Fig. 3: ERK1/2 promote CDK12 activation in BRAF-mutated melanoma cells