCE-tau-RD-LM). (C) Averaged mean particle size results from three replicates of NiPSCE and NiPSCE-tau-RD-LM suspensions (NiPSCE, 151.7 ± 9.849 vs. NiPSCE-tau-RD-LM, 114.2 ± 0.7182 nm; Unpaired t-test * = P < 0.05). (D) Non-exosome (non-exo) and exosomal fractions were probed against various tau species. Representative western blot demonstrates that human tau (KJ9A) is only detected in the exosomal fraction derived from NiPSCE-tau-RD-LM, while the non-exo and exosomal fractions were all negative for phosphorylated tau species as recognized by PHF-1 and AT8 antibodies (data not shown). Exosome surface marker, CD63, was used as a loading control. (E) Representative photomicrographs of hippocampal sections obtained from wild-type, C57/Bl6 mice that were injected with NiPSCE-tau-RD-LM suspensions. At one-month post injection, mice injected with NiPSCE-tau-RD-LM display modest enhancement of human tau immunoreactivity in the CA1 region of the hippocampus (n = 5/group).

Article Snippet: To investigate this, we applied a novel human induced pluripotent stem cell (hiPSC) system we have developed to serve as a human neuronal model. We introduced the tau repeat domain (tau-RD) with P301L and V337M (tau-RD-LM) mutations into hiPSC-derived neurons and observed expression of tau-RD at levels similar to total tau in postmortem AD brains.

Techniques: Derivative Assay, Injection, Concentration Assay, Cell Culture, Western Blot, Marker

Journal: Cell stem cell

Article Title: Smooth muscle cell reprogramming in aortic aneurysms

doi: 10.1016/j.stem.2020.02.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Monoclonal anti-Mac2/Galectin-3-153Eu (clone M3/38) , Fluidigm , Cat# 3153026B, RRID:AB_2814900.

Techniques: Recombinant, Multiplex Assay, Staining, In Situ, Marker, Expressing, Plasmid Preparation, shRNA, Software

Journal: Reviews in Cardiovascular Medicine

Article Title: The Role of Soluble Suppression of Tumorigenicity-2 and Galectin-3 in Heart Failure With Preserved Ejection Fraction: A Scoping Review of the Literature

doi: 10.31083/RCM51830

Figure Lengend Snippet: Diagnostic and prognostic value of sST2 and Gal-3 .

Article Snippet: Kanukurti et al. [ ] , Cross-sectional (Diagnosis) , India 83 (75.9%) , - , Referral by cardiologist , Gal-3 >10.1 ng/mL ELISA (Elabscience Human ELISA kit, Houston, TX, USA) , - , - , - , -.

Techniques: Diagnostic Assay, Sensitivity, Marker

Journal: bioRxiv

Article Title: Expression and role of Galectin-3 in the postnatal development of the cerebellum

doi: 10.1101/364760

Figure Lengend Snippet: (A) Lgals3 mRNA expression relative to Rpl13a measured by RTqPCR in the cerebellum during development. E17: embryonic day 17. P0, P7, P14, and P21: postnatal day 0, 7, 14, and 21. (B) Lgals3 knockout (KO) mice present no defects in cerebellar morphology. Parasagittal cerebellar slices of wild-type (WT) and Lgals3 KO adult mice were stained using the nuclear marker Hoechst.

Article Snippet: The following primary antibodies were used: mouse monoclonal anti-CABP (1:1000; swant, Cat#300), rabbit polyclonal anti-CABP (1:1000; swant, Cat#9.03), mouse monoclonal anti-GFAP (1:500; millipore, Cat#MAB360), rabbit polyclonal anti-GLURδ1/2 (1:1000; millipore, Cat#AB2285), goat polyclonal anti-LGALS3 (1:200; R&D Systems, Cat#AF1197), mouse monoclonal anti-OLIG2 (1:500; millipore, Cat#MABN50), guinea pig polyclonal anti-VGLUT1 (1:5000; millipore, Cat#AB5905) and guinea pig polyclonal anti-VGLUT2 (1:5000; millipore, Cat#AB2251).

Techniques: Expressing, Knock-Out, Staining, Marker

Journal: bioRxiv

Article Title: Expression and role of Galectin-3 in the postnatal development of the cerebellum

doi: 10.1101/364760

Figure Lengend Snippet: (A and B) Immunostaining for the Purkinje cell marker CABP (green) and the presynaptic marker VGLUT2 (red) was performed on sections from Lgals3 WT and KO mice at P15 (A) and at P65 (B). (C and D) Immunostaining for the Purkinje cell marker CABP (green) and the presynaptic marker VGLUT1 (red) specific for mature parallel fiber synapses was performed on sections from Lgals3 WT and KO mice at P15 (C) and at P65 (D). Scale bar: 25μm. Data are representative of three independent experiments.

