Review

mouse anti-human clusterin b-5 (Santa Cruz Biotechnology)

Name: mouse anti-human clusterin b-5
Brand: Santa Cruz Biotechnology
Rating: 90 (1 reviews)

Santa Cruz Biotechnology is a verified supplier

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

Santa Cruz Biotechnology mouse anti-human clusterin b-5

Glomerular <t>clusterin</t> expression is increased in patients with DN. ( a , b ) Representative images of kidney sections obtained from a healthy subject (Control; a ) and a patient with DN (DN; b ) stained for clusterin; the scale bars represent 200 µm. ( c , d ) Representative high-magnification images of a and b; the scale bars represent 50 µm. ( e , f ) The high-magnification views of the rectangles in ( c , d ). Clusterin staining was present along the outer side of the GBM at the place where the podocytes are located (arrowheads; the square shows a zoomed in image of a clusterin-positive podocyte). ( g ) Summary of clusterin staining (semi-quantitative score) in the glomeruli of healthy subjects (Control; n = 10) and patients with DN (DN; n = 12); *** P < 0.001 vs. control (Student’s t-test). ( h ) Summary of CLU mRNA measured in isolated glomeruli from healthy subjects (Control; n = 11) and patients with DN (DN; n = 24); ** P < 0.01 vs. control (Student’s t-test).

Mouse Anti Human Clusterin B 5, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse anti-human clusterin b-5/product/Santa Cruz Biotechnology
Average 90 stars, based on 1 article reviews

mouse anti-human clusterin b-5 - by Bioz Stars, 2026-02

90/100 stars

Images

1) Product Images from "Glomerular clusterin expression is increased in diabetic nephropathy and protects against oxidative stress-induced apoptosis in podocytes"

Article Title: Glomerular clusterin expression is increased in diabetic nephropathy and protects against oxidative stress-induced apoptosis in podocytes

Journal: Scientific Reports

doi: 10.1038/s41598-020-71629-z

Glomerular clusterin expression is increased in patients with DN. ( a , b ) Representative images of kidney sections obtained from a healthy subject (Control; a ) and a patient with DN (DN; b ) stained for clusterin; the scale bars represent 200 µm. ( c , d ) Representative high-magnification images of a and b; the scale bars represent 50 µm. ( e , f ) The high-magnification views of the rectangles in ( c , d ). Clusterin staining was present along the outer side of the GBM at the place where the podocytes are located (arrowheads; the square shows a zoomed in image of a clusterin-positive podocyte). ( g ) Summary of clusterin staining (semi-quantitative score) in the glomeruli of healthy subjects (Control; n = 10) and patients with DN (DN; n = 12); *** P < 0.001 vs. control (Student’s t-test). ( h ) Summary of CLU mRNA measured in isolated glomeruli from healthy subjects (Control; n = 11) and patients with DN (DN; n = 24); ** P < 0.01 vs. control (Student’s t-test).

Figure Legend Snippet: Glomerular clusterin expression is increased in patients with DN. ( a , b ) Representative images of kidney sections obtained from a healthy subject (Control; a ) and a patient with DN (DN; b ) stained for clusterin; the scale bars represent 200 µm. ( c , d ) Representative high-magnification images of a and b; the scale bars represent 50 µm. ( e , f ) The high-magnification views of the rectangles in ( c , d ). Clusterin staining was present along the outer side of the GBM at the place where the podocytes are located (arrowheads; the square shows a zoomed in image of a clusterin-positive podocyte). ( g ) Summary of clusterin staining (semi-quantitative score) in the glomeruli of healthy subjects (Control; n = 10) and patients with DN (DN; n = 12); *** P < 0.001 vs. control (Student’s t-test). ( h ) Summary of CLU mRNA measured in isolated glomeruli from healthy subjects (Control; n = 11) and patients with DN (DN; n = 24); ** P < 0.01 vs. control (Student’s t-test).

Techniques Used: Expressing, Control, Staining, Isolation

Clusterin expression is increased in the glomeruli of mice with type 1 diabetes and co-localises with podocytes. ( a , b ) Representative images of mouse kidney sections obtained from control mice (Control; a ) and STZ-treated mice 15 weeks after diabetes was induced (STZ; b ); the scale bars represent 100 µm. ( c , d ) The high-magnification views of the rectangles in ( a, b ); the scale bars represent 50 µm. ( e , f ) Representative images of the adjacent sections stained for clusterin ( e ) and WT1 ( f ), showing co-localisation of clusterin with WT1 in the glomeruli (arrowheads). ( g ) Averaged clusterin-positive area (measured by using ImageJ) in the glomeruli of control and STZ-treated mice 15 weeks after diabetes was induced; ** P < 0.01 vs. control (Student’s t-test).

Figure Legend Snippet: Clusterin expression is increased in the glomeruli of mice with type 1 diabetes and co-localises with podocytes. ( a , b ) Representative images of mouse kidney sections obtained from control mice (Control; a ) and STZ-treated mice 15 weeks after diabetes was induced (STZ; b ); the scale bars represent 100 µm. ( c , d ) The high-magnification views of the rectangles in ( a, b ); the scale bars represent 50 µm. ( e , f ) Representative images of the adjacent sections stained for clusterin ( e ) and WT1 ( f ), showing co-localisation of clusterin with WT1 in the glomeruli (arrowheads). ( g ) Averaged clusterin-positive area (measured by using ImageJ) in the glomeruli of control and STZ-treated mice 15 weeks after diabetes was induced; ** P < 0.01 vs. control (Student’s t-test).

Techniques Used: Expressing, Control, Staining

Clusterin expression in podocytes cultured under various diabetic conditions. ( a–d ) Human podocytes were cultured for the indicated times in PAN (30 µg/mL) ( a ), Glucose (5 mM or 25 mM) ( b ), Angiotensin II (1 µM) ( c ), or the indicated concentrations of MGO for 24 h ( d ). The CLU mRNA expression after these treatments was measured, the mRNA levels were normalised to the respective control. In ( a – c ), * P < 0.05 vs. the respective control at the same time point (Student’s t-test); in ( d ), * P < 0.05 vs. control, and † P < 0.05 vs. 0.5 mM MGO (one-way ANOVA). PAN puromycin aminonucleoside, MGO methylglyoxal.

