- PA1-24931 - Invitrogen Antibodies CFL1 antibody

Burat B, Faucher Q, Čechová P, Arnion H, Di Meo F, Sauvage FL, Marquet P, Essig M
FASEB bioAdvances 2019 Sep;1(9):561-578

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Cofilin was achieved by transfecting HeLa with Cofilin specific siRNAs (Silencer® select Product # s2037, s2936 ). Western blot analysis (Fig. a) was performed using Whole cell extracts from the untransfected cells (lane 1), non-targeting scrambled siRNA transfected cells (lane 2) and,Cofilin knockdown cells (lane 3). The blot was probed with Cofilin Polyclonal Antibody (Product # PA1-24931, 1:10000 dilution) 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 Cofilin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-Cofilin Polyclonal Antibody(Product # PA1-24931) and an 18kDa band corresponding to Cofilin was observed across cell lines tested. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), A-431 (Lane 2), Hep G2 (Lane 3), PC-12 (Lane 4) and NIH/3T3 (Lane 5) were electrophoresed using NuPAGE™ 12% Bis-Tris Protein Gel (Product # NP0341BOX). 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 (1:10000 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).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Cofilin 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 Cofilin Polyclonal Antibody (Product # PA1-24931) at 1:1000 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034), (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 merged image showing cytoskeleton, plasma membrane, 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: Figure 3 Cyclosporine A (CsA), but not tacrolimus (Tac), induced changes in cofilin (CFL) concentration-to-actin. A, Western blot detection of formaldehyde-cross-linked CFL oligomers in Lilly Laboratories Porcine Kidney-1 lysates. B, Quantification of relative protein abundance of CFL oligomeric forms. Mean +- SEM. One-way ANOVA plus Dunnett's post-test (* P < .05, ** P < .01). Drug condition: 5 umol/L CsA, 0.05 umol/L Tac. Exposure time: 24 h (n = 5)

PA1-24931