- PA5-29171 - Invitrogen Antibodies YY1 antibody

Submitted references Upregulation of Yy1 Suppresses Dilated Cardiomyopathy caused by Ttn insufficiency.

Liao D, Chen W, Tan CY, Wong JX, Chan PS, Tan LW, Foo R, Jiang J
Scientific reports 2019 Nov 8;9(1):16330

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of YY1 using 30 µg of A431 lysate. Samples were loaded onto a 10% SDS-PAGE gel and probed with a YY1 polyclonal antibody (Product # PA5-29171) at a dilution of 1:2000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot using YY1 Polyclonal Antibody (Product # PA5-29171). Sample (30 µg of whole cell lysate). Lane A: NIH-3T3. 10% SDS PAGE. YY1 Polyclonal Antibody (Product # PA5-29171) diluted at 1:2,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: YY1 Polyclonal Antibody detects YY1 protein by Western blot analysis. A. 30 µg PC-12 whole cell lysate/extract. B. 30 µg Rat2 whole cell lysate/extract.10 % SDS-PAGE. YY1 Polyclonal Antibody (Product # PA5-29171) dilution: 1:1,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot using YY1 Polyclonal Antibody (Product # PA5-29171). Various whole cell extracts (30 µg) were separated by 10% SDS-PAGE, and the membrane was blotted with YY1 Polyclonal Antibody (Product # PA5-29171) diluted at 1:2,000. 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: Knockdown of YY1 was achieved by transfecting DU 145 cells with YY1 specific siRNAs (Silencer® select Product # s14958, s14960). Western blot analysis (Fig. a) was performed using modified whole cell extracts from the YY1 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with YY1 Rabbit Polyclonal Antibody (Product # PA5-29171, 1:3000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP (Product # A27036, 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 YY1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-YY1 Polyclonal Antibody (Product # PA5-29171) and a 55 kDa band corresponding to YY1 was observed across cell lines tested. Modified whole cell extracts (1% SDS) (30 µg lysate) of DU 145 (Lane 1) and HCT 116 (Lane 2) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). 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 (1:3000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: YY1 Polyclonal Antibody detects YY1 protein at nucleus by immunofluorescent analysis. Sample: HeLa cells were fixed in 4% paraformaldehyde at RT for 15 min. Green: YY1 stained by YY1 Polyclonal Antibody (Product # PA5-29171) diluted at 1:1,000. Red: phalloidin, a cytoskeleton marker, diluted at 1:200. Scale bar= 10 µm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of YY1 was performed using DU 145 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 YY1 Rabbit Polyclonal Antibody (Product # PA5-29171) at 1:100 dilution in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731) 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 localization of YY1. 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 paraffin-embedded 59T xenograft, using YY1 (Product # PA5-29171) 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: Immunohistochemical analysis of paraffin-embedded C2C12 xenograft, using YY1 (Product # PA5-29171) 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: Chromatin Immunoprecipitation (ChIP) assay of endogenous YY1 protein using YY1 Antibody: ChIP was performed using YY1 Polyclonal Antibody (Product # PA5-29171, 5 µg) on sheared chromatin from DU 145 cells using the MAGnify ChIP System kit (Product # 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by qPCR using primers binding to CXXC1-Promoter, CXXC1- Exon1, FAM193B and NR3C1- Promoter (active) and SAT alpha and SAT2 satellite repeats (Inactive). Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Upregulation of Yy1 promotes cardiac cell cycle in dilated cardiomyopathy induced by Ttn shRNA. ( A ) Quantitative real-time PCR analysis of Ccnd1 and Ccnd2 expression in mouse heart tissue from control, Ttn shRNA and Yy1 treated groups. Virus does, 0.2E + 13 vg/kg. Mice were harvest four weeks after transduction. n >= 10. ( B ) Paraffin sections (left) stained with DAPI (blue), cTnI (green) and Ccnd1 (red), representing Non-CM and CM with positive Ccnd1 signal (arrowed); quantification of Ccnd1 + Non-CM and CM (right) from control, Ttn shRNA and Yy1 treated groups, n >= 4. ( C ) Paraffin section (left) and quantification (right) of Ccnd2 + in Non-CM and CM respectively, n >= 4. ( D ) Western blot and quantitative analysis ( E ) of Yy1, Ccnd1 and Ccnd2 protein levels in mouse heart tissue, n = 4. Data were normalized by Lamin B1 antibody. Full-length blots were presented in Supplementary Fig. S6 . ( F ) Quantitative real-time PCR analysis of Ccnd1 and Ccnd2 expression in isolated CM from control, Ttn shRNA and Yy1 treated groups four weeks after virus transduction, n >= 3. ( G ) Paraffin section (left) and quantification (right) of EdU + in Non-CM and CM respectively, n = 4. ( H ) Paraffin section (left) and quantification (right) of pH3 + in non-CM, n >= 4. In ( B ), ( C ), ( G ) and ( H ), magnification = 100x, scale bar = 10 um. Data were shown as mean +- SD and were normalized to total nucleus number labelled by DAPI.

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