Figure Legend Snippet: Clusterin expression in podocytes cultured under various diabetic conditions. ( a–d ) Human podocytes were cultured for the indicated times in PAN (30 µg/mL) ( a ), Glucose (5 mM or 25 mM) ( b ), Angiotensin II (1 µM) ( c ), or the indicated concentrations of MGO for 24 h ( d ). The CLU mRNA expression after these treatments was measured, the mRNA levels were normalised to the respective control. In ( a – c ), * P < 0.05 vs. the respective control at the same time point (Student’s t-test); in ( d ), * P < 0.05 vs. control, and † P < 0.05 vs. 0.5 mM MGO (one-way ANOVA). PAN puromycin aminonucleoside, MGO methylglyoxal.

Techniques Used: Expressing, Cell Culture, Control

Recombinant human clusterin protein protects podocytes against methylglyoxal-induced oxidative stress and apoptosis. ( a ) Cell viability was measured in podocytes incubated with the indicated concentrations of rhCLU for 24 h. ( b ) Cell viability was measured in podocytes pre-incubated for 4 h with the indicated concentrations of rhCLU, followed by 24 h in the presence or absence of 1.5 mM MGO. Cell viability in the presence of MGO was normalised to cells stimulated in the absence of rhCLU; * P < 0.05 vs. 0 µg/mL rhCLU + 1.5 mM MGO (one-way ANOVA). ( c ) Representative images of podocytes treated with Milli-Q water (Control) or podocytes pre-treated for 4 h with 2.0 µg/mL rhCLU, 2 h with 1.5 mM MGO, or both, followed by MitoSOX Red staining. The scale bars indicate 20 µm and the boxed areas are shown at higher magnification in the bottom row. ( d ) Representative images of podocytes treated as described in ( c ), followed by DCFDA staining. The scale bars indicate 100 µm. ( e ) Caspase3/7 activity in podocytes treated with Milli-Q water (Control) or MGO (1.5 mM) for 12 h with or without pre-treated with 2.0 µg/mL rhCLU for 4 h. The measurement was normalised to the control; ** P < 0.01 vs. control, †† P < 0.01 vs. rhCLU, and ‡‡ P < 0.01 vs. MGO (one-way ANOVA). (f) Podocytes treated with Milli-Q water (Control) or MGO (1.5 mM) for 2 h with or without pre-treatment with 2.0 µg/mL rhCLU for 4 h, followed by measuring BAX and BCL2 mRNA expression. The BAX/BCL2 ratio was calculated and normalised to the control; ** P < 0.01 vs. control, †† P < 0.01 vs. rhCLU, and ‡ P < 0.05 vs. MGO (one-way ANOVA). ( g , h ) Podocytes treated with Milli-Q water (Control) or MGO (1.5 mM) for 2 h with or without pre-treated with 2.0 µg/mL rhCLU for 4 h, followed by measuring ZO-1 ( g ) and NEPH1 ( h ) mRNA expression; the mRNA levels were normalised to the respective control; * P < 0.05 vs. control, ** P < 0.01 vs. control *** P < 0.001 vs. control, † P < 0.05 vs. rhCLU, †† P < 0.01 vs. rhCLU, and ‡ P < 0.05 vs. MGO (one-way ANOVA). MGO methylglyoxal, rhCLU recombinant human clusterin protein.

Figure Legend Snippet: Recombinant human clusterin protein protects podocytes against methylglyoxal-induced oxidative stress and apoptosis. ( a ) Cell viability was measured in podocytes incubated with the indicated concentrations of rhCLU for 24 h. ( b ) Cell viability was measured in podocytes pre-incubated for 4 h with the indicated concentrations of rhCLU, followed by 24 h in the presence or absence of 1.5 mM MGO. Cell viability in the presence of MGO was normalised to cells stimulated in the absence of rhCLU; * P < 0.05 vs. 0 µg/mL rhCLU + 1.5 mM MGO (one-way ANOVA). ( c ) Representative images of podocytes treated with Milli-Q water (Control) or podocytes pre-treated for 4 h with 2.0 µg/mL rhCLU, 2 h with 1.5 mM MGO, or both, followed by MitoSOX Red staining. The scale bars indicate 20 µm and the boxed areas are shown at higher magnification in the bottom row. ( d ) Representative images of podocytes treated as described in ( c ), followed by DCFDA staining. The scale bars indicate 100 µm. ( e ) Caspase3/7 activity in podocytes treated with Milli-Q water (Control) or MGO (1.5 mM) for 12 h with or without pre-treated with 2.0 µg/mL rhCLU for 4 h. The measurement was normalised to the control; ** P < 0.01 vs. control, †† P < 0.01 vs. rhCLU, and ‡‡ P < 0.01 vs. MGO (one-way ANOVA). (f) Podocytes treated with Milli-Q water (Control) or MGO (1.5 mM) for 2 h with or without pre-treatment with 2.0 µg/mL rhCLU for 4 h, followed by measuring BAX and BCL2 mRNA expression. The BAX/BCL2 ratio was calculated and normalised to the control; ** P < 0.01 vs. control, †† P < 0.01 vs. rhCLU, and ‡ P < 0.05 vs. MGO (one-way ANOVA). ( g , h ) Podocytes treated with Milli-Q water (Control) or MGO (1.5 mM) for 2 h with or without pre-treated with 2.0 µg/mL rhCLU for 4 h, followed by measuring ZO-1 ( g ) and NEPH1 ( h ) mRNA expression; the mRNA levels were normalised to the respective control; * P < 0.05 vs. control, ** P < 0.01 vs. control *** P < 0.001 vs. control, † P < 0.05 vs. rhCLU, †† P < 0.01 vs. rhCLU, and ‡ P < 0.05 vs. MGO (one-way ANOVA). MGO methylglyoxal, rhCLU recombinant human clusterin protein.

