Mouse Clusterin Antibody

Detection of Mouse Clusterin/APOJ by Western Blot

Extracellular clusterin limits the uptake of alpha‐synuclein preformed fibrils (pffs) by murine primary astrocytes. (a) Cell lysates from clusterin wild‐type (WT) and knock‐out (KO) astrocytes were subjected to immunoblotting using clusterin and GAPDH antibodies. Asterisk indicates clusterin precursor. (b) Maximum intensity Z‐projection confocal images of clusterin WT and KO astrocytes treated with * alpha‐synuclein pffs for 4 hr. Scale bar 5 μm. (c) Quantification of * alpha‐synuclein pffs is shown as mean of fluorescence intensity from six independent experiments (~50 cells per experiment). Quantification of * alpha‐synuclein pffs is calculated as fluorescence intensity divided by the cell area (μm2). Data are expressed as the mean ± SD. Data were analyzed using unpaired t test; *p = .0315. (d) Cell lysates from clusterin WT and KO primary astrocytes treated with pffs for 16 hr were subjected to immunoblotting using alpha‐synuclein and GAPDH antibodies. (e) Quantification of intracellular alpha‐synuclein is normalized to GAPDH protein. Data are representative of eight independent experiments and are expressed as the mean ± SD. Data were analyzed using unpaired t test; *p = .0239. (f) Cell lysates from clusterin KO primary astrocytes treated with pffs for 16 hr in a medium containing or not containing extracellular clusterin were subjected to immunoblotting using alpha‐synuclein, clusterin and GAPDH antibodies. (g) Quantification of intracellular alpha‐synuclein is normalized to GAPDH protein. Data are representative of four independent experiments and are expressed as the mean ± SD. Data were analyzed using unpaired t test; *p = .0134. (h) Cell lysates from clusterin WT and KO primary astrocytes treated with pffs and chloroquine (CQ) for 16 hr were subjected to immunoblotting using alpha‐synuclein and GAPDH antibodies. (i) Quantification of intracellular alpha‐synuclein normalized to GAPDH protein. Data are representative of six independent experiments and are expressed as the mean ± SD. Data were analyzed using unpaired t test; *p = .0377. Individual points of the graphs represent each single experiment [Color figure can be viewed at wileyonlinelibrary.com] Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/33045109), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Clusterin/APOJ by Western Blot

Clusterin protein levels are increased in two different animal models of Parkinson's disease. (a) Lysates from contralateral and ipsilateral ventral midbrain of mice injected with adeno‐associated virus (AAV)‐h alpha‐synuclein and sacrificed after 8 weeks were subjected to immunoblotting using clusterin and GAPDH antibodies. (b) Quantification of total cleaved clusterin is normalized to GAPDH protein. Data are representative of five animals and are expressed as the mean ± SD. Data were analyzed using one‐sample t test; *p = .0253. (c) Representative images of clusterin‐GFAP double labeling in the ipsilateral and contralateral striatum of mice injected with AAV‐h alpha‐synuclein and sacrificed after 8 weeks. Scale bar 20 μm. (d) Representative images of clusterin‐S100 beta double labeling in the substantia nigra of mice injected with alpha‐synuclein preformed fibrils (pffs) and sacrificed after 4 weeks. Scale bar 20 μm. (e) Representative images of clusterin‐S100 beta double labeling in the striatum of mice injected with alpha‐synuclein pffs and sacrificed after 4 weeks. Scale bar 20 μm [Color figure can be viewed at wileyonlinelibrary.com] Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/33045109), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Clusterin/APOJ by Western Blot

