- PA5-54031 - Invitrogen Antibodies UPF1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of UPF1 in A-549 cells transfected with control siRNA, target specific siRNA probe #1 and #2, using a UPF1 Polyclonal Antibody (Product # PA5-54031). Remaining relative intensity is presented. Loading control: Anti-GAPDH.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of UPF1 in human cell line A-549 using a UPF1 Polyclonal Antibody (Product # PA5-54031).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of UPF1 in mouse cell line NIH-3T3 and rat cell line NBT-II using a UPF1 Polyclonal Antibody (Product # PA5-54031).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of UPF1 was achieved by transfecting PC-3 with UPF1 specific siRNAs (Silencer® select Product # S11926, S11927). Western Blot analysis (Fig. a) was performed using Whole cell extracts from the UPF1 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The Blot was probed with UPF1 Polyclonal Antibody (Product # PA5-54031, 0.02 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant 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 UPF1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot was performed using Anti-UPF1 Polyclonal Antibody (Product # PA5-54031) and a ~130 kDa band corresponding to UPF1 was observed along with two uncharacterised band (*) at ~110 kDa and ~80 kDa across cell lines and tissues tested. Whole cell extracts (30 µg lysate) of A549 (Lane 1), SH-SY5Y (Lane 2), PC-3 (Lane 3), HEK-293 (Lane 4), NIH/3T3 (Lane 5), PC-12 (Lane 6), Mouse Testis (Lane 7), Mouse Spleen (Lane 8) and Rat Testis (Lane 9) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # LC2001) by iBlot® 2 Dry Blotting System (Product # IB21001). The Blot was probed with the primary antibody (0.02 µg/mL) 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).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent staining of UPF1 in human cell line U-2 OS shows positivity in nucleus but excluded from the nucleoli. Samples were probed using an UPF1 Polyclonal Antibody (Product # PA5-54031).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of UPF1 was performed using 70% confluent log phase PC-3 cells. The cells were fixed with 4% paraformaldehyde for 5 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with UPF1 Polyclonal Antibody (Product # PA5-54031) at 1 µg/mL 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 Nucleus and cytoplasm 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 staining of UPF1 in human duodenum tissue shows strong granular positivity in glandular cells. Samples were probed using an UPF1 Polyclonal Antibody (Product # PA5-54031).

PA5-54031