Techniques Used: Recombinant, Incubation, Control, Staining, Activity Assay, Expressing

Image Search Results

The C7 and clusterin association was confirmed with a native-restricted anti-C7 mAb. (A) The M7-HB2H mAb binding target is located in the C-terminal domains (highlighted in grey) of C7. These domains participate in the formation of the MAC via interaction with the C345C domain of C5, hence, the M7-HB2H antibody would likely bind to an exposed epitope in the native form of C7 (i.e. C7 that is not involved in the assembly of the MAC). The C7 gene contains 18 exons, each exon codes for a specific region of the C7 domain, which corresponds to the exon coding region displayed above. The boundary phase refers to the phase of the corresponding intron. (B) C7 purified from NHS-BioIVT with anti-C7 mAb, M7-HB2H, was separated by SDS-PAGE under non-reducing conditions and the immunoblot was probed with M7-HB2H. Protein composition of the bands visualized in (B) was analzyed by mass spectrometry and protein abundance was quantified for the (C) 100 kDa band and the (D) 75 kDa band. (E) The C7 aa residues detected in the 75 kDa band (3B) are in bold and underlined, along with an illustration of the screened residues in the C7 polypeptide chain highlighted in grey. The screened aa residues are 47% of the total C7 polypeptide chain. Black arrows indicate the exon/intron boundaries. Western blot (B) is representative of at least three independent experiments. Images were taken using Proxima C16 Phi. Figure (A) modified from Massri et al., 2022 .

Journal: Frontiers in Immunology

Article Title: Complement C7 and clusterin form a complex in circulation

doi: 10.3389/fimmu.2024.1330095

Figure Lengend Snippet: The C7 and clusterin association was confirmed with a native-restricted anti-C7 mAb. (A) The M7-HB2H mAb binding target is located in the C-terminal domains (highlighted in grey) of C7. These domains participate in the formation of the MAC via interaction with the C345C domain of C5, hence, the M7-HB2H antibody would likely bind to an exposed epitope in the native form of C7 (i.e. C7 that is not involved in the assembly of the MAC). The C7 gene contains 18 exons, each exon codes for a specific region of the C7 domain, which corresponds to the exon coding region displayed above. The boundary phase refers to the phase of the corresponding intron. (B) C7 purified from NHS-BioIVT with anti-C7 mAb, M7-HB2H, was separated by SDS-PAGE under non-reducing conditions and the immunoblot was probed with M7-HB2H. Protein composition of the bands visualized in (B) was analzyed by mass spectrometry and protein abundance was quantified for the (C) 100 kDa band and the (D) 75 kDa band. (E) The C7 aa residues detected in the 75 kDa band (3B) are in bold and underlined, along with an illustration of the screened residues in the C7 polypeptide chain highlighted in grey. The screened aa residues are 47% of the total C7 polypeptide chain. Black arrows indicate the exon/intron boundaries. Western blot (B) is representative of at least three independent experiments. Images were taken using Proxima C16 Phi. Figure (A) modified from Massri et al., 2022 .

Article Snippet: Antibodies: goat anti-human C7 polyclonal antibody [P7] ( ); mouse anti-human C7 monoclonal antibody [M7-WU4-15] (cat# HM2277, Hycult Biotech, Uden, Netherlands); mouse anti-human C7 monoclonal antibody [M7-HB2H] (cat# HM2421, Hycult Biotech), goat anti-human clusterin polyclonal antibody [PC] (Sigma-Aldrich, St. Louis, MO, USA); mouse anti-human clusterin monoclonal antibody [MC] (Sinobiological, Beijing, China), mouse anti-human clusterin monoclonal antibody [MC-2D5] (cat# HM2435, Hycult Biotech); goat anti-mouse HRP-conjugated IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Binding Assay, Purification, SDS Page, Western Blot, Mass Spectrometry, Modification

Clusterin is the most abundant protein detected in the 75 kDa band of purified C7. Protein composition of the bands visualized for C7 purified with pAb P7 ( <xref ref-type=

Figure 2A ) was analzyed by mass spectrometry and protein abundance was quantified for the (A) 100 kDa band and the (B) 75 kDa band. Protein composition of the bands visualized for C7 purified with mAb M7-WU4-15 ( Figure 2B ) was analyzed by mass spectrometry and protein abundance was quantified in the (C) 100 kDa band and the (D) 75 kDa band. " width="100%" height="100%">

Journal: Frontiers in Immunology

Article Title: Complement C7 and clusterin form a complex in circulation

doi: 10.3389/fimmu.2024.1330095

Figure Lengend Snippet: Clusterin is the most abundant protein detected in the 75 kDa band of purified C7. Protein composition of the bands visualized for C7 purified with pAb P7 ( Figure 2A ) was analzyed by mass spectrometry and protein abundance was quantified for the (A) 100 kDa band and the (B) 75 kDa band. Protein composition of the bands visualized for C7 purified with mAb M7-WU4-15 ( Figure 2B ) was analyzed by mass spectrometry and protein abundance was quantified in the (C) 100 kDa band and the (D) 75 kDa band.

Techniques: Purification, Mass Spectrometry

Clusterin purified from NHS with polyclonal and monoclonal antibodies contained low levels of C7. (A) Clusterin purified from NHS-MUI with anti-clusterin pAb, PC, was separated by SDS-PAGE under non-reducing conditions and the immunoblot was probed with the anti-clusterin mAb, MC. The clusterin band on the left was from the 1 st eluate acquired from the antibody-coupled column, while the band on the right was from a 2 nd , consecutive elution step. (B) Coomassie staining of clusterin purified from NHS-MUI with the anti-clusterin pAb, PC, and separated by SDS-PAGE gel under non-reducing conditions. (C) Protein composition of the 75 kDa band visualized in (A) was analyzed by mass spectrometry and protein abundance was quantified. (D) Protein compostion of the 75 kDa band (not shown) from C7 purified with the anti-clusterin mAb, MC-2D5, was analyzed by mass spectrometry and protein abundance was quantified. Results from purified clusterin in (B, C) were acquiered from the 1 st eluate. Western blot and coomassie staining are representative of at least three independent experiments. Images were taken using the ImageQuant™ LAS 400 camera system.

