recombinant clu  (R&D Systems)

Journal: Acta Neuropathologica Communications

Article Title: Clusterin ameliorates tau pathology in vivo by inhibiting fibril formation

doi: 10.1186/s40478-020-01079-1

Figure Lengend Snippet: CLU binds to tau and prevents tau aggregation. a In vitro tau fibrilization assay was performed with the 4R0N tau isoform in the presence or absence of recombinant human CLU and thioflavine T intensity was monitored during 24-h time course. b Quantification of thioflavine T signal at the 24-h time point. Four replicates were used. Data present as mean ± S.E.M. and analyzed by Student’s t test *** p < 0.001. c Western blot analysis of soluble (supernatant, s) and insoluble (pellet, p) fractions following tau fibril assembly in the presence or absence of exogenous CLU. d Tau assembly assay performed with 4R0N recombinant tau, tau PHFs from AD brains, and increasing concentrations of recombinant CLU. Four replicates were used. Data present as mean ± S.E.M. and analyzed by one-way ANOVA, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Tau filament assembly was performed with 5 μM 4R0N isoform in assembly buffer (10 mMHEPES (pH7.4), 33 mM NaCl, 1 mM MgCl2, 1.5 mM EGTA, 60 μM EDTA, 5uM Thioflavin-T and 20ug/ml Heparin) with and without 4 μM recombinant CLU (R&D #2937-HS-050).

Techniques: In Vitro, Recombinant, Western Blot

Journal: PLoS ONE

Article Title: Clusterin Seals the Ocular Surface Barrier in Mouse Dry Eye

doi: 10.1371/journal.pone.0138958

Figure Lengend Snippet: The standard desiccating stress (DS) protocol was applied, while eyes were left untreated (UT) or treated topically 4 times/day with 1 uL of CLU formulated in PBS, or with PBS control. Non-stressed (NS) mice housed under normal ambient conditions served as a baseline control. After the indicated time period, barrier integrity was assayed by measuring corneal epithelial uptake of fluorescein (FU = Fluorescence Units at 521 nm). Values are expressed as the mean ± SD. (A) The desiccating stress (DS) protocol was applied for 5 days while also treating with rhCLU at 10 or 100 ug/mL. *P<0.0001 (n = 9). (B) The desiccating stress (DS) protocol was applied for 7 days while also treating with rhCLU at 1 or 10 ug/mL. *P<0.0001 (n = 4). (C) The desiccating stress (DS) protocol was applied for 5 days while also treating with human plasma CLU (pCLU) at 2 ug/mL *P<0.0001 (n = 4). (D) The desiccating stress (DS) protocol was applied for 5 days while also treating with recombinant mouse CLU (rmCLU) at 2 ug/mL. *P<0.0001 (n = 4)

Article Snippet: The secreted form of recombinant human CLU (rhCLU) and recombinant mouse CLU (rmCLU), both of which contain a polyhistidine-tag (His6 tag) at the C-terminus, were purchased from R&D Systems (Minneapolis, MN).

Techniques: Fluorescence, Recombinant

Journal: PLoS ONE

Article Title: Clusterin Seals the Ocular Surface Barrier in Mouse Dry Eye

doi: 10.1371/journal.pone.0138958

Figure Lengend Snippet: The standard desiccating stress (DS) protocol was applied, while eyes were left untreated (UT) or treated topically 4 times/day with 1 uL of CLU formulated in PBS, or with PBS control. Non-stressed (NS) mice housed under normal ambient conditions served as a baseline control. After the indicated time period, barrier integrity was assayed by measuring corneal epithelial uptake of fluorescein (FU = Fluorescence Units at 521 nm). Values are expressed as the mean ± SD. (A) Dose response experiment. The desiccating stress (DS) protocol was applied for 5 days while also treating with (Left) recombinant human CLU (rhCLU) at the indicated 10-fold dilutions (n = 6), (Middle) recombinant human CLU (rhCLU) at 0.1, 0.3, 0.6, or 1 ug/mL (n = 6), or (Right) recombinant mouse CLU (rmCLU) at 0.3, 0.6, and 1 ug/mL (n = 4). *P<0.0001. (B) Experiment comparing CLU with BSA. The desiccating stress (DS) protocol was applied for 5 days while also treating with recombinant human CLU (rhCLU) and BSA, individually or in combination, as indicated. *P<0.0001 (n = 4) . (C) Stress reduction experiment. The standard desiccating stress (DS) protocol was applied for 5 days while eyes were also treated with recombinant human CLU (rhCLU) at 0.01, 0.1, and 1 ug/mL. Using a subset (n = 4) of each treatment group the effect of each rhCLU dose on integrity of the ocular surface barrier was confirmed by the fluorescein uptake test at day 5. Then the rest of the mice in each treatment group were subjected for two more days to a more moderate desiccating stress by continuing with the air draft and heat, but omitting scopolamine and CLU treatments. The fluorescein uptake test was then performed on these remaining mice. *P = 0.004 (n = 4); **P = 0.05 (n = 4)

