- MA5-15155 - Invitrogen Antibodies IKBKG antibody

Submitted references Placental Expression of NEMO Protein in Normal Pregnancy and Preeclampsia.

Sakowicz A, Lisowska M, Biesiada L, Płuciennik E, Gach A, Rybak-Krzyszkowska M, Huras H, Sakowicz B, Romanowicz H, Piastowska-Ciesielska AW, Grzesiak M, Pietrucha T
Disease markers 2019;2019:8418379

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of IKK-gamma in extracts from HeLa cells, untreated or treated with TNF-alpha (20 ng/mL) and a phosphatase inhibitor (50 nM), using IKK-gamma monoclonal antibody (Product # MA5-15155) (bottom) and a Phospho-IKK-gamma pSer376 polyclonal antibody (top).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of IKKG was achieved by transfecting HEK 293 cells with IKKG specific siRNAs (Silencer® select Product # s16186). Western blot analysis (Fig. a) was performed using whole cell extracts from the IKKG knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with IKKG Polyclonal Antibody (Product # MA5-515155,1:1000 dilution) and chemiluminescence Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 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 IKKG.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using anti-IKKG Polyclonal Antibody (Product # MA5-15155) and a 48 kDa corresponding to IKKG was observed across the cell lines tested. Whole cell extracts of (30ug lysate) of U-87 MG (Lane 1), HEK-293 (Lane 2) and HeLa (Lane 3) 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 with Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution). 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: Immunofluorescence analysis of IKK gamma was performed using HEK-293 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 IKK gamma Mouse Monoclonal Antibody (Product # MA5-15155) at 1:100 dilution in 0.1% BSA and incubated overnight at 4 degree and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 conjugate (Product # A32766) at a dilution of 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 composite image showing cytoplasmic localization of IKK gamma in HEK-293 cells. 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)
Main image
Experimental details: Figure 4 Western blot and immunohistochemical staining analyses of the NEMO protein in placentas from early, late preeclampsia, and noncomplicated pregnancies. Western blot analyses of the NEMO protein for total homogenates (a), cytoplasmic fraction (b), and nuclear fraction (c). The western blot analyses are presented as mean +- SEM ( n = 16 for each subfraction). Asterisks indicate significant differences ( * p value < 0.05) between late preeclampsia and control. Immunostaining of NEMO in early preeclampsia (d), late preeclampsia (e), and control placentas (f). The histograms (g, h) represent the median value and interquartile range of the optical density (OD) of the NEMO protein between whole preeclamptic and control groups (g) and between the early preeclamptic group, late preeclamptic group, and control (h). The OD density is given on an arbitrary scale in arbitrary units (a.u.). Asterisks indicate significant differences ( ** p < 0.001).

MA5-15155

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