Journal: Frontiers in Immunology

Article Title: Complement C7 and clusterin form a complex in circulation

doi: 10.3389/fimmu.2024.1330095

Figure Lengend Snippet: Clusterin purified from NHS with polyclonal and monoclonal antibodies contained low levels of C7. (A) Clusterin purified from NHS-MUI with anti-clusterin pAb, PC, was separated by SDS-PAGE under non-reducing conditions and the immunoblot was probed with the anti-clusterin mAb, MC. The clusterin band on the left was from the 1 st eluate acquired from the antibody-coupled column, while the band on the right was from a 2 nd , consecutive elution step. (B) Coomassie staining of clusterin purified from NHS-MUI with the anti-clusterin pAb, PC, and separated by SDS-PAGE gel under non-reducing conditions. (C) Protein composition of the 75 kDa band visualized in (A) was analyzed by mass spectrometry and protein abundance was quantified. (D) Protein compostion of the 75 kDa band (not shown) from C7 purified with the anti-clusterin mAb, MC-2D5, was analyzed by mass spectrometry and protein abundance was quantified. Results from purified clusterin in (B, C) were acquiered from the 1 st eluate. Western blot and coomassie staining are representative of at least three independent experiments. Images were taken using the ImageQuant™ LAS 400 camera system.

Techniques: Purification, SDS Page, Western Blot, Staining, Mass Spectrometry

Western blot analysis reveals strong association between C7 and clusterin. (A, B) Indicated proteins were separated by SDS-PAGE under (A) non-reducing or (B) reducing conditions and immunoblots were probed with the anti-C7 mAb, M7-HB2H. (C, D) Indicated proteins were separated by SDS-PAGE under (C) non-reducing or (D) reducing conditions and immunoblots were probed with the anti-clusterin mAb, MC-2D5. Western blots are representative of at least three independent experiments. Images were taken using Proxima C16 Phi.

Journal: Frontiers in Immunology

Article Title: Complement C7 and clusterin form a complex in circulation

doi: 10.3389/fimmu.2024.1330095

Figure Lengend Snippet: Western blot analysis reveals strong association between C7 and clusterin. (A, B) Indicated proteins were separated by SDS-PAGE under (A) non-reducing or (B) reducing conditions and immunoblots were probed with the anti-C7 mAb, M7-HB2H. (C, D) Indicated proteins were separated by SDS-PAGE under (C) non-reducing or (D) reducing conditions and immunoblots were probed with the anti-clusterin mAb, MC-2D5. Western blots are representative of at least three independent experiments. Images were taken using Proxima C16 Phi.

Techniques: Western Blot, SDS Page

$C7 and CLU co-elute in high molecular weight complexes. (A) Fractions of pooled NHS (F30-F62) were obtained by size exclusion chromatography and subsequently separated by SDS-PAGE under non-reducing conditions and immunoblots were probed with (B) anti-C7 mAb, M7-HB2H or (C) anti-clusterin mAb, MC-2D5. (D) Summary of (B, C) illustrating the fractions that were either positive or negative for C7 and clusterin. ± indicates fractions where a very faint band was detected due to minute amounts of the protein. (E) Concentration of the C7-CLU complex and native C7 in serum fractions. Note the difference between the y-axis scales, which translates to small concentrations of complexed C7, relative to native C7. NHS, normal human serum.$

Journal: Frontiers in Immunology

Article Title: Complement C7 and clusterin form a complex in circulation

doi: 10.3389/fimmu.2024.1330095

Figure Lengend Snippet: C7 and CLU co-elute in high molecular weight complexes. (A) Fractions of pooled NHS (F30-F62) were obtained by size exclusion chromatography and subsequently separated by SDS-PAGE under non-reducing conditions and immunoblots were probed with (B) anti-C7 mAb, M7-HB2H or (C) anti-clusterin mAb, MC-2D5. (D) Summary of (B, C) illustrating the fractions that were either positive or negative for C7 and clusterin. ± indicates fractions where a very faint band was detected due to minute amounts of the protein. (E) Concentration of the C7-CLU complex and native C7 in serum fractions. Note the difference between the y-axis scales, which translates to small concentrations of complexed C7, relative to native C7. NHS, normal human serum.

Techniques: Molecular Weight, Size-exclusion Chromatography, SDS Page, Western Blot, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Complement C7 and clusterin form a complex in circulation

doi: 10.3389/fimmu.2024.1330095

Techniques: Binding Assay, Purification, SDS Page, Western Blot, Mass Spectrometry, Modification

Journal: Frontiers in Immunology

Article Title: Complement C7 and clusterin form a complex in circulation

doi: 10.3389/fimmu.2024.1330095

Techniques: Purification, Mass Spectrometry

Journal: Frontiers in Immunology

Article Title: Complement C7 and clusterin form a complex in circulation

doi: 10.3389/fimmu.2024.1330095

Techniques: Purification, SDS Page, Western Blot, Staining, Mass Spectrometry

Journal: Frontiers in Immunology

Article Title: Complement C7 and clusterin form a complex in circulation

doi: 10.3389/fimmu.2024.1330095

Techniques: Western Blot, SDS Page

Journal: Frontiers in Immunology

Article Title: Complement C7 and clusterin form a complex in circulation

doi: 10.3389/fimmu.2024.1330095

Techniques: Molecular Weight, Size-exclusion Chromatography, SDS Page, Western Blot, Concentration Assay

Fig. 3. MetO-Clusterin levels are predominantly expressed in postmortem AD-model mouse brains as a function of age. A. Postmortem mouse brain samples at 4 months of age (Lanes 1 & 3) and at 8 months of age; Lanes 2 & 4). Lanes 3 & 4 represent protein loads of 5xFAD mouse model of AD and Lanes 1 & 2 represent protein loads of the corresponding controls. Whole brains (n = 5 per strains per age) were extracted in PBS in the presence of protease inhibitors (Sigma-Aldrich). The resulting extracted protein moiety contains both cellular and extracellular proteins in each brain. Equal amounts of protein extracts per brain were subjected to immunoprecipitation (IP), using the rabbit anti-MetO antibody; followed by western-blot (WB) analysis, using a primary goat anti-mouse clusterin antibody (Pro teintech). The WB depicts representative data for one brain per strain per age. kDa, molecular mass markers. B. Western blot analysis of whole brain protein extracts obtained for the brains described in panel A (using the same goat anti-mouse clusterin primary antibody). (Lanes 1 & 3) and at 8 months of age; Lanes 2 & 4). Lanes 3 & 4 represent protein loads of 5xFAD mouse model of AD and Lanes 1 & 2 represent protein loads of the corresponding controls. The WB depicts representative data for one brain per strain per age (total n = 5 per strainper age). The β-actin expression levels serve as loading controls (using anti-β-actin primary antibody, Thermo- Fisher Scientific). kDa, molecular mass markers. C. Quantification of the clusterin and MetO-Clusterin bands shown in the WB in Fig. 3 A&B (using the NIH-Image-J program). The data showed that the 5xFAD brains had higher MetO-Clusterin levels relative to the non-AD brains (*, t-test, P<0.001) after correction for the total expressed levels of clusterin in panel B.

