- MA5-25317 - Invitrogen Antibodies HDAC6 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of HDAC6 in HepG2, HeLa, HT29, A549, COS7, Jurkat, MDCK, PC12, MCF7 cells using 35 µg per lane. Samples were probed with HDAC6 (Product # MA5-25317) monoclonal antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of HDAC6 was achieved by transfecting A549 with HDAC6 specific siRNAs (Silencer® select Product # s19459). Western blot analysis (Fig. a) was performed using whole cell extracts from the HDAC6 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with HDAC6 Monoclonal Antibody (OTI4C5) (Product # MA5-25317, 1:1000 dilution) and Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP conjugate (Product # A28177, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to HDAC6.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-HDAC6 Monoclonal Antibody (OTI4C5) (Product # MA5-25317) and a 160kDa band corresponding to HDAC6 was observed in the cell lines tested. Whole cell extracts of A549 (Lane 1), HeLa (Lane 2), MCF7 (Lane 3) and HT-29 (Lane 4) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP conjugate (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of HDAC6 in COS7 cells. Cells were transfected with a plasmid overexpressing HDAC6 and probed with a HDAC6 monoclonal antibody (Product # MA5-25317).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of HDAC6 in COS7 cells. Cells were transfected with a plasmid overexpressing HDAC6 and probed with a HDAC6 monoclonal antibody (Product # MA5-25317).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of HDAC6 in COS7 cells. Cells were transfected with a plasmid overexpressing HDAC6 and probed with a HDAC6 monoclonal antibody (Product # MA5-25317).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of HDAC6 was performed using 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with HDAC6 Mouse Monoclonal Antibody (Product # MA5-23517) at 1:100 dilution in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the composite image showing nuclear and cytoplasmic localization of HDAC6. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of HDAC6 in paraffin-embedded human liver tissue using a HDAC6 monoclonal antibody (Product # MA5-25317) at a dilution of 1:150.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of HDAC6 in paraffin-embedded adenocarcinoma of human ovary tissue using a HDAC6 monoclonal antibody (Product # MA5-25317) at a dilution of 1:150.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of HDAC6 in paraffin-embedded human colon tissue using a HDAC6 monoclonal antibody (Product # MA5-25317) at a dilution of 1:150.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemical analysis of HDAC6 in paraffin-embedded human kidney tissue using a HDAC6 monoclonal antibody (Product # MA5-25317) at a dilution of 1:150.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Chromatin Immunoprecipitation (ChIP) assay of endogenous HDAC6 protein using Anti-HDAC6 Antibody: ChIP was performed using Anti-HDAC6 Mouse Monoclonal Antibody (Product # MA5-25317, 5 µg) on sheared chromatin from HeLa cells using the MAGnify ChIP System kit (Product # 49-2024). Normal Mouse IgG was used as a negative IP control. The purified DNA was analyzed by qPCR using primers binding to IGFBP4 promoter (-4.2kb), SMARCA2 transcriptional start site, AXIN2 exon1 and GAPDH promoter. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

MA5-25317