- PA5-20018 - Invitrogen Antibodies IRAK4 antibody

Molagoda IMN, Athapaththu AMGK, Choi YH, Park C, Jin CY, Kang CH, Lee MH, Kim GY
Antioxidants (Basel, Switzerland) 2021 Jul 28;10(8)

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot Validation in Human Cell Lines. Loading: 15 µg/ of lysates per lane. Antibodies: IRAK4 Polyclonal Antibody (Product # PA5-20018) (1 µg/mL), 1h incubation at RT in 0.05 NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10,000 dilution.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot Validation in Human HeLa Cell Lines. Loading: 15 µg of lysates per lane. Antibodies: IRAK4 Polyclonal Antibody (Product # PA5-20018) 1h incubation at RT in 0.05 NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10,000 dilution. Lane 1: 1 µg/mL Lane 2: 2 µg/mL Lane 3: 4 µg/mL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of IRAK4 was achieved by transfecting THP-1 with IRAK4 specific siRNAs (Silencer® select Product # s535019, s535020). Western blot analysis (Fig. a) was performed using whole cell extracts from the IRAK4 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with IRAK4 Polyclonal Antibody (Product # PA5-20018, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (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 IRAK4.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-IRAK4 Polyclonal Antibody, (Product # PA5-20018) and a 52 kDa band corresponding to IRAK4 was observed in the cell lines and tissues tested. Whole cell extracts (30 µg lysate) of THP-1 (Lane 1), RAW 264.7 (Lane 2) and Mouse spleen (Lane 3) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX). 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 ug/ml) and detected by chemiluminescence Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant 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: Immunofluorescent analysis of K562 cells using a IRAK-4 polyclonal antibody (Product # PA5-20018) at a 10 µg/mL dilution.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunocytochemistry of K562 cells using IRAK4 Polyclonal Antibody (Product # PA5-20018) at 10 µg/mL. Cells were fixed with formaldehyde and blocked with 0.1 serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4°C. A goat anti-rabbit IgG H&L (HRP) at 1:250 was used as secondary. Counter stained with Hematoxylin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of 4% paraformaldehyde-fixed K562 Cells labeling IRAK4 with IRAK4 Polyclonal Antibody (Product # PA5-20018) at 10 µg/mL, followed by goat anti-rabbit IgG secondary antibody at 1:500 dilution (red).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of IRAK4 was performed using RAW 264.7 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 1 hour at room temperature. The cells were labeled with IRAK4 Polyclonal Antibody (Product # PA5-20018) at 5 µg/mL concentration in 0.1% BSA, incubated at 4 degree Celsius overnight and then with Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant 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 represents the merged image showing staining in cytoplasm. 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 2 Fisetin probably binds to TLR4 and inhibits the TLR4-mediated intracellular signaling pathway. ( A ) Molecular docking of fisetin (pose 1) with the TLR4/MD2 complex (PDB ID: 3FXI). ( B ) BV2 microglial cells were seeded on 3% gelatin-coated coverslips and treated with the indicated concentrations of fisetin (0-5 uM) for 2 h prior to stimulation with 1 ug/mL LPS for 2 h and subsequent stimulation with 1 mM ATP (LPS/ATP) for an additional 2 h. Cells were fixed with 4% paraformaldehyde and immunostained for TLR4 using Alexa Fluor 647-conjugated secondary antibody. The images were captured using a CELENA S Digital Imaging System. ( C ) In a parallel experiment, the total proteins were extracted and western blotting was performed for detecting the expression of MyD88 and IRAK4 proteins. beta-Actin was used as a loading control. The results indicate the mean +- standard error median (SEM), and is representative of the results obtained from three independent experiments. ### p < 0.001 vs. untreated cells; *** p < 0.001 and ** p < 0.01 vs. LPS/ATP-treated cells.

PA5-20018