NF-κB信号通路-免疫学专题- ABclonal

NF-κB信号通路

NF-κB是一个高度保守的多功能转录因子家族，包含五个成员，分别为p65（RelA）、RelB、c-Rel、NF-κB1和NF-κB2，它们会组成不同的同源或异源二聚体而发挥基因转录调节作用，进而调节许多重要的细胞行为，特别是炎症反应、应激反应、细胞生长、细胞存活和凋亡[1]。与其他家族成员不同的是，NF-κB1和NF-κB2被合成为前体（p105和p100），并分别可以被加工成p50和p52[2]。在大多数静止细胞中，这些二聚体的转录调节活性受到NF-κB抑制分子（IκB）的限制，或在加工为成熟形式前受其自身内部抑制基序的限制（比如p105或p100）。当细胞受到各种刺激，含有IKKα、IKKβ和IKKγ（NF-κB必需调节物，也称为NEMO）的IκB激酶复合物可以被多种上游信号通路激活，并介导IκB的磷酸化和蛋白酶体降解。然后NF-κB同源或异源二聚体可以转移到细胞核并激活靶基因转录[2]。NF-κB激活的另一种途径，也被称为替代途径，在包括BAFFR、CD40、RANK或LTβR的下游存在，导致NF-κB诱导激酶（NIK）活化并激活IKKα[3]。IKKα能磷酸化p100并导致p100的多泛素化及其随后经蛋白酶体被加工为p52。然后，p52-RelB二聚体可以激活靶基因的转录。除了上述两种激活途径，NF-κB还可以在特定情况下被其他信号通路或激酶激活，称为非典型激活途径 [4]。NF-κB转录调控系统的复杂性也体现在如下几个方面：1）不同的NF-κB二聚体对DNA结合序列的变化有不同的偏好。因此，不同的靶基因由不同的NF-κB二聚体在不同程度上被激活[5]； 2）NF-κB家族成员和调节因子还含有磷酸化和其他翻译后修饰的位点，这些位点对NF-κB的调节和与其他信号通路的串话很重要[4,6]；3）在不同的细胞环境下，NF-κB信号通路与其他信号通路之间或NF-κB与其他转录因子之间的串话，增加了NF-κB调控和功能的复杂性 [2, 7-14]。

ABclonal针对NF-κB信号通路研究，提供以下常见核心靶点的产品

IκBα Rabbit pAb

Western blot analysis of various lysates using IκBα Rabbit pAb (A11397) at 1:1000 dilution.
Secondary antibody: HRP-conjugated Goat anti-Rabbit IgG (H+L) (AS014) at 1:10000 dilution.
Lysates/proteins: 25μg per lane.
Blocking buffer: 3% nonfat dry milk in TBST.
Detection: ECL Enhanced Kit (RM00021).
Exposure time: 180s.

Western blot analysis of lysates from Rat liver, using IκBα Rabbit pAb (A11397) at 1:1000 dilution.
Secondary antibody: HRP-conjugated Goat anti-Rabbit IgG (H+L) (AS014) at 1:10000 dilution.
Lysates/proteins: 25μg per lane.
Blocking buffer: 3% nonfat dry milk in TBST.
Detection: ECL Basic Kit (RM00020).
Exposure time: 180s.

[KO Validated] NF-kB p65/RelA Rabbit mAb

Western blot analysis of lysates from wild type (WT) and NF-kB p65/RelA knockout (KO) HeLa cells using [KO Validated] NF-kB p65/RelA Rabbit mAb (A19653) at 1:10000 dilution incubated overnight at 4℃.
Secondary antibody: HRP-conjugated Goat anti-Rabbit IgG (H+L) (AS014) at 1:10000 dilution.
Lysates/proteins: 25 μg per lane.
Blocking buffer: 3% nonfat dry milk in TBST.
Detection: ECL Basic Kit (RM00020).
Exposure time: 30s.

Western blot analysis of lysates from PC-12 cells using [KO Validated] NF-kB p65/RelA Rabbit mAb (A19653) at 1:10000 dilution incubated overnight at 4℃.
Secondary antibody: HRP-conjugated Goat anti-Rabbit IgG (H+L) (AS014) at 1:10000 dilution.
Lysates/proteins: 25 μg per lane.
Blocking buffer: 3% nonfat dry milk in TBST.
Detection: ECL Basic Kit (RM00020).
Exposure time: 30s.

