Cell-Based assays for c-Jun phosphorylation

The cell-based kinase assays for c-Jun phosphorylation were carried out by using the LanthaScreen c-Jun (1 ~ 79) HeLa cell lines which stably expressed GFP-c-Jun 1 ~ 79 and GFP-ATF2 19 ~ 106, respectively. Phosphorylation was determined by measuring the TR-FRET between a terbium-labeled phospho-c-Jun specific antibody and GFP. The cells were plated in white tissue culture treated 384 well plates at a density of 10,000 cells per well in 32 μL assay medium (Opti-MEM, supplemented with 0.5% charcoal/dextran-treated FBS, 100 U/mL Penicillin, 100 μg/mL Streptomycin, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 25 mM HEPES [pH 7.3] and lacking phenol red). After overnight incubation, cells were pretreated for 90 mins with JNK-IN-7 (at indicated concentration) diluted in 4 μL assay buffer followed by 30 mins of stimulation with 5 ng/mL of TNF-α in 4 μL assay buffer (final assay volume was 40 μL). The medium was then removed by aspiration and the cells were lysed by adding 20 μL of lysis buffer (20 mM Tris-HCl [pH 7.6], 5 mM EDTA, 1% Nonidet P-40 substitute, 5 mM NaF, 150 mM NaCl and 1:100 protease and phosphatase inhibitor mix, P8340 and P2850, respectively). The lysis buffer included 2 nM of the terbium-labeled anti-c-Jun (pSer73) detection antibodies. After allowing the assay to equilibrate for 60 mins at room temperature, TR-FRET emission ratios were determined on a BMG Pherastar fluorescence plate reader using the following parameters: excitation at 340 nm, emission 520 and 490 nm; 100 ms lag time; 200 μs integration time; emission ratio = Em 520/Em 490. All data were analyzed and plotted using GraphPad Prism 4.

Cell lines

Human IL-1R cells and RAW264.7 macrophages

Preparation method

Soluble in DMSO. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reaction Conditions

0.1, 1 and 10 mM; 1 hr

Applications

In human IL-1R cells, JNK-IN-7 inhibited IL-1β-stimulated phosphorylation of c-Jun and the activation of Pellino 1. In Pam3CSK4-stimulated RAW macrophages, JNK-IN-7 also inhibited the phosphorylation of c-Jun.

References:

[1]. Zhang T, Inesta-Vaquera F, Niepel M, et al. Discovery of potent and selective covalent inhibitors of JNK. Chem Biol, 2012, 19(1): 140-154.

[2]. Goh ET, Arthur JS, Cheung PC, et al. Identification of the protein kinases that activate the E3 ubiquitin ligase Pellino 1 in the innate immune system. Biochem J, 2012, 441(1): 339-346.