Journal: Neuroscience letters

Article Title: Methionine oxidation of clusterin in Alzheimer's disease and its effect on clusterin's binding to beta-amyloid.

doi: 10.1016/j.neulet.2024.137874

Figure Lengend Snippet: Fig. 3. MetO-Clusterin levels are predominantly expressed in postmortem AD-model mouse brains as a function of age. A. Postmortem mouse brain samples at 4 months of age (Lanes 1 & 3) and at 8 months of age; Lanes 2 & 4). Lanes 3 & 4 represent protein loads of 5xFAD mouse model of AD and Lanes 1 & 2 represent protein loads of the corresponding controls. Whole brains (n = 5 per strains per age) were extracted in PBS in the presence of protease inhibitors (Sigma-Aldrich). The resulting extracted protein moiety contains both cellular and extracellular proteins in each brain. Equal amounts of protein extracts per brain were subjected to immunoprecipitation (IP), using the rabbit anti-MetO antibody; followed by western-blot (WB) analysis, using a primary goat anti-mouse clusterin antibody (Pro teintech). The WB depicts representative data for one brain per strain per age. kDa, molecular mass markers. B. Western blot analysis of whole brain protein extracts obtained for the brains described in panel A (using the same goat anti-mouse clusterin primary antibody). (Lanes 1 & 3) and at 8 months of age; Lanes 2 & 4). Lanes 3 & 4 represent protein loads of 5xFAD mouse model of AD and Lanes 1 & 2 represent protein loads of the corresponding controls. The WB depicts representative data for one brain per strain per age (total n = 5 per strainper age). The β-actin expression levels serve as loading controls (using anti-β-actin primary antibody, Thermo- Fisher Scientific). kDa, molecular mass markers. C. Quantification of the clusterin and MetO-Clusterin bands shown in the WB in Fig. 3 A&B (using the NIH-Image-J program). The data showed that the 5xFAD brains had higher MetO-Clusterin levels relative to the non-AD brains (*, t-test, P<0.001) after correction for the total expressed levels of clusterin in panel B.

Article Snippet: A.S. Smith et al. Neuroscience Letters 836 (2024) 137874 amounts of the resulting extracellular protein moiety from each brain were subjected to immunoprecipitation (IP), using the rabbit anti-MetO antibody [1]; followed by western-blot (WB) analysis, using a primary mouse anti-human clusterin antibody (Proteintech, Rosemont, IL).

Techniques: Immunoprecipitation, Western Blot, Expressing

Fig. 2. MetO-Clusterin levels are predominantly expressed in postmortem brains of late-stage AD patients. A. Postmortem human brain samples of late stages of AD (hippocampus region, n = 5) and at late stages of non-AD brains (hippocampus region, n = 5) were incubated in PBS in the presence of 0.32 M sucrose and protease inhibitors (Sigma-Aldrich) for 4 h at 4 ◦C. Equal amounts of the resulting extracellular protein moiety from each brain were subjected to immunoprecipitation (IP), using the rabbit anti-MetO antibody; followed by western-blot (WB) analysis, using a primary mouse anti-human clusterin antibody (Proteintech). Lanes 1–3, loads of one processed extracted brain per each lane (representatives out of the total brains’ number). B. Western blot analysis of extracellular brain protein extracts obtained for the brains described in panel A (using the same mouse anti-human clusterin antibody). Equal amounts of protein extracts were loaded per each lane. C. Quantification of the clusterin and MetO-Clusterin bands shown in the WB in Fig. 2 (using the NIH-Image-J program) showed that the AD brains had about 3.5-fold higher MetO-Clusterin levels relative to the non-AD brains (*, t-test, P<0.001) after correction for the total expressed levels of clusterin in panel B.

Journal: Neuroscience letters

Article Title: Methionine oxidation of clusterin in Alzheimer's disease and its effect on clusterin's binding to beta-amyloid.

doi: 10.1016/j.neulet.2024.137874

Figure Lengend Snippet: Fig. 2. MetO-Clusterin levels are predominantly expressed in postmortem brains of late-stage AD patients. A. Postmortem human brain samples of late stages of AD (hippocampus region, n = 5) and at late stages of non-AD brains (hippocampus region, n = 5) were incubated in PBS in the presence of 0.32 M sucrose and protease inhibitors (Sigma-Aldrich) for 4 h at 4 ◦C. Equal amounts of the resulting extracellular protein moiety from each brain were subjected to immunoprecipitation (IP), using the rabbit anti-MetO antibody; followed by western-blot (WB) analysis, using a primary mouse anti-human clusterin antibody (Proteintech). Lanes 1–3, loads of one processed extracted brain per each lane (representatives out of the total brains’ number). B. Western blot analysis of extracellular brain protein extracts obtained for the brains described in panel A (using the same mouse anti-human clusterin antibody). Equal amounts of protein extracts were loaded per each lane. C. Quantification of the clusterin and MetO-Clusterin bands shown in the WB in Fig. 2 (using the NIH-Image-J program) showed that the AD brains had about 3.5-fold higher MetO-Clusterin levels relative to the non-AD brains (*, t-test, P<0.001) after correction for the total expressed levels of clusterin in panel B.