Western blot analysis of various lysates using [KO Validated] NF-kB p65/RelA Rabbit mAb (A19653) at 1:10000 dilution incubated overnight at 4℃.
Secondary antibody: HRP-conjugated Goat anti-Rabbit IgG (H+L) (AS014) at 1:10000 dilution.
Lysates/proteins: 25 μg per lane.
Blocking buffer: 3% nonfat dry milk in TBST.
Detection: ECL Basic Kit (RM00020).
Exposure time: 60s.

Immunohistochemistry analysis of paraffin-embedded HT-1080 cell lines(untreated and treated with TNF-α) using [KO Validated] NF-kB p65/RelA Rabbit mAb (A19653) at a dilution of 1:3000 (40x lens). High pressure antigen retrieval performed with 0.01M Tris-EDTA Buffer (pH 9.0) prior to IHC staining.

Immunohistochemistry analysis of paraffin-embedded Human colon tissue using [KO Validated] NF-kB p65/RelA Rabbit mAb (A19653) at a dilution of 1:3000 (40x lens). High pressure antigen retrieval performed with 0.01M Tris-EDTA Buffer (pH 9.0) prior to IHC staining.

Immunohistochemistry analysis of paraffin-embedded Human lung cancer tissue using [KO Validated] NF-kB p65/RelA Rabbit mAb (A19653) at a dilution of 1:3000 (40x lens). High pressure antigen retrieval performed with 0.01M Tris-EDTA Buffer (pH 9.0) prior to IHC staining.

Confocal imaging of HT-1080 cells (treated with TNF-α) and HT-1080 cells (untreated) cells using [KO Validated] NF-kB p65/RelA Rabbit mAb (A19653, dilution 1:2100) followed by a further incubation with Cy3 Goat Anti-Rabbit IgG (H+L) (AS007, dilution 1:500) (Red). The cells were counterstained with α-Tubulin Mouse mAb (AC012, dilution 1:400) followed by incubation with ABflo® 488-conjugated Goat Anti-Mouse IgG (H+L) Ab (AS076, dilution 1:500) (Green). DAPI was used for nuclear staining (Blue). Objective: 100x.

Confocal imaging of NIH/3T3 cells (treated with TNF-α) and NIH/3T3 cells (untreated) cells using [KO Validated] NF-kB p65/RelA Rabbit mAb (A19653, dilution 1:2100) followed by a further incubation with Cy3 Goat Anti-Rabbit IgG (H+L) (AS007, dilution 1:500) (Red). The cells were counterstained with α-Tubulin Mouse mAb (AC012, dilution 1:400) followed by incubation with ABflo® 488-conjugated Goat Anti-Mouse IgG (H+L) Ab (AS076, dilution 1:500) (Green). DAPI was used for nuclear staining (Blue). Objective: 100x.

Confocal imaging of PC-12 cells (treated with TNF-α) and PC-12 cells (untreated) cells using [KO Validated] NF-kB p65/RelA Rabbit mAb (A19653, dilution 1:2100) followed by a further incubation with Cy3 Goat Anti-Rabbit IgG (H+L) (AS007, dilution 1:500) (Red). The cells were counterstained with α-Tubulin Mouse mAb (AC012, dilution 1:400) followed by incubation with ABflo® 488-conjugated Goat Anti-Mouse IgG (H+L) Ab (AS076, dilution 1:500) (Green). DAPI was used for nuclear staining (Blue). Objective: 100x.

Chromatin immunoprecipitation was performed with 10 μg of cross-linked chromatin from HT-1080 cells treated by TNF-α (20 ng/ml) at 37℃ for 30 minutes, using 5 μg of [KO Validated] NF-kB p65/RelA Rabbit mAb (A19653) and Rabbit IgG isotype control (AC042). The enrichment of immunoprecipitated DNA at different genomic loci was examined by quantitative PCR. The histogram compares the ratio of the immunoprecipitated DNA to the input at given loci.

Phospho-NF-kB p65/RelA-S536 Rabbit mAb

Western blot analysis of various lysates, using Phospho-NF-kB p65/RelA-S536 Rabbit mAb (AP1294) at1:1000 dilution (upper) or [KO Validated] NF-kB p65/RelA Rabbit mAb (A22331) at1:50000 dilution (lower) incubated overnight at 4℃. HeLa cells were treated with Calyculin A (100 nM) at 37℃ for 30 minutes after serum-starvation overnight.C6 cells were treated with TNF-α (20 ng/ml) and Calyculin A (50 nM) at 37℃ for 30 minutes after serum-starvation overnight.
Secondary antibody: HRP-conjugated Goat anti-Rabbit IgG (H+L) (AS014) at 1:10000 dilution.
Lysates/proteins: 30 μg per lane.
Blocking buffer: 3% nonfat dry milk in TBST.
Detection: ECL Basic Kit (RM00020).
Exposure time: 10s.

