IKKepsilon antibody | knockout validation | Cell Signaling Technology 5483

This is a knockout-validated antibody summary, based on the publications "The Golgi apparatus acts as a platform for TBK1 activation after viral RNA sensing", as cited below [1] for immmunocytochemistry, "Cytosolic DNA Promotes Signal Transducer and Activator of Transcription 3 (STAT3) Phosphorylation by TANK-binding Kinase 1 (TBK1) to Restrain STAT3 Activity" [2] (see figure 3a from the article) for western blot, and "Priming Phosphorylation of TANK-Binding Kinase 1 by IκB Kinase β Is Essential in Toll-Like Receptor 3/4 Signaling" for western blot knockout validation (figure 2e) [3]. Labome curates formal publications to compile a list of antibodies with unambiguous specificity within Validated Antibody Database (VAD).

IKKepsilon antibody | knockout validation | Cell Signaling Technology 5483 figure 1
Figure 1. TBK1+/+ and TBK1–/– mouse embryonic fibroblasts detected with phosphoTBK1 antibody. From Figure 1f in [1]. According to the authors, this figure was obtained with Abcam ab109272; however, Cell Signaling Technology 5483 displayed the same results.
Antibody information

Rabbit monoclonal IgG

Company: Cell Signaling Technology

Antibody: phosphorylated IKKε/TBK1

Catalog number: 5483

Summary: The immunogen is a synthetic phosphopeptide corresponding to residues surrounding Ser172 of human TBK1. Species Reactivity: Human, Mouse.

Validation Method

Immmunocytochemistry (this article). Western blot knockout-validation in [2] (see figure 3a in the article).

Sample

Cells grown on coverslips were fixed in 4 % paraformaldehyde in phosphate buffered saline (PBS) for 10 minutes, and then permeabilized by incubation with 0.15 % Triton X-100 in PBS for 15 minutes.

Non-specific binding sites were blocked by incubating cells in a solution of 2 % BSA in PBS for 1 hour.

Primary incubation

1 hour at room temperature or overnight at 4 °C.

Detection

A Leica SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with a 63× oil immersion fluorescence objective.

References
  1. Pourcelot M, Zemirli N, Silva da Costa L, Loyant R, Garcin D, Vitour D, et al. The Golgi apparatus acts as a platform for TBK1 activation after viral RNA sensing. BMC Biol. 2016;14:69 pubmed publisher
  2. Hsia H, Hutti J, Baldwin A. Cytosolic DNA Promotes Signal Transducer and Activator of Transcription 3 (STAT3) Phosphorylation by TANK-binding Kinase 1 (TBK1) to Restrain STAT3 Activity. J Biol Chem. 2017;292:5405-5417 pubmed publisher
  3. Abe H, Satoh J, Shirasaka Y, Kogure A, Kato H, Ito S, et al. Priming Phosphorylation of TANK-Binding Kinase 1 by IκB Kinase β Is Essential in Toll-Like Receptor 3/4 Signaling. Mol Cell Biol. 2020;40: pubmed publisher