Techniques: Incubation, Immunoprecipitation, Western Blot

Fig. 4. Oxidation of active recombinant ApoJ and the binding kinetics of re combinant ApoJ (clusterin) and its oxidized form (MetO-ApoJ) to Aβ42, in the presence of ThT. A. Methionine oxidation of an active ApoJ protein (50 µg) was performed by incubating the protein with 50 mM H2O2 for 3 h at room tem perature. The residual H2O2 was quenched by multiple washes in PBS and concentration using an Amicon Ultra-0.5 Centrifugal Filter Unit (3000 Da cut- off). The oxidation of the methionine moiety of ApoJ was confirmed by both dot-blot (right panel) and western blot (left panel) analyses using the anti-MetO antibody (as the primary antibody) and 1µg of non-oxidized and oxidized ApoJ. N, non-oxidized ApoJ; Ox, oxidized ApoJ; kDa, molecular mass markers in kilo- Daltons. B. Effects of ApoJ and MetO-ApoJ on the kinetics of Aβ42 fibril for mation. Freshly prepared Aβ42 was diluted at the indicated concentration in 20 mM phosphate buffer (pH 8.0) containing 0.02 % NaN3 and 200 µM EDTA. Then the Aβ42 was incubated, in the presence and absence of active recombi nant human ApoJ or MetO-ApoJ, under quiescent conditions at 37 ◦C in microplate 96-wells Microplate. in the presence of 20 µM ThT. ThT fluorescence was measured every 4.0 min for 18 h, using an M200 Infinity plate reader. A parallel analysis was performed using BSA instead ApoJ, at the indicated con centration, serving as a non-chaperone control protein. The ThT dye was excited at 440 nm, and the emission was measured at 495 nm. Fluorescence data are reported after normalization on the corresponding maximal ThT value after subtracting ThT basal background signal value. The figure shows a representative data of three repetitive independent experiments. Abeta, Aβ42.

Journal: Neuroscience letters

Article Title: Methionine oxidation of clusterin in Alzheimer's disease and its effect on clusterin's binding to beta-amyloid.

doi: 10.1016/j.neulet.2024.137874

Figure Lengend Snippet: Fig. 4. Oxidation of active recombinant ApoJ and the binding kinetics of re combinant ApoJ (clusterin) and its oxidized form (MetO-ApoJ) to Aβ42, in the presence of ThT. A. Methionine oxidation of an active ApoJ protein (50 µg) was performed by incubating the protein with 50 mM H2O2 for 3 h at room tem perature. The residual H2O2 was quenched by multiple washes in PBS and concentration using an Amicon Ultra-0.5 Centrifugal Filter Unit (3000 Da cut- off). The oxidation of the methionine moiety of ApoJ was confirmed by both dot-blot (right panel) and western blot (left panel) analyses using the anti-MetO antibody (as the primary antibody) and 1µg of non-oxidized and oxidized ApoJ. N, non-oxidized ApoJ; Ox, oxidized ApoJ; kDa, molecular mass markers in kilo- Daltons. B. Effects of ApoJ and MetO-ApoJ on the kinetics of Aβ42 fibril for mation. Freshly prepared Aβ42 was diluted at the indicated concentration in 20 mM phosphate buffer (pH 8.0) containing 0.02 % NaN3 and 200 µM EDTA. Then the Aβ42 was incubated, in the presence and absence of active recombi nant human ApoJ or MetO-ApoJ, under quiescent conditions at 37 ◦C in microplate 96-wells Microplate. in the presence of 20 µM ThT. ThT fluorescence was measured every 4.0 min for 18 h, using an M200 Infinity plate reader. A parallel analysis was performed using BSA instead ApoJ, at the indicated con centration, serving as a non-chaperone control protein. The ThT dye was excited at 440 nm, and the emission was measured at 495 nm. Fluorescence data are reported after normalization on the corresponding maximal ThT value after subtracting ThT basal background signal value. The figure shows a representative data of three repetitive independent experiments. Abeta, Aβ42.

Techniques: Recombinant, Binding Assay, Concentration Assay, Dot Blot, Western Blot, Incubation, Fluorescence, Control

Fig. 5. In-Silico analyses for the location of clusterin’s conserved methionine residues and their associated hydrophobicity regions. The number indicates the position of a methionine residue in the sequence alignment. The intensity of the red color indicates the level of hydrophobicity, and the intensity of the blue color indicates the level of hydrophilicity (a denser color-coded amino acids corelates with either stronger hydrophobic or hydrophilic cluster, respectively). The Uniprot program and the align tool were used to acquire the clusterin sequences of the indicated organisms. The amino acid numbers are based on the longest Clusterin sequence in the indicated species.

Journal: Neuroscience letters

Article Title: Methionine oxidation of clusterin in Alzheimer's disease and its effect on clusterin's binding to beta-amyloid.

doi: 10.1016/j.neulet.2024.137874

Figure Lengend Snippet: Fig. 5. In-Silico analyses for the location of clusterin’s conserved methionine residues and their associated hydrophobicity regions. The number indicates the position of a methionine residue in the sequence alignment. The intensity of the red color indicates the level of hydrophobicity, and the intensity of the blue color indicates the level of hydrophilicity (a denser color-coded amino acids corelates with either stronger hydrophobic or hydrophilic cluster, respectively). The Uniprot program and the align tool were used to acquire the clusterin sequences of the indicated organisms. The amino acid numbers are based on the longest Clusterin sequence in the indicated species.

Techniques: In Silico, Residue, Sequencing

The 25 most abundantly represented genes in the pterygium cDNA library.

Journal: Molecular Vision

Article Title: Expression analysis of human pterygium shows a predominance of conjunctival and limbal markers and genes associated with cell migration

doi:

Figure Lengend Snippet: The 25 most abundantly represented genes in the pterygium cDNA library.

Article Snippet: For confocal immunolocalization studies, 5% normal serum was used as the blocking reagent for 30 min at 23 ºC, followed by incubation for 1 h with the following primary antibodies: rabbit anti-cytokeratin 13 (polyclonal, 1:50; Protein Group, Inc., Chicago, IL), mouse anti-cytokeratin 12 (monoclonal, 1:25; Biogenesis, Poole, UK), mouse anti-human clusterin (monoclonal, 1:50; BD Bioscience, San Jose, CA), mouse anti-S100A6 (calcyclin; monoclonal 1:1,000; Sigma®, Saint Louis, MO), mouse anti-SAT (spermidine/spermine N1 acetyltransferase; polyclonal, 1:200), mouse-anti-annexin II (3D5/4; monoclonal, 1:50; Gene-Tex Inc., San Antonio, TX).

