Fig. 4.

MAPK15 induces PKC-dependent NRF2 Serine 40 phosphorylation and protein stabilization. (A) 293T cells were transiently co-transfected with NRF2-MYC-FLAG plus empty vector or MAPK15_WT. After 24 h, they were treated with 50 μM Cycloheximide (CHX), for indicated times. Lysates were analyzed by WB for the indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. (B) 293T cells were transiently co-overexpressed with NRF2-MYC-FLAG plus empty vector or MAPK15_WT or MAPK15_KD. After 24 h, samples were treated with 300 μM H₂O₂, for 1 h, and the lysates were subjected to SDS-PAGE followed by WB and analyzed for indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. (C) 293T cells were transiently co-transfected with NRF2-MYC-FLAG or NRF2-MYC-FLAG S40A, plus empty vector or MAPK15_WT. After 24 h. Lysates were analyzed by WB for the indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. (D) 293T cells were transfected with siSCR or siMAPK15. Then, after 24 h, they were transfected with NRF2-MYC-FLAG. After additional 48 h, cells were treated with 300 μM H₂O₂ for 1 h and lysates were analyzed by WB for indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. (E) 293T cells were transfected with siSCR or siMAPK15 and, after 24 h, transfected with empty vector or NRF2-MYC-FLAG. After a total of 72h, samples were treated with 300 μM H₂O₂, for 1 h, then fixed and subjected to immunofluorescence analysis. Scale bars correspond to 10 μm. The accompanied graph shows intensitometric analysis of phospho-NRF2 S40A fluorescence from five representative microscopy fields signal per cell ± SD. (F) 293T cells were transiently transfected with empty vector or MAPK15_WT or treated with 300 μM H₂O₂ or 200 nM TPA for 1 h, as positive controls. Lysates were analyzed by WB for the indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. (G) 293T cells were transiently transfected with NRF2-MYC-FLAG or NRF2-MYC-FLAG_S40A plus empty vector or HA-MAPK15_WT. After 24 h, samples were treated, as indicated, with 10 μM GO6983 for 2 h. Lysates were next immunoprecipitated with anti-MYC antibodies and subjected to SDS-PAGE followed by WB to detect phospho-NRF2-S40 protein. Total lysates were also analyzed for expression of indicated proteins. One experiment, representative of 3 independent experiments, is shown. (H) 293T cells were transfected with ARE luciferase reporter vector plus empty vector or MAPK15_WT. Twenty-four hours after transfection, samples were treated with 5 μM or 10 μM GO6983, for 2 h. Then, samples were lysed, and luciferase activity was measured in cell extracts. Data are represented as fold induction of the normalized luciferase activity compared to control cells transfected with GFP. All luciferase results represent the average ± S.D. of three independent experiments. All samples were read in triplicate.