- PA5-30442 - Invitrogen Antibodies U2AF2 antibody

Submitted references Alternatively spliced ANLN isoforms synergistically contribute to the progression of head and neck squamous cell carcinoma.

Guo E, Mao X, Wang X, Guo L, An C, Zhang C, Song K, Wang G, Duan C, Zhang X, Yang X, Yuan Z, Sun J, Li X, Yang W, Meng H, Miao S
Cell death & disease 2021 Aug 3;12(8):764

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot using U2AF2 Polyclonal Antibody (Product # PA5-30442). Sample (30 µg of whole cell lysate). Lane A: A431 . 7.5% SDS PAGE. U2AF2 Polyclonal Antibody (Product # PA5-30442) diluted at 1:10,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: KD of U2AF2 was achieved by transfecting Hep G2 with U2AF2 specific siRNAs (Silencer® select Product # S22366). Western blot analysis (Fig. a) was performed using whole cell extracts from theU2AF2 KD cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with U2AF2 Polyclonal Antibody (Product # PA5-30442, 1:10000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25µg/mL, 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 U2AF2..

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-U2AF2 Polyclonal Antibody (Product # PA5-30442) and a 55kDa band corresponding to U2AF2 was observed in all the cell lines and tissues tested. Modified whole cell lysates (1% SDS) (30 µg lysate) of A-431 (Lane 1), HCT 116 (Lane 2), Hep G2 (Lane 3), Jurkat (Lane 4), B16-F10 (Lane 5), A549 (Lane 6), and tissue extract (30 µg lysate) of Mouse Spleen (Lane 7) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris 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 (1:1000 dilution) and detected by chemiluminescence Goat Anti-Rabbit IgG Secondary Antibody, HRP conjugate (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).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: U2AF2 Polyclonal Antibody detects U2AF65 protein at nucleus by immunofluorescent analysis. Sample: A431 cells were fixed in 4% paraformaldehyde at RT for 15 min. Green: U2AF65 stained by U2AF2 Polyclonal Antibody (Product # PA5-30442) diluted at 1:500. Red: alpha Tubulin, a cytoskeleton marker, stained by alpha Tubulin Polyclonal Antibody [GT114] (Product # MA5-31466) diluted at 1:1,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of U2AF2 was performed using 70% confluent A-431 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with U2AF2 Polyclonal Antibody (Product # PA5-30442) at 5µg/mL concentration in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d and e 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: Immunohistochemical analysis of paraffin-embedded human ovarian cancer, using U2AF65 (Product # PA5-30442) antibody at 1:250 dilution. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of U2AF65 showing staining in the nucleus of HCT 116 cells. HCT 116 cells were fixed in 4% paraformaldehyde at RT for 15 min and stained using a U2AF65 polyclonal antibody (Product # PA5-30442) diluted at 1:500. Blue: Hoechst 33342 staining. Scale bar = 10µm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: RNA Immunoprecipitation (RIP) assay of endogenous U2AF2 protein using Anti-U2AF2 Antibody: RIP assay was performed using Anti-U2AF2 Recombinant Rabbit Polyclonal Antibody (Product # PA5-30442, 5 ug) on whole cell lysate from Hep G2 cells. Normal Rabbit IgG was used as a negative IP control. RNA purified by RiboPure™ RNA Purification Kit (Product # AM1924) was analyzed by RT-PCR using the Power SYBR® Green RNA-to-CT™ 1-Step Kit (Product # 4389986) with the primers pairs over ACTB, SMN2 mRNA and MALAT1 non-coding RNA. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

PA5-30442

Antibody data