Conservation of clusterin‐dependent alpha‐synuclein preformed fibrils (pffs) uptake from mouse to human astrocytes. (a) Representative image of hiAstrocytes (27 days of differentiation) treated with * alpha‐synuclein pffs for 48 hr. Scale bar 10 μm. (b) Cell lysates from hiAstrocytes (27 days of differentiation) treated with alpha‐synuclein pffs, or monomeric alpha‐synuclein (m), for 48 hr were subjected to immunoblotting using alpha‐synuclein and GAPDH antibodies. Asterisks in the alpha‐synuclein immunoblot indicate alpha‐synuclein nonspecific bands. (c) Medium from hiAstrocytes (27 days of differentiation) treated with alpha‐synuclein pffs, or monomeric alpha‐synuclein (M), for 48 hr were subjected to immunoblotting using clusterin antibody. Ponceau‐S staining was used as loading control. (d) Quantification of clusterin is normalized to GAPDH protein of cell lysates. Data are representative of three independent experiments and are expressed as the mean ± SD. Data were analyzed using unpaired t test; *p = .0446. (e) Cell lysates from hiAstrocytes (31 days of differentiation) transfected with negative control (NC) or clusterin siRNA for 24 hr were subjected to immunoblotting using clusterin and GAPDH antibodies. Asterisk in the clusterin immunoblot indicates clusterin nonspecific band. (f) Quantification of clusterin precursor is normalized to GAPDH. Data are representative of three independent experiments and are expressed as the mean ± SD. Data were analyzed using unpaired t test; *p = .0165. (g) Representative images of hiAstrocytes (35 days of differentiation) transfected with NC or clusterin siRNA and treated with * alpha‐synuclein pffs for 24 hr. Scale bar 10 μm. (h) Quantification of * alpha‐synuclein pffs is shown as mean of fluorescence intensity from three independent experiments (~50 cells per experiment). Quantification of * alpha‐synuclein pffs is calculated as fluorescence intensity divided by the cell area (μm2). Data are expressed as the mean ± SD. Data were analyzed using unpaired t test; *p = .0178. Individual points of the graphs represent each single experiment [Color figure can be viewed at wileyonlinelibrary.com] Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/33045109), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Clusterin/APOJ by Western Blot

Extracellular clusterin limits the uptake of alpha‐synuclein preformed fibrils (pffs) by murine primary astrocytes. (a) Cell lysates from clusterin wild‐type (WT) and knock‐out (KO) astrocytes were subjected to immunoblotting using clusterin and GAPDH antibodies. Asterisk indicates clusterin precursor. (b) Maximum intensity Z‐projection confocal images of clusterin WT and KO astrocytes treated with * alpha‐synuclein pffs for 4 hr. Scale bar 5 μm. (c) Quantification of * alpha‐synuclein pffs is shown as mean of fluorescence intensity from six independent experiments (~50 cells per experiment). Quantification of * alpha‐synuclein pffs is calculated as fluorescence intensity divided by the cell area (μm2). Data are expressed as the mean ± SD. Data were analyzed using unpaired t test; *p = .0315. (d) Cell lysates from clusterin WT and KO primary astrocytes treated with pffs for 16 hr were subjected to immunoblotting using alpha‐synuclein and GAPDH antibodies. (e) Quantification of intracellular alpha‐synuclein is normalized to GAPDH protein. Data are representative of eight independent experiments and are expressed as the mean ± SD. Data were analyzed using unpaired t test; *p = .0239. (f) Cell lysates from clusterin KO primary astrocytes treated with pffs for 16 hr in a medium containing or not containing extracellular clusterin were subjected to immunoblotting using alpha‐synuclein, clusterin and GAPDH antibodies. (g) Quantification of intracellular alpha‐synuclein is normalized to GAPDH protein. Data are representative of four independent experiments and are expressed as the mean ± SD. Data were analyzed using unpaired t test; *p = .0134. (h) Cell lysates from clusterin WT and KO primary astrocytes treated with pffs and chloroquine (CQ) for 16 hr were subjected to immunoblotting using alpha‐synuclein and GAPDH antibodies. (i) Quantification of intracellular alpha‐synuclein normalized to GAPDH protein. Data are representative of six independent experiments and are expressed as the mean ± SD. Data were analyzed using unpaired t test; *p = .0377. Individual points of the graphs represent each single experiment [Color figure can be viewed at wileyonlinelibrary.com] Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/33045109), licensed under a CC-BY license. Not internally tested by R&D Systems.