Western blot analysis of lysates from NIH/3T3 cells using Phospho-NF-kB p65/RelA-S536 Rabbit mAb (AP1294) at 1:1000 dilution incubated overnight at 4℃. NIH/3T3 cells were treated with TNF-α (20 ng/ml) at 37℃ for 30 minutes after serum-starvation overnight.
Secondary antibody: HRP-conjugated Goat anti-Rabbit IgG (H+L) (AS014) at 1:10000 dilution.
Lysates/proteins: 30 μg per lane.
Blocking buffer: 3 % nonfat dry milk in TBST.
Detection: ECL Basic Kit (RM00020).
Exposure time: 180s.

Western blot analysis of lysates from HeLa cells using Phospho-NF-kB p65/RelA-S536 Rabbit mAb (AP1294) at 1:19000 dilution incubated at room temperature for 1.5 hours. HeLa cells were treated with TNF-α (20 ng/ml) and Calyculin A (100 nM) at 37℃ for 30 minutes after serum-starvation overnight.
Secondary antibody: HRP-conjugated Goat anti-Rabbit IgG (H+L) (AS014) at 1:10000 dilution.
Lysates/proteins: 30 μg per lane.
Blocking buffer: 3 % nonfat dry milk in TBST.
Detection: ECL Basic Kit (RM00020).
Exposure time: 30s.

Western blot analysis of lysates from C6 cells using Phospho-NF-kB p65/RelA-S536 Rabbit mAb (AP1294) at 1:19000 dilution incubated at room temperature for 1.5 hours. C6 cells were treated with Calyculin A (100 nM) at 37℃ for 30 minutes after serum-starvation overnight.
Secondary antibody: HRP-conjugated Goat anti-Rabbit IgG (H+L) (AS014) at 1:10000 dilution.
Lysates/proteins: 30 μg per lane.
Blocking buffer: 3 % nonfat dry milk in TBST.
Detection: ECL Basic Kit (RM00020).
Exposure time: 30s.

IκBα Rabbit mAb

Western blot analysis of lysates from MCF7 cells, using IκBα Rabbit mAb (A19714) at 1:1000 dilution.
Secondary antibody: HRP-conjugated Goat anti-Rabbit IgG (H+L) (AS014) at 1:10000 dilution.
Lysates/proteins: 25μg per lane.
Blocking buffer: 3% nonfat dry milk in TBST.
Detection: ECL Basic Kit (RM00020).
Exposure time: 30s.

Western blot analysis of lysates from NIH/3T3 cells, using IκBα Rabbit mAb (A19714) at 1:1000 dilution.
Secondary antibody: HRP-conjugated Goat anti-Rabbit IgG (H+L) (AS014) at 1:10000 dilution.
Lysates/proteins: 25μg per lane.
Blocking buffer: 3% nonfat dry milk in TBST.
Detection: ECL Basic Kit (RM00020).
Exposure time: 60s.

Immunohistochemistry analysis of paraffin-embedded Rat kidney using IκBα Rabbit mAb (A19714) at dilution of 1:100 (40x lens). Microwave antigen retrieval performed with 0.01M PBS Buffer (pH 7.2) prior to IHC staining.

Immunohistochemistry analysis of paraffin-embedded Human colon using IκBα Rabbit mAb (A19714) at dilution of 1:100 (40x lens). Microwave antigen retrieval performed with 0.01M PBS Buffer (pH 7.2) prior to IHC staining.

Immunohistochemistry analysis of paraffin-embedded Mouse kidney using IκBα Rabbit mAb (A19714) at dilution of 1:100 (40x lens). Microwave antigen retrieval performed with 0.01M PBS Buffer (pH 7.2) prior to IHC staining.

NFKB1 Rabbit pAb

Western blot analysis of lysates from Mouse lung, using NFKB1 Rabbit pAb (A6667) at 1:500 dilution.
Secondary antibody: HRP-conjugated Goat anti-Rabbit IgG (H+L) (AS014) at 1:10000 dilution.
Lysates/proteins: 25μg per lane.
Blocking buffer: 3% nonfat dry milk in TBST.
Detection: ECL Basic Kit (RM00020).
Exposure time: 20s.

