- PA5-22369 - Invitrogen Antibodies ATXN3 antibody

Cheema MU, Poulsen ET, Enghild JJ, Hoorn EJ, Fenton RA, Praetorius J
Physiological reports 2013 Sep;1(4):e00064

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Ataxin3 was performed by loading 30 ug of HEK-293 control (Lane 1), HEK-293 - Ataxin3 knockout (Lane 2) whole cell lysates. The blot was probed with Ataxin 3 Polyclonal Antibody (Product # PA5-22369, 1:3000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/ml 1:4000 dilution). Loss of signal upon CRISPR mediated knockout (KO) confirms that antibody is specific to Ataxin3.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot analysis of Ataxin 3 was performed by separating 30 µg of various whole cell extracts by 10% SDS-PAGE. Proteins were transferred to a membrane and probed with a Ataxin 3 Polyclonal Antibody (Product # PA5-22369) at a dilution of 1:1000 and a HRP-conjugated anti-rabbit IgG secondary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot using Ataxin 3 Polyclonal Antibody (Product # PA5-22369). Various whole cell extracts (30 µg) were separated by 7.5% SDS-PAGE, and the membrane was blotted with Ataxin 3 Polyclonal Antibody (Product # PA5-22369) diluted at 1:1,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Ataxin 3 Polyclonal Antibody detects Ataxin 3 protein by western blot analysis. Mouse tissue extracts (50 µg) was separated by 10% SDS-PAGE, and blotted with Ataxin 3 Polyclonal Antibody (Product # PA5-22369) diluted by 1:500. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Ataxin 3 Polyclonal Antibody detects Ataxin 3 protein by western blot analysis.293T mock transfected (-) or transfected with a construct expressing human Ataxin 3 (+).10% SDS-PAGE. Ataxin 3 Polyclonal Antibody (Product # PA5-22369) dilution: 1:5,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extract (30 µg lysate) of HCT116 (Lane 1), HCT116 treated with thapsigargin (2uM, 6 hrs) (Lane 2), SH-SY5Y (Lane 3), and U-87MG (Lane 4). The blot was probed with Anti-Ataxin 3 Polyclonal Antibody (Product # PA5-22369, 1:3000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/ml, 1:4000 dilution). A 42 kDa band corresponding to Ataxin 3 was detected in cell lines tested.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunocytochemistry-Immunofluorescence analysis of Ataxin 3 was performed in SK N SH cells fixed in 4% paraformaldehyde at RT for 15 min. Green: Ataxin 3 Polyclonal Antibody (Product # PA5 22369) diluted at 1:250. Blue: Hoechst 33342 staining.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunocytochemistry-Immunofluorescence analysis of Ataxin 3 was performed in 293T cells fixed in 4% paraformaldehyde at RT for 15 min. Green: Ataxin 3 Polyclonal Antibody (Product # PA5 22369) diluted at 1:500. Blue: Hoechst 33342 staining.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of paraffin-embedded H661 xenograft, using Ataxin 3 (Product # PA5-22369) antibody at 1:500 dilution. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Protein aggregation in renal proximal tubules after angiotensin II treatment. Angiotensin was administered to rats to increase cellular protein turnover in proximal tubules, and aggregation-prone protein content was analyzed by immunohistochemistry. Angiotensin II was elevated by either infusion from subcutaneous minipumps or by adrenalectomy with glucocorticoid substitution. (A) Representative example of the RPL22/27 immunostaining in the kidney cortex from a control and angiotensin II (AngII)-treated rats, as indicated. PT denotes proximal tubules, while DT marks distal tubules and connecting tubules. (B) Similar RPL22/27 staining in sections from dexamethasone-treated adrenalectomized rats (ADX) and from similar rats with aldosterone and dexamethasone coadministration (ADX+Aldo). (C) Micrographs comparing keratin 5 immunoreactivity in renal cortex from control and angiotensin II-treated rats. (D) Immunolabeling for ataxin-3 in the angiotensin II versus vehicle administration model, as indicated. (E) Similar staining in model of dexamethasone-corrected adrenalectomy with and without angiotensin II administration (ADX+AngII), as indicated.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 Effect of aldosterone on expression of proteins involved in aggregate handling. For transfer of aggregated proteins to aggresomes, a partial deubiquitination by ataxin-3 must first occur. (A) Ataxin-3 immunoreactivity in control and aldosterone (Aldo)-treated rat kidney sections. Proximal tubules (PT); distal tubules and connecting tubules (DT). (B) Micrographs comparing dynactin p62 labeling in renal cortex from control and aldosterone-treated rats. (C) LC3 immunoreactivity in proximal tubules in control and aldosterone-treated rat kidney sections. (D) Bar graph showing the densitometry of the 17 kDa ataxin-3 band from the inserted immunoblot ( P > 0.05, n = 5). (E) Bar graph shows the semi-quantitation of immunoblot analysis of the 53 kDa dynactin p62 band (insert). (F) The bar graphs show the quantitation of the number, the mean area, and the fluorescence intensity of the renal cortical LC3 staining in the two experimental groups. (G) Quantitative analysis of the proximal tubular colocalization of LC3 with dynactin p62 and HDAC6, respectively, in aldosterone-treated rats.

PA5-22369