Cell Signaling Technology
XP Monoclonal Antibody

Product Pathways - NF-kB Signaling

Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb #5483

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP IF-IC F H (M) (R) (Mk) (X) (B) (Dg) Endogenous 84 Rabbit IgG

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  X=Xenopus  B=Bovine  Dg=Dog
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

Phospho-TBK1 (Ser172) (D52C2) XP® Rabbit mAb detects endogenous levels of TBK1 only when phosphorylated at Ser172. This antibody may cross-react with phospho-IKKε.

Source / Purification

Monoclonal antibody is prepared from animals immunized with a synthetic phosphopeptide corresponding to residues surrounding Ser172 of human TBK1.

Western Blotting

Western Blotting

Western blot analysis of extracts from THP-1 cells differentiated with TPA #4174 (80 nM, overnight) followed by treatment with LPS (1 μg/ml), up to 24h, using Phospho-TBK1 (Ser172) (D52C2) XP® Rabbit mAb (upper), or total TBK1/NAK (D1B4) Rabbit mAb #3504 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from THP-1 cells differentiated with TPA #4174 (80 nM, overnight) followed by treatment with LPS (1 μg/ml, 1 hour), with or without phosphatase treatment using Phospho-TBK1 (Ser172) (D52C2) XP® Rabbit mAb (upper), or total TBK1/NAK (D1B4) Rabbit mAb #3504 (lower).

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of THP-1 cells differentiated with TPA #9905, untreated (blue) or LPS-treated (green), using Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb.


IF-IC

IF-IC

Confocal immunofluorescent analysis of THP-1 cells differentiated with TPA #4174 (80nM, overnight) (left), followed by treatment with LPS (1μg/ml, 1 hour) (center) or LPS with λ phosphatase treatment (right) using Phospho-TBK1/NAK (Ser172) (D52C2) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 Phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Background

TBK1 (TANK-binding kinase 1)/NAK (NF-κB activating kinase) is an IκB kinase (IKK)-activating kinase and can activate IKK through direct phosphorylation (1). TBK1 was identified through association with the TRAF binding protein, TANK, and found to function upstream of NIK and IKK in the activation of NF-κB (2). TBK1/NAK induces IκB degradation and NF-κB activity through IKKβ. NAK may mediate IKK and NF-κB activation in response to growth factors that stimulate PKCε activity (1). TBK1 plays a pivotal role in the activation of IRF3 in the innate immune response (3).

TBK1 is phosphorylated at Ser172 within its activation loop, which is necessary of its kinase activity including the downstream phosphorylation of IRF3 (4,5).

  1. Tojima, Y. et al. (2000) Nature 404, 778-82.
  2. Pomerantz, J.L. and Baltimore, D. (1999) EMBO J 18, 6694-704.
  3. Fitzgerald, K.A. et al. (2003) Nat Immunol 4, 491-6.
  4. Kishore, N. et al. (2002) J Biol Chem 277, 13840-7.
  5. McCoy, C.E. et al. (2008) J Biol Chem 283, 14277-85.

Application References

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For Research Use Only. Not For Use In Diagnostic Procedures.

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