Immunohistochemistry analysis of paraffin-embedded Human colon carcinoma using NFKB1 Rabbit pAb (A6667) at dilution of 1:50 (40x lens). High pressure antigen retrieval performed with 0.01M Citrate buffer (pH 6.0) prior to IHC staining.

Immunohistochemistry analysis of paraffin-embedded Mouse intestin using NFKB1 Rabbit pAb (A6667) at dilution of 1:50 (40x lens). High pressure antigen retrieval performed with 0.01M Citrate buffer (pH 6.0) prior to IHC staining.

Immunohistochemistry analysis of paraffin-embedded Rat lung using NFKB1 Rabbit pAb (A6667) at dilution of 1:50 (40x lens). High pressure antigen retrieval performed with 0.01M Citrate buffer (pH 6.0) prior to IHC staining.

Immunofluorescence analysis of A-549 cells using NFKB1 Rabbit pAb (A6667) at dilution of 1:50 (40x lens). Secondary antibody: Cy3-conjugated Goat anti-Rabbit IgG (H+L) (AS007) at 1:500 dilution. Blue: DAPI for nuclear staining.

Immunofluorescence analysis of NIH/3T3 cells using NFKB1 Rabbit pAb (A6667) at dilution of 1:50 (40x lens). Secondary antibody: Cy3-conjugated Goat anti-Rabbit IgG (H+L) (AS007) at 1:500 dilution. Blue: DAPI for nuclear staining.

Immunofluorescence analysis of U2OS cells using NFKB1 Rabbit pAb (A6667) at dilution of 1:50 (40x lens). Secondary antibody: Cy3-conjugated Goat anti-Rabbit IgG (H+L) (AS007) at 1:500 dilution. Blue: DAPI for nuclear staining.

IKKβ Rabbit mAb

Confocal imaging of HeLa cells using IKKβ Rabbit mAb (A19606,dilution 1:100)(Red). The cells were counterstained with α-Tubulin Mouse mAb (AC012,dilution 1:400) (Green). DAPI was used for nuclear staining (blue). Objective: 100x.

IKKε Rabbit mAb

Western blot analysis of various lysates using IKKε Rabbit mAb (A3463) at 1：1000 dilution.
Secondary antibody: HRP-conjugated Goat anti-Rabbit IgG (H+L) (AS014) at 1:10000 dilution.
Lysates/proteins: 25μg per lane.
Blocking buffer: 3% nonfat dry milk in TBST.
Detection: ECL Basic Kit (RM00020).
Exposure time: 30s.

Immunohistochemistry analysis of paraffin-embedded Mouse heart using IKKε Rabbit mAb (A3463) at dilution of 1:100 (40x lens). Microwave antigen retrieval performed with 0.01M PBS Buffer (pH 7.2) prior to IHC staining.

Immunohistochemistry analysis of paraffin-embedded Human colon tissue using IKKε Rabbit mAb (A3463) at a dilution of 1:200 (40x lens). High pressure antigen retrieval performed with 0.01M Citrate buffer (pH 6.0) prior to IHC staining.

Immunohistochemistry analysis of paraffin-embedded Human tonsil tissue using IKKε Rabbit mAb (A3463) at a dilution of 1:200 (40x lens). High pressure antigen retrieval performed with 0.01M Citrate buffer (pH 6.0) prior to IHC staining.

Immunohistochemistry analysis of paraffin-embedded Rat colon tissue using IKKε Rabbit mAb (A3463) at a dilution of 1:200 (40x lens). High pressure antigen retrieval performed with 0.01M Citrate buffer (pH 6.0) prior to IHC staining.

Immunohistochemistry analysis of paraffin-embedded Rat spleen tissue using IKKε Rabbit mAb (A3463) at a dilution of 1:200 (40x lens). High pressure antigen retrieval performed with 0.01M Citrate buffer (pH 6.0) prior to IHC staining.

NF-κB2 Rabbit mAb

Western blot analysis of various lysates using NF-κB2 Rabbit mAb (A19605) at 1:1000 dilution.
Secondary antibody: HRP-conjugated Goat anti-Rabbit IgG (H+L) (AS014) at 1:10000 dilution.
Lysates/proteins: 25μg per lane.
Blocking buffer: 3% nonfat dry milk in TBST.
Detection: ECL Basic Kit (RM00020).
Exposure time: 3s.

Immunohistochemistry analysis of paraffin-embedded Human appendix using NF-κB2 Rabbit mAb (A19605) at dilution of 1:100 (40x lens). Microwave antigen retrieval performed with 0.01M PBS Buffer (pH 7.2) prior to IHC staining.