- 702410 - Invitrogen Antibodies FXR1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of FXR1 was achieved by transfecting NIH3T3 cells with FXR1 specific siRNA (Silencer® select Cat # s15610 and s15612). Western blot analysis (Fig a) was performed using Whole cell extract from the FXR1 knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-FXR1 Recombinant Rabbit Monoclonal Antibody (Product # 702410, 1-3 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4µg/mL, 1:2500 dilution). Densitometric analysis of this Western blot is shown in histogram (Fig b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to FXR1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on Whole cell extracts (30 µg lysate) of C2C12 (Lane 1), NIH/3T3 (Lane 2), HEK-293 (Lane 3) and tissue extract (30 µg lysate) of Mouse Skeletal Muscle (Lane 4). The blots were probed with Anti-FXR1 Recombinant Rabbit Monoclonal Antibody (Product # 702410, 2.5 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:4000 dilution). 71 and 80 kDa bands corresponding to FXR1 was observed across the cell lines and tissue tested. Known quantity of protein samples were electrophoresed using Novex®NuPAGE®4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: For immunofluorescence analysis, SH-SY5Y cells were fixed and permeabilized for detection of endogenous FXR1 using Anti-FXR1 Recombinant Rabbit Monoclonal Antibody (Product # 702410, 5µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Nuclei (blue) is stained using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938) and cytoskeletal F-actin (red) staining using Rhodamine Phalloidin (Product # R415, 1:300).Panel a-d) shows representative cells in normal growth conditions that were stained for detection and localization of FXR1 protein (green) with diffused cytoplasmic signal. Panel e-h) clearly demonstrate translocation of FXR1 to cytoplasmic stress granules in cells treated with MG132 (10 µg/mL 3h). The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: RNA iImmunoprecipitation (RIP) western of FXR1 was performed on K562 cells. Antigen-antibody complexes were formed by incubating approximately 500 µg whole cell lysate with 5 µg of FXR1 monoclonal antibody (Product # 702410) rotating 60 min at RT. The immune complexes were captured on 625 µg of anti-rabbit coated Dynabeads (Product # 11204D), washed extensively, and eluted with NuPAGE™ LDS Sample Buffer (Product # NP0007). Samples were resolved onto NuPAGE™ 4-12% Bis-Tris gel (Product # NP0335BOX). Lanes 1 and 3 are input and lanes 2 and 4 are IP. Proteins were transferred to PVDF membrane (Product # IB23001). Membrane was blocked in 5% milk. Target was detected using a FXR1 monoclonal antibody (Product # 702410) at a dilution of 1:2000, followed by a 1:4000 dilution of secondary antibody. Chemiluminescent detection was performed using ECL Western Blotting Substrate (Product # 32106). Data courtesy of the Yeo lab as part of the ENCODE project (www.encodeproject.org).

702410