- PA5-78253 - Invitrogen Antibodies SMARCA5 antibody

Submitted references Tau Modulates mRNA Transcription, Alternative Polyadenylation Profiles of hnRNPs, Chromatin Remodeling and Spliceosome Complexes.

Montalbano M, Jaworski E, Garcia S, Ellsworth A, McAllen S, Routh A, Kayed R
Frontiers in molecular neuroscience 2021;14:742790

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of SMARCA5 in HepG2 cells using 30 µg of protein. Samples were separated with 5% SDS-PAGE and incubated with SMARCA5 polyclonal antibody (Product # PA5-78253) using a dilution of 1:10,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot using SMARCA5 Polyclonal Antibody (Product # PA5-78253). HepG2 whole cell and nuclear extracts (30 µg) were separated by 5% SDS-PAGE, and the membrane was blotted with SMARCA5 Polyclonal Antibody (Product # PA5-78253) diluted at 1:10,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-SMARCA5 Polyclonal Antibody (Product # PA5-78253) and a 130kDa band corresponding to SMARCA5 was observed across all cell lines tested. Nuclear enriched extracts (30 µg lysate) of Jurkat (Lane 1), K-562 (Lane 2), HeLa (Lane 3), Raji (Lane 4), U-2 OS (Lane 5), Hep G2 (Lane 6) and MOLT-4 (Lane 7) 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 (1:10,000 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 Chemiluminescent Substrate Reagent Kit (Product # WP20005). An uncharacterized band was also observed at ~45kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: SMARCA5 Polyclonal Antibody detects SNF2H protein at nucleus by immunofluorescent analysis. Sample: HeLa cells were fixed in 4% paraformaldehyde at RT for 15 min. Green: SNF2H protein stained by SMARCA5 Polyclonal Antibody (Product # PA5-78253) diluted at 1:500. Red: phalloidin, a cytoskeleton marker, diluted at 1:50.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of SMARCA5 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 45 minutes at room temperature. The cells were labeled with SMARCA5 Polyclonal Antibody (Product # PA5-78253) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000 dilution), 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 merged image showing Nuclear localization. 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: SMARCA5 Polyclonal Antibody detects SNF2H protein at nucleus in rat lung by immunohistochemical analysis. Sample: Paraffin-embedded rat lung. SMARCA5 Polyclonal Antibody (Product # PA5-78253) diluted at 1:400. Antigen Retrieval: Citrate buffer, pH 6.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Chromatin Immunoprecipitation (ChIP) assay of endogenous SMARCA5 protein using Anti-SMARCA5 Antibody: ChIP was performed using Anti-SMARCA5 Polyclonal Antibody (Product # PA5-78253) 5 µg, on sheared chromatin from MCF-7 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 promoter of CCND1, ERALPHA and SAT2 satellite repeats. 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 3 WT tau modulates gene expression of chromatin organization and remodeling factors. (A) Enrichment plot for GO Chromatin organization. (B) Enrichment plot for GO Chromatin remodeling. (C) Enrichment plot for GO-Covalent Chromatin modification. (D) IB of Up-regulated genes: RCC1, DNAJC2 and Histone 1.2 (red box) and Down-regulated genes: SMARCC1, SMARCA5 and HMGB1 (blue box) in cytoplasm and nuclear fractions from WT and P301L tau iHEK. Histone 3 and beta-Actin has been used as loading control for nuclear and cytoplasmatic fractions, respectively. (E) Immunoblot quantification of RCC1 (* p < 0.01), DNAJC2 (* p < 0.01), Histone1.2 (* p < 0.01), cytoplasmic HMGB1 (* p < 0.01), nuclear HMGB1 and SMARCA5 proteins in cytoplasmatic and nuclear fractions of WT tau iHEK. (F) Representative Tau 13 (magenta) and Histone 1.2 (green) Co-IF of control (-Tet) and treated WT tau iHEK. (G) Histone 1.2 integrated density quantification in control and + Tet cells (Unpaired t -test, **** p < 0.0001). (H) Representative Tau 13 (magenta) and Ki67 (green) Co-IF of control (-Tet) and treated WT tau iHEK. (I) Ki67 integrated density quantification in control and + Tet cells (Unpaired t -test, **** p < 0.0001). (J) Representative Tau 13 (magenta) and SMARCC1 (green) Co-IF of control (-Tet) and treated WT tau iHEK. (K) SMARCC1 integrated density quantification in control and + Tet cells (Unpaired t -test, **** p < 0.0001). (L) Representative Tau 13 (magenta) and SMARCA5 (green) Co-IF of control

PA5-78253

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