Toll样受体信号通路-免疫学专题- ABclonal

Toll样受体信号通路

作为机体的第一道防线，天然免疫系统是通过模式识别受体(Pattern Recognition Receptors , PRRs)或信号感受器识别病原体相关分子模式（Pathogen-associated Molecular Patterns, PAMPs）和内源性损伤相关分子模式(Damage-associated Molecular Patterns, DAMPs)而被启动的 [1]。其中，Toll样受体（Toll-like Receptors,TLRs）因与果蝇中Toll蛋白结构相似而得名，是最早被鉴定并在从昆虫到人类的多种动物中被广泛研究的模式识别受体[2]。
TLRs在包括免疫细胞和非免疫细胞的多种细胞类型上表达，它们是跨膜受体，可以定位于细胞表面或内体[3]。他们被外源性和可能的内源性配体结合，在连接天然免疫和炎症的各种免疫细胞中触发促炎信号级联。结合配体后，形成TLR同源或异源二聚体，并通过MyD88、TIRAP、TRAM和/或TRIF等不同接头蛋白激活MyD88依赖和/或MyD88非依赖的信号途径[4]。除TLR3外，所有的TLRs都可以通过MyD88介导下游信号，而TLR3信号通过TRIF介导下游信号。然而，TLR2和TLR4需要通过TIRAP招募MyD88。TLR4比较特别，因为它可以同时利用MyD88和TRIF作为信号接头分子启动下游信号，但它还需要接头分子TRAM来招募TRIF和TIRAP来招募MyD88。在MyD88依赖的信号途径中，MyD88招募IRAK并造成这些IRAK被磷酸化，然后招募TRAF泛素连接酶。通常情况下，TRAF6与TAK和TAB形成复合物，TAK通过磷酸化修饰激活下游IKK-NF-κB和MAPK级联，进而导致转录因子NF-κB和AP-1的激活，并控制促炎细胞因子和其他免疫相关基因的表达[5]。也有报道发现，在特殊情况下TRAF6和MyD88可以通过激活IRF1或IRF5介导这个过程[6]。而TRAF3招募TBK1和IKKε，磷酸化和激活转录因子IRF(主要是IRF-3和IRF-7)并导致I型干扰素的产生。此外，在病毒感染时，TRAF6可以与IRF7、MyD88、IRAK4和IRAK1形成复合体[7]。 MyD88非依赖的信号途径存在于TLR3和TLR4下游，是由TRIF介导并通过RIPK1、TRAF6或TRAF3激活类似的下游信号[4]。
此外，TLRs与其他PRRs（如C型凝集素受体、NOD样受体和RIG-I样受体）的串话，使多种信号转导整合为一个网络，在感染和免疫中发挥协同作用[8]。TLRs还与PRRs以外的一些信号通路相互作用，例如，与低氧依赖通路共同参与肿瘤微环境中代谢重新编程的调控[9]，与NRF2通路[10]和PPARs[11]共同参与炎症调节。

ABclonal针对Toll样受体信号通路研究，提供以下常见核心靶点的产品

TLR4 Rabbit pAb

Western blot analysis of various lysates, using TLR4 Rabbit pAb (A5258) at 1:2000 dilution.
Secondary antibody: HRP 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.

Western blot analysis of lysates from Mouse liver, using TLR4 Rabbit pAb (A5258) at 1:700 dilution.
Secondary antibody: HRP Goat Anti-Rabbit IgG (H+L) (AS014) at 1:10000 dilution.
Lysates/proteins: 25ug per lane.
Blocking buffer: 3% nonfat dry milk in TBST.
Detection: ECL Enhanced Kit (RM00021).
Exposure time: 60s.

Immunofluorescence analysis of RAW264.7 cells using TLR4 Rabbit pAb (A5258) at dilution of 1:200 (40x lens).Secondary antibody: Cy3 Goat Anti-Rabbit IgG (H+L) (AS007) at 1:500 dilution. Blue: DAPI for nuclear staining.

Immunofluorescence analysis of HepG2 cells using TLR4 Rabbit pAb (A5258) at dilution of 1:200 (40x lens).Secondary antibody: Cy3 Goat Anti-Rabbit IgG (H+L) (AS007) at 1:500 dilution. Blue: DAPI for nuclear staining.

Immunofluorescence analysis of THP-1 cells using TLR4 Rabbit pAb (A5258) at dilution of 1:200 (40x lens).Secondary antibody: Cy3 Goat Anti-Rabbit IgG (H+L) (AS007) at 1:500 dilution. Blue: DAPI for nuclear staining.

Phospho-c-Jun-S73 Rabbit pAb

Western blot analysis of lysates from PC14 cells using Phospho-c-Jun-S73 Rabbit pAb (AP0119).
Secondary antibody: HRP Goat Anti-Rabbit IgG (H+L) (AS014) at 1:10000 dilution.
Lysates/proteins: 25μg per lane.
Blocking buffer: 3% BSA.

