Fig. 7: MKK7 is a direct c-Myc-S62 kinase.

a Representative immunoblots of the indicated proteins in total lysates of RIPK2/MKK7-DKO HEK293T cells, which were transiently co-transfected with 0.5 µg MYC, 0.5 µg RIPK2m4, and 0.1 µg MAP2K7 or vector plasmids and then treated with vehicle control, MKK7-COV-3 (25 µM), or JNK-IN-8 (1 µM) in 10% FBS-containing medium for 2 h. b Representative images (upper) and quantification (lower) of PLA detecting the association of endogenous MKK7 and c-Myc in PC3 cells (n = 7 biologically independent samples per group). Scale bars, 10 μm. c Representative images of immunofluorescence colocalization of MKK7 and c-Myc in RIPK2/MKK7-DKO 22Rv1 cells, which were transiently co-transfected with RIPK2m4, MKK7-His, and MYC plasmids. Scale bars, 10 μm. d Representative immunoblots of the indicated proteins after in vitro kinase reactions. e Representative immunoblots of the indicated proteins in total lysates of RIPK2/MKK7-DKO HEK293T cells, which were transiently transfected with the indicated forms of MKK7 plasmids or vector control. f Representative immunoblots of the indicated proteins in total lysates, cytoplasmic fraction, and nuclear fraction of RIPK2/MKK7-DKO HEK293T cells, which were transiently co-transfected with RIPK2m4, MYC, and MKK7 or vector control. WCL: whole-cell lysate; Cyto: cytoplasmic; Nuc: nuclear. g Representative images (upper) and quantification (lower) of PLA detecting the MKK7-c-Myc complex in RIPK2-KO HEK293T cells, which were transiently transfected with MYC and RIPK2m4 or vector control (n = 5 biologically independent samples per group). Scale bars, 20 μm. Nominal p-values were determined by unpaired two-tailed Student’s t-test (b, g). Data are Mean ± SEM (b, g). Experiments were repeated three times (a, d) or twice (e, f) independently with similar results.