Techniques: Clone Assay, Binding Assay

The abundance of clusterin in pterygium is illustrated by immunofluorecence. Labeling for clusterin is in red, nuclei are blue (DAPI). The arrow indicates cells enveloping the body of the pterygium that are clusterin negative.

Journal: Molecular Vision

Article Title: Expression analysis of human pterygium shows a predominance of conjunctival and limbal markers and genes associated with cell migration

doi:

Figure Lengend Snippet: The abundance of clusterin in pterygium is illustrated by immunofluorecence. Labeling for clusterin is in red, nuclei are blue (DAPI). The arrow indicates cells enveloping the body of the pterygium that are clusterin negative.

Techniques: Labeling

Number of urinary extracellular vesicles (EVs) carrying cellular adhesion/inflammatory and renal injury molecules from primary hyperoxaluria type 1 patients without and with nephrocalcinosis (NC) or kidney stones (KS)

Journal: Orphanet Journal of Rare Diseases

Article Title: Specific populations of urinary extracellular vesicles and proteins differentiate type 1 primary hyperoxaluria patients without and with nephrocalcinosis or kidney stones

doi: 10.1186/s13023-020-01607-1

Figure Lengend Snippet: Number of urinary extracellular vesicles (EVs) carrying cellular adhesion/inflammatory and renal injury molecules from primary hyperoxaluria type 1 patients without and with nephrocalcinosis (NC) or kidney stones (KS)

Article Snippet: PE conjugated mouse anti-human clusterin antibody purchased from Novus Biologicals, LLC, Centennial, CO, USA.

Techniques:

Journal: Scientific Reports

Article Title: Glomerular clusterin expression is increased in diabetic nephropathy and protects against oxidative stress-induced apoptosis in podocytes

doi: 10.1038/s41598-020-71629-z

Figure Lengend Snippet: Glomerular clusterin expression is increased in patients with DN. ( a , b ) Representative images of kidney sections obtained from a healthy subject (Control; a ) and a patient with DN (DN; b ) stained for clusterin; the scale bars represent 200 µm. ( c , d ) Representative high-magnification images of a and b; the scale bars represent 50 µm. ( e , f ) The high-magnification views of the rectangles in ( c , d ). Clusterin staining was present along the outer side of the GBM at the place where the podocytes are located (arrowheads; the square shows a zoomed in image of a clusterin-positive podocyte). ( g ) Summary of clusterin staining (semi-quantitative score) in the glomeruli of healthy subjects (Control; n = 10) and patients with DN (DN; n = 12); *** P < 0.001 vs. control (Student’s t-test). ( h ) Summary of CLU mRNA measured in isolated glomeruli from healthy subjects (Control; n = 11) and patients with DN (DN; n = 24); ** P < 0.01 vs. control (Student’s t-test).

Article Snippet: Mouse kidney sections were stained with the following primary antibodies: rabbit anti-mouse clusterin (1:800, H-330, Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit anti-mouse Wilms’ tumour protein (1:1,000, WT1; Santa Cruz Biotechnology); human kidney sections were stained with mouse anti-human clusterin (1:9,000, clone B-5, Santa Cruz Biotechnology).

Techniques: Expressing, Control, Staining, Isolation

Journal: Scientific Reports

Article Title: Glomerular clusterin expression is increased in diabetic nephropathy and protects against oxidative stress-induced apoptosis in podocytes

doi: 10.1038/s41598-020-71629-z

Figure Lengend Snippet: Clusterin expression is increased in the glomeruli of mice with type 1 diabetes and co-localises with podocytes. ( a , b ) Representative images of mouse kidney sections obtained from control mice (Control; a ) and STZ-treated mice 15 weeks after diabetes was induced (STZ; b ); the scale bars represent 100 µm. ( c , d ) The high-magnification views of the rectangles in ( a, b ); the scale bars represent 50 µm. ( e , f ) Representative images of the adjacent sections stained for clusterin ( e ) and WT1 ( f ), showing co-localisation of clusterin with WT1 in the glomeruli (arrowheads). ( g ) Averaged clusterin-positive area (measured by using ImageJ) in the glomeruli of control and STZ-treated mice 15 weeks after diabetes was induced; ** P < 0.01 vs. control (Student’s t-test).

Techniques: Expressing, Control, Staining

Journal: Scientific Reports

Article Title: Glomerular clusterin expression is increased in diabetic nephropathy and protects against oxidative stress-induced apoptosis in podocytes

doi: 10.1038/s41598-020-71629-z

Figure Lengend Snippet: Clusterin expression in podocytes cultured under various diabetic conditions. ( a–d ) Human podocytes were cultured for the indicated times in PAN (30 µg/mL) ( a ), Glucose (5 mM or 25 mM) ( b ), Angiotensin II (1 µM) ( c ), or the indicated concentrations of MGO for 24 h ( d ). The CLU mRNA expression after these treatments was measured, the mRNA levels were normalised to the respective control. In ( a – c ), * P < 0.05 vs. the respective control at the same time point (Student’s t-test); in ( d ), * P < 0.05 vs. control, and † P < 0.05 vs. 0.5 mM MGO (one-way ANOVA). PAN puromycin aminonucleoside, MGO methylglyoxal.