Immunohistochemistry analysis of paraffin-embedded Rat brain using Phospho-c-Jun-S73 Rabbit pAb (AP0119) at dilution of 1:100 (40x lens). Perform high pressure antigen retrieval with 10 mM citrate buffer pH 6.0 before commencing with IHC staining protocol.

Immunohistochemistry analysis of paraffin-embedded Human colon carcinoma using Phospho-c-Jun-S73 Rabbit pAb (AP0119) at dilution of 1:100 (40x lens). Perform high pressure antigen retrieval with 10 mM citrate buffer pH 6.0 before commencing with IHC staining protocol.

Immunohistochemistry analysis of paraffin-embedded Mouse kidney using Phospho-c-Jun-S73 Rabbit pAb (AP0119) at dilution of 1:100 (40x lens). Perform high pressure antigen retrieval with 10 mM citrate buffer pH 6.0 before commencing with IHC staining protocol.

IRAK4 Rabbit pAb

Western blot analysis of lysates from Rat lung, using IRAK4 Rabbit pAb (A6208) 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: 60s.

Immunohistochemistry analysis of paraffin-embedded Rat brain using IRAK4 Rabbit pAb (A6208) at dilution of 1:50 (40x lens). High pressure antigen retrieval performed with 0.01M Citrate Bufferr (pH 6.0) prior to IHC staining.

Immunohistochemistry analysis of paraffin-embedded Rat heart using IRAK4 Rabbit pAb (A6208) at dilution of 1:50 (40x lens). High pressure antigen retrieval performed with 0.01M Citrate Bufferr (pH 6.0) prior to IHC staining.

Immunohistochemistry analysis of paraffin-embedded Human lung cancer using IRAK4 Rabbit pAb (A6208) at dilution of 1:50 (40x lens). High pressure antigen retrieval performed with 0.01M Citrate Bufferr (pH 6.0) prior to IHC staining.

Immunohistochemistry analysis of paraffin-embedded Human hepatobiliary duct using IRAK4 Rabbit pAb (A6208) at dilution of 1:50 (40x lens). High pressure antigen retrieval performed with 0.01M Citrate Bufferr (pH 6.0) prior to IHC staining.

Immunofluorescence analysis of NIH/3T3 cells using IRAK4 Rabbit pAb (A6208) at dilution of 1:100 (40x lens). Secondary antibody: Cy3-conjugated Goat anti-Rabbit IgG (H+L) (AS007) at 1:500 dilution. Blue: DAPI for nuclear staining.

IRF3 Rabbit pAb

Western blot analysis of lysates from 293T cells using IRF3 Rabbit pAb (A11118) at 1:1000 dilution.
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: 1s.

Immunohistochemistry analysis of paraffin-embedded Human lung cancer using IRF3 Rabbit pAb (A11118) 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 esophageal cancer using IRF3 Rabbit pAb (A11118) at dilution of 1:100 (40x lens). Microwave antigen retrieval performed with 0.01M PBS Buffer (pH 7.2) prior to IHC staining.

Immunofluorescence analysis of HeLa cells using IRF3 Rabbit pAb (A11118) at dilution of 1:100 (40x lens). Secondary antibody: ABflo® 488-conjugated Goat Anti-Rabbit IgG (H+L) (AS073) at 1:500 dilution. Blue: DAPI for nuclear staining.

Immunoprecipitation analysis of 300 μg extracts of 293T cells using 3 μg IRF3 antibody (A11118). Western blot was performed from the immunoprecipitate using IRF3 (A11118) at a dilution of 1:1000.

Chromatin immunoprecipitation analysis of extracts of HCT116 cell line, using IRF3 rabbit polyclonal antibody (A11118) and rabbit IgG. The amount of immunoprecipitated DNA was checked by quantitative PCR. Histogram was constructed by the ratios of the immunoprecipitated DNA to the input.

TBK1/NAK Rabbit mAb

Western blot analysis of various lysates using TBK1/NAK Rabbit mAb (A3458) 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.

Western blot analysis of various lysates using TBK1/NAK Rabbit mAb (A3458) 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.

Confocal imaging of MCF7 cells using TBK1/NAK Rabbit mAb (A3458,at dilution of 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.

Confocal imaging of NIH/3T3 cells using TBK1/NAK Rabbit mAb (A3458,at dilution of 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.

Immunoprecipitation analysis of 300 μg extracts from 293T cells using 3 μg TBK1/NAK Rabbit mAb (A3458). Western blot was performed from the immunoprecipitate using TBK1/NAK Rabbit mAb (A3458) at a dilution of 1:1000.

