Phospho-MKK4 (Ser257) cellular kit HTRF®

The Phospho-MKK4 (Ser257) assay measures MKK4 when phosphorylated at Ser257. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses 2 antibodies, one labeled with a donor fluorophore and the other with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein's phosphorylation state under a no-wash assay format.

Human SH-SY5Y cells were plated at 25,000 cells/well in a 96-well plate. After 24 h incubation at 37 °C, 5% CO2, the cells were incubated in differentiation medium containing 10 µM of RA for 1 week in the dark. Note that due to the poor stability of RA, cell culture medium containing fresh RA was renewed daily. Once differentiated, SH-SY5Y were stimulated with increasing concentrations of anisomycin for 45 min. Then the medium was removed and 50 µL of supplemented lysis buffer 1X were added. After 30min lysis at RT under gentle shaking, 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho-MKK4 (Ser257) or total MKK4 detection reagents were added. The HTRF signal was recorded after an overnight incubation.

As described elsewhere, a dose dependent phosphorylation of MKK4 by Ser257 was induced by anisomycin, whereas total MKK4 slightly decreased under the same experimental conditions.

Human SH-SY5Y cells were plated at 400,000 cells/well in a 6-well plate. After 24 h incubation at 37 °C, 5% CO2, the cells were incubated in differentiation medium containing 10 µM of RA for 1 week in the dark. Note that due to the poor stability of RA, cell culture medium containing fresh RA was renewed daily. Once differentiated, SH-SY5Y were stimulated with 0.5 mM of H2O2 for 1h. Then the medium was removed and 500 µL of supplemented lysis buffer 1X were added. After 30 min lysis at RT under gentle shaking, 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho-MKK4 (Ser257) or total MKK4 detection reagents were added. The HTRF signal was recorded after an overnight incubation.

H2O2 induced phosphorylation MKK4 on Ser257 residue whereas the MKK4 expression level remained almost stable under the same experimental conditions.

Human HepG2 cells were plated at 100,000 cells/well in a 96-well plate. After an incubation of 24 h at 37 °C, 5% CO2, the cells were stimulated with increasing concentrations of anisomycin for 45min. Then the medium was removed and 50 µL of supplemented lysis buffer 1X were added. After 30 min lysis at RT under gentle shaking, 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho-MKK4 (Ser257) or total MKK4 detection reagents were added. The HTRF signal was recorded after an overnight incubation. The same amount of lysate was analyzed by Western Blot in a side by side experiment.

As shown on the graphs, both HTRF and Western Blot indicated an increase of MKK4 phosphorylation associated with a decrease of MKK4 expression.

The human HepG2 cell line was seeded in a T175 flask and incubated a 37 °C, 5% CO2. The cells were then stimulated with Sorbitol (1 M) for 30 min before lysis.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF phospho-MKK4 (Ser257) detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

Using the HTRF phospho-MKK4 (Ser257) assay, 310 cells/well were enough to detect a signal, while 5,000 cells were needed using Western Blot which relies on a Chemiluminescence signal. These results indicate that the HTRF phospho-MKK4 (Ser257) assay is 16 times more sensitive than the Western Blot.

MKK4 (Mitogen-activated Kinase Kinase 4) is a member of MAP kinase kinase family that is activated by phosphorylation on Ser257 following activation of different MKKKs, for example TAK1 or ASK1, in response to stimuli such as GPCR activation, Growth factors, cellular stresses, or inflammatory cytokines. In turn, activated MKK4 phosphorylates JNK or p38 in order to activate c-jun, p53, or ATF2 and induce inflammation, cell survival/apoptosis, proliferation, or differentiation by regulating gene transcriptions.