Article Snippet: The secreted form of recombinant human CLU (rhCLU) and recombinant mouse CLU (rmCLU), both of which contain a polyhistidine-tag (His6 tag) at the C-terminus, were purchased from R&D Systems (Minneapolis, MN).

Techniques: Fluorescence, Recombinant

Journal: PLoS ONE

Article Title: Clusterin Seals the Ocular Surface Barrier in Mouse Dry Eye

doi: 10.1371/journal.pone.0138958

Figure Lengend Snippet: (Left). The standard desiccating stress (DS) protocol was applied for 5-days to create ocular surface disruption. Non-stressed (NS) mice housed under normal ambient conditions served as a baseline control. (Left) After the indicated time period, barrier disruption was confirmed by measuring corneal epithelial uptake of fluorescein (FU = Fluorescence Units at 521 nm) in a subset of mice. Values are expressed as the mean ± SD. *p<0.0001 (n = 4). (Right) The same desiccating stress (DS) protocol was continued for another 5 days while eyes with desiccating stress were treated topically with 1 uL of recombinant human CLU (rhCLU) formulated in PBS at 2 ug/mL, or with PBS control, 4 times/day. The fluorescein uptake test was then performed on these remaining mice. Values are expressed as the mean ± SD. *p<0.0001(n = 4).

Article Snippet: The secreted form of recombinant human CLU (rhCLU) and recombinant mouse CLU (rmCLU), both of which contain a polyhistidine-tag (His6 tag) at the C-terminus, were purchased from R&D Systems (Minneapolis, MN).

Techniques: Disruption, Fluorescence, Recombinant

Journal: PLoS ONE

Article Title: Clusterin Seals the Ocular Surface Barrier in Mouse Dry Eye

doi: 10.1371/journal.pone.0138958

Figure Lengend Snippet: The standard desiccating stress (DS) protocol was applied for 5-days to create ocular surface disruption. Non-stressed (NS) mice housed under normal ambient conditions served as a baseline control. Eyes with desiccating stress were then treated topically, a single time, with 1 uL of CLU formulated in PBS, 1 uL of BSA formulated in PBS for comparison, or 1 uL of PBS control. Barrier disruption was assayed by measuring corneal epithelial uptake of fluorescein (FU = Fluorescence Units at 521 nm). Values are expressed as the mean ± SD. (A) Eyes were treated a single time with recombinant human CLU (rhCLU) at 1, 3, 6 or 10 ug/mL, BSA at 10 ug/mL, or PBS. Fifteen minutes later, the fluorescein uptake test was performed, before there was time for barrier repair to occur. *P<0.0001 (n = 4). (B) Images of central cornea from the experiment shown in (A), obtained using laser scanning confocal microscopy at 10X magnification. One representative image out of two independent experiments is shown. Scale bar = 100 um. (C) Eyes were treated a single time with recombinant human CLU (rhCLU) at 10 ug/mL (right eyes) or PBS (left eyes). Then the mice were kept further for 2 h or 16 h while continuing with the same desiccating stress protocol. The fluorescein uptake test was performed following the indicated time period to assess the time length of CLU treatment effect. *p<0.0001 (n = 4)

Article Snippet: The secreted form of recombinant human CLU (rhCLU) and recombinant mouse CLU (rmCLU), both of which contain a polyhistidine-tag (His6 tag) at the C-terminus, were purchased from R&D Systems (Minneapolis, MN).