Article Snippet: The following primary antibodies were used: mouse monoclonal anti-CABP (1:1000; swant, Cat#300), rabbit polyclonal anti-CABP (1:1000; swant, Cat#9.03), mouse monoclonal anti-GFAP (1:500; millipore, Cat#MAB360), rabbit polyclonal anti-GLURδ1/2 (1:1000; millipore, Cat#AB2285), goat polyclonal anti-LGALS3 (1:200; R&D Systems, Cat#AF1197), mouse monoclonal anti-OLIG2 (1:500; millipore, Cat#MABN50), guinea pig polyclonal anti-VGLUT1 (1:5000; millipore, Cat#AB5905) and guinea pig polyclonal anti-VGLUT2 (1:5000; millipore, Cat#AB2251).

Techniques: Immunostaining, Marker

Journal: bioRxiv

Article Title: Expression and role of Galectin-3 in the postnatal development of the cerebellum

doi: 10.1101/364760

Figure Lengend Snippet: (A) Immunohistochemistry for LGALS3 show the specificity of the LGALS3 antibody. WT: wild-type mice; KO: knockout mice. Positive cells in the molecular layer are marked with a white arrowhead, scale bar 50μm. (B) Immunohistochemistry for the Purkinje cell marker CABP (green) and LGALS3 (red) on parasagittal cerebellar sections from P15 and P22 wild-type mice. Scale bar=100μm. Data are representative of three independent experiments. Legends: 4V, 4 th ventricle; chp, choroid plexus; pcg, pontine central gray and mv, medial vestibular nucleus.

Article Snippet: The following primary antibodies were used: mouse monoclonal anti-CABP (1:1000; swant, Cat#300), rabbit polyclonal anti-CABP (1:1000; swant, Cat#9.03), mouse monoclonal anti-GFAP (1:500; millipore, Cat#MAB360), rabbit polyclonal anti-GLURδ1/2 (1:1000; millipore, Cat#AB2285), goat polyclonal anti-LGALS3 (1:200; R&D Systems, Cat#AF1197), mouse monoclonal anti-OLIG2 (1:500; millipore, Cat#MABN50), guinea pig polyclonal anti-VGLUT1 (1:5000; millipore, Cat#AB5905) and guinea pig polyclonal anti-VGLUT2 (1:5000; millipore, Cat#AB2251).

Techniques: Immunohistochemistry, Knock-Out, Marker

Journal: bioRxiv

Article Title: Expression and role of Galectin-3 in the postnatal development of the cerebellum

doi: 10.1101/364760

Figure Lengend Snippet: (A) LGALS3 expression in oligodendrocytes in the cerebellar white matter. Co-immunostaining of parasagittal cerebellar slices using anti-LGALS3 (red), oligodendrocyte marker anti-OLIG-2 (green) and the nuclear marker Hoechst (blue). (B) LGALS3 expression in astrocytes in the cerebellar white matter. Co-immunostaining of parasagittal cerebellar slices using anti-LGALS3 (red), astrocyte marker anti-GFAP (green) and the nuclear marker Hoechst (blue). (C) LGALS3 expression in some microglial cells of the molecular layer. Immunostaining anti-LGALS3 (red) in parasagittal sections of mice CX 3 CR1 eGFP/eGFP , microglial CX 3 CR1-GFP (green) and the nuclear marker Hoechst (blue). Scale bar 50μm and 20μm for the magnification. Data are representative of three independent experiments. Legends: 4V, 4 th ventricle; GL, granular layer; ML, molecular layer and WM, white matter.

Article Snippet: The following primary antibodies were used: mouse monoclonal anti-CABP (1:1000; swant, Cat#300), rabbit polyclonal anti-CABP (1:1000; swant, Cat#9.03), mouse monoclonal anti-GFAP (1:500; millipore, Cat#MAB360), rabbit polyclonal anti-GLURδ1/2 (1:1000; millipore, Cat#AB2285), goat polyclonal anti-LGALS3 (1:200; R&D Systems, Cat#AF1197), mouse monoclonal anti-OLIG2 (1:500; millipore, Cat#MABN50), guinea pig polyclonal anti-VGLUT1 (1:5000; millipore, Cat#AB5905) and guinea pig polyclonal anti-VGLUT2 (1:5000; millipore, Cat#AB2251).

Techniques: Expressing, Immunostaining, Marker