Phospho-TBK1/NAK-S172 Rabbit mAb

Western blot analysis of various lysates using Phospho-TBK1/NAK-S172 Rabbit mAb (AP1026) at 1:1000 dilution. Both HeLa cells and NIH/3T3 cells were treated by 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: 25μg per lane.
Blocking buffer: 3% BSA.
Detection: ECL Basic Kit (RM00020).
Exposure time: 1min.

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.

Phospho-JNK1/2/3-T183/T183/T221 Rabbit mAb

Western blot analysis of lysates from HeLa cells, using Phospho-JNK1/2/3-T183/T183/T221 Rabbit mAb (AP0631) at 1:3000 dilution. HeLa cells were treated by Anisomycin (25 μg/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: 25μg per lane.
Blocking buffer: 3% nonfat dry milk in TBST.
Detection: ECL Basic Kit (RM00020).
Exposure time: 180s.

Western blot analysis of lysates from 293T cells, using Phospho-JNK1/2/3-T183/T183/T221 Rabbit mAb (A23099) at 1:1000 dilution. 293T cells were treated by Anisomycin (25 μg/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: 25μg per lane.
Blocking buffer: 3% nonfat dry milk in TBST.
Detection: ECL Basic Kit (RM00020).
Exposure time: 90s.

Western blot analysis of lysates from 293T cells, using Phospho-JNK1/2/3-T183/T183/T221 Rabbit mAb (A23099) at 1:1000 dilution. 293T cells were treated by UV at room temperature for 15-30 minutes.
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: 90s.

Western blot analysis of lysates from C6 cells, using Phospho-JNK1/2/3-T183/T183/T221 Rabbit mAb (A23099) at 1:1000 dilution. C6 cells were treated by UV at room temperature for 15-30 minutes.
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: 90s.

Western blot analysis of lysates from C6 cells, using Phospho-JNK1/2/3-T183/T183/T221 Rabbit mAb (A23099) at 1:1000 dilution. C6 cells were treated by Anisomycin (25 μg/mL) at 37℃ for 20 minutes.
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.

Immunohistochemistry analysis of paraffin-embedded Human colon carcinoma using Phospho-JNK1/2/3-T183/T183/T221 Rabbit mAb (AP0631) at dilution of 1:200 (40x lens). High pressure antigen retrieval performed with 0.01M Citrate Bufferr (pH 6.0) prior to IHC staining.

Immunohistochemistry analysis of paraffin-embedded Human colon using Phospho-JNK1/2/3-T183/T183/T221 Rabbit mAb (AP0631) at dilution of 1:200 (40x lens). High pressure antigen retrieval performed with 0.01M Citrate Bufferr (pH 6.0) prior to IHC staining.

Immunohistochemistry analysis of paraffin-embedded Human lung squamous carcinoma tissue using Phospho-JNK1/2/3-T183/T183/T221 Rabbit mAb (AP0631) at dilution of 1:200 (40x lens). High pressure antigen retrieval performed with 0.01M Citrate Bufferr (pH 6.0) prior to IHC staining.

Immunohistochemistry analysis of paraffin-embedded Human tonsil using Phospho-JNK1/2/3-T183/T183/T221 Rabbit mAb (AP0631) at dilution of 1:200 (40x lens). High pressure antigen retrieval performed with 0.01M Citrate Bufferr (pH 6.0) prior to IHC staining.

Immunohistochemistry analysis of paraffin-embedded Mouse colon using Phospho-JNK1/2/3-T183/T183/T221 Rabbit mAb (AP0631) at dilution of 1:200 (40x lens). High pressure antigen retrieval performed with 0.01M Citrate Bufferr (pH 6.0) prior to IHC staining.

Immunohistochemistry analysis of paraffin-embedded Rat kidney using Phospho-JNK1/2/3-T183/T183/T221 Rabbit mAb (AP0631) at dilution of 1:200 (40x lens). High pressure antigen retrieval performed with 0.01M Citrate Bufferr (pH 6.0) prior to IHC staining.

Immunohistochemistry analysis of paraffin-embedded Rat liver using Phospho-JNK1/2/3-T183/T183/T221 Rabbit mAb (AP0631) at dilution of 1:200 (40x lens). High pressure antigen retrieval performed with 0.01M Citrate Bufferr (pH 6.0) prior to IHC staining.

Immunofluorescence analysis of NIH-3T3 cells using Phospho-JNK1/2/3-T183/T183/T221 Rabbit mAb (AP0631).NIH-3T3 cells were treated by Anisomycin (25 μg/mL) at 37℃ for 30 minutes after serum-starvation overnight. Secondary antibody: Cy3-conjugated Goat anti-Rabbit IgG (H+L) (AS007) at 1:500 dilution. Blue: DAPI for nuclear staining.