Techniques: Disruption, Comparison, Fluorescence, Recombinant, Confocal Microscopy

Journal: PLoS ONE

Article Title: Clusterin Seals the Ocular Surface Barrier in Mouse Dry Eye

doi: 10.1371/journal.pone.0138958

Figure Lengend Snippet: (A) The standard desiccating stress (DS) protocol was applied for 5-days to create ocular surface disruption. Non-stressed (NS) mice housed under normal ambient conditions were included for comparison. Eyes were treated with CF-594-anti-His antibody that binds to the His tag of recombinant human CLU (rhCLU), or with a complex of the antibody-rhCLU for 15 min, followed by confocal imaging of central cornea. Images were taken at 10X magnification. Scale bar = 100 um. (B) A DS eye was treated with a complex of the antibody-rhCLU (red) as in (A), as well as a fluorescent membrane tracer DiO (green). Images were taken at 20X magnification. In the left panel only CLU was projected. The right three panels show one Z-section plane with cross-sections oriented to the XY, YZ, and XZ axes, generated using Image J software. Yellow indicates regions of co-localization of the red and green signal. Scale bar = 100 um. (C) LGALS3-Sepharose affinity column chromatography. 1.5 ug rhCLU was applied to a 300 uL LGALS3 affinity column equilibrated in PBS containing 0.1% Triton X-100 (PBST) and the column was washed with PBST. To test sugar-binding specificity, the column was then treated sequentially with a non-competing disaccharide, sucrose (0.1 M), and then a competing disaccharide, 0.1 M lactose, dissolved in PBST. Western blotting was used to quantify CLU in the resulting fractions. Loading of the “Lac” lane represents a 1:10 dilution of the input and the “Beads” lane is a 1:4 dilution of the input, thus ~2.5X more CLU was Lac-eluted than retained on the beads. FT = flow-through; Suc = sucrose; Lac = lactose

Article Snippet: The secreted form of recombinant human CLU (rhCLU) and recombinant mouse CLU (rmCLU), both of which contain a polyhistidine-tag (His6 tag) at the C-terminus, were purchased from R&D Systems (Minneapolis, MN).

Techniques: Disruption, Comparison, Recombinant, Imaging, Membrane, Generated, Software, Affinity Column, Chromatography, Binding Assay, Western Blot

Journal: PLoS ONE

Article Title: Clusterin Seals the Ocular Surface Barrier in Mouse Dry Eye

doi: 10.1371/journal.pone.0138958

Figure Lengend Snippet: (A) The standard desiccating stress (DS) protocol was applied, while eyes were left untreated (UT) or treated topically, 4 times/day, with 1 uL of recombinant human CLU (rhCLU) formulated in PBS, or with 1 uL of PBS control. Non-stressed (NS) mice housed under normal ambient conditions were included as a control for PBS treatment. At the end of the experiment, eyes were removed and embedded for frozen sectioning at 10-um thickness. TUNEL staining was performed and nuclei were counterstained with DAPI. Images were taken at 20X magnification. Arrows indicate apoptotic cells in the apical ocular surface epithelium of DS+PBS eyes. (B) The standard desiccating stress (DS) protocol was applied, while eyes were left untreated (UT) or treated topically, 4 times/day, with 1 uL of recombinant human CLU (rhCLU) formulated in PBS, or with 1 uL of PBS control. Non-stressed (NS) mice housed under normal ambient conditions were included as a control for PBS treatment. Desiccating stress was applied to 7 mice per treatment group for 5 days (OCLN) or 9 days (LGALS3) while treated with PBS or CLU at 1 ug/mL. Then total proteins were extracted from the ocular surface epithelia using TRIzol, pooled among the same treatment groups, and subjected to Western blotting with anti-LGALS3 and anti-OCLN antibodies. The protein band image was obtained by Fuji Doc digital camera. “F” indicates full length LGALS3 protein, and “C” is the cleaved product of LGALS3. A digital image analyzer built into the camera was used to quantify the density of individual protein bands. The relative cleavage of LGALS3 was calculated by ratio of the C over the total (F+C) LGALS3 protein. The relative amount of OCLN was normalized to the loading control (ACTB) in each gel lane. (C) Stratified HCLE cells were treated with TNFA (5 ng/mL), alone or with recombinant human CLU (rhCLU) (4 ug/mL) or BSA (40 ug/mL) for 24 h. the conditioned media were subject to gelatin zymography and the developed MMP9 image were analyzed by Image J software. *P<0.05 (n = 3, student’s t-test)

Article Snippet: The secreted form of recombinant human CLU (rhCLU) and recombinant mouse CLU (rmCLU), both of which contain a polyhistidine-tag (His6 tag) at the C-terminus, were purchased from R&D Systems (Minneapolis, MN).

Techniques: Recombinant, TUNEL Assay, Staining, Western Blot, Zymography, Software