Techniques: Expressing, Cell Culture, Control

Journal: Scientific Reports

Article Title: Glomerular clusterin expression is increased in diabetic nephropathy and protects against oxidative stress-induced apoptosis in podocytes

doi: 10.1038/s41598-020-71629-z

Figure Lengend Snippet: Recombinant human clusterin protein protects podocytes against methylglyoxal-induced oxidative stress and apoptosis. ( a ) Cell viability was measured in podocytes incubated with the indicated concentrations of rhCLU for 24 h. ( b ) Cell viability was measured in podocytes pre-incubated for 4 h with the indicated concentrations of rhCLU, followed by 24 h in the presence or absence of 1.5 mM MGO. Cell viability in the presence of MGO was normalised to cells stimulated in the absence of rhCLU; * P < 0.05 vs. 0 µg/mL rhCLU + 1.5 mM MGO (one-way ANOVA). ( c ) Representative images of podocytes treated with Milli-Q water (Control) or podocytes pre-treated for 4 h with 2.0 µg/mL rhCLU, 2 h with 1.5 mM MGO, or both, followed by MitoSOX Red staining. The scale bars indicate 20 µm and the boxed areas are shown at higher magnification in the bottom row. ( d ) Representative images of podocytes treated as described in ( c ), followed by DCFDA staining. The scale bars indicate 100 µm. ( e ) Caspase3/7 activity in podocytes treated with Milli-Q water (Control) or MGO (1.5 mM) for 12 h with or without pre-treated with 2.0 µg/mL rhCLU for 4 h. The measurement was normalised to the control; ** P < 0.01 vs. control, †† P < 0.01 vs. rhCLU, and ‡‡ P < 0.01 vs. MGO (one-way ANOVA). (f) Podocytes treated with Milli-Q water (Control) or MGO (1.5 mM) for 2 h with or without pre-treatment with 2.0 µg/mL rhCLU for 4 h, followed by measuring BAX and BCL2 mRNA expression. The BAX/BCL2 ratio was calculated and normalised to the control; ** P < 0.01 vs. control, †† P < 0.01 vs. rhCLU, and ‡ P < 0.05 vs. MGO (one-way ANOVA). ( g , h ) Podocytes treated with Milli-Q water (Control) or MGO (1.5 mM) for 2 h with or without pre-treated with 2.0 µg/mL rhCLU for 4 h, followed by measuring ZO-1 ( g ) and NEPH1 ( h ) mRNA expression; the mRNA levels were normalised to the respective control; * P < 0.05 vs. control, ** P < 0.01 vs. control *** P < 0.001 vs. control, † P < 0.05 vs. rhCLU, †† P < 0.01 vs. rhCLU, and ‡ P < 0.05 vs. MGO (one-way ANOVA). MGO methylglyoxal, rhCLU recombinant human clusterin protein.

Techniques: Recombinant, Incubation, Control, Staining, Activity Assay, Expressing

$( A ) Empty vector (pCMV6), V5-tagged pCMV6-human CLU Exon 2–9, or Exon 3–9 constructs were transfected into human SH-SY5Y cells. Cell lysates were harvested and probed for CLU immunoreactivity using anti-V5 (left panel) or anti-Clusterin (right panel). ( B : left panel) pCMV6-human CLU Exon 2–9 or Exon 3–9 constructs were transfected into human SH-SY5Y cells. The resulting cell lysates were treated with PNGase F or Endo H and probed for CLU immunoreactivity using anti-V5. ( B : right panel) Cell lysates harvested from pCMV6-human CLU Exon 3–9-transfected SH-SY5Y cells were treated with O -glycosidase, neuraminidase, or a combination of the two and probed for expression of CLU_45 kDa protein using anti-V5. ( C ) pCMV6-human CLU Exon 3–9-transfected SH-SY5Y cells were subjected to cellular fractionation using the Qiagen Qproteome Mitochondrial Isolation Kit and analyzed for CLU immunoreactivity using anti-V5. Fraction isolation was confirmed using calnexin (ER), Gapdh (cytosol), lamin A/C and lamin B1 (nucleus), and VDAC and Tom20 (mitochondria).$

Journal: eLife

Article Title: Brain clusterin protein isoforms and mitochondrial localization

doi: 10.7554/eLife.48255

Figure Lengend Snippet: ( A ) Empty vector (pCMV6), V5-tagged pCMV6-human CLU Exon 2–9, or Exon 3–9 constructs were transfected into human SH-SY5Y cells. Cell lysates were harvested and probed for CLU immunoreactivity using anti-V5 (left panel) or anti-Clusterin (right panel). ( B : left panel) pCMV6-human CLU Exon 2–9 or Exon 3–9 constructs were transfected into human SH-SY5Y cells. The resulting cell lysates were treated with PNGase F or Endo H and probed for CLU immunoreactivity using anti-V5. ( B : right panel) Cell lysates harvested from pCMV6-human CLU Exon 3–9-transfected SH-SY5Y cells were treated with O -glycosidase, neuraminidase, or a combination of the two and probed for expression of CLU_45 kDa protein using anti-V5. ( C ) pCMV6-human CLU Exon 3–9-transfected SH-SY5Y cells were subjected to cellular fractionation using the Qiagen Qproteome Mitochondrial Isolation Kit and analyzed for CLU immunoreactivity using anti-V5. Fraction isolation was confirmed using calnexin (ER), Gapdh (cytosol), lamin A/C and lamin B1 (nucleus), and VDAC and Tom20 (mitochondria).

Article Snippet: Primary antibodies used in the studies presented herein include: rabbit polyclonal actin beta (β-actin, Pierce, Cat. #PA5-16914), mouse monoclonal beta-tubulin [TBN06 (Tub 2.5)] (β-tubulin, Pierce, Cat. #MA5-11732), rabbit polyclonal calnexin (H70) (Santa Cruz, Cat. #sc-11397), rabbit monoclonal calreticulin (D3E6) (Cell Signaling, Cat. #12238), rabbit polyclonal clusterin (CLU H-330; Santa Cruz, Cat. #sc-8354), goat polyclonal clusterin-α (CLU M18; Santa Cruz, Cat. #sc-6420), mouse monoclonal clusterin-α (CLU B-5; Santa Cruz, Cat. #sc-5289), mouse monoclonal human clusterin antibody (CLU MAB2937; R and D Systems, Cat. #MAB2937), goat polyclonal human clusterin isoform one antibody (CLU AF2937; R and D Systems, Cat. #AF2937), rabbit monoclonal COX IV (3E11) (Cell Signaling, Cat. #4850), rabbit monoclonal cytochrome C (Cyt.

Techniques: Plasmid Preparation, Construct, Transfection, Expressing, Cell Fractionation, Isolation

Review

Structured Review

Images

1) Product Images from "Glomerular clusterin expression is increased in diabetic nephropathy and protects against oxidative stress-induced apoptosis in podocytes"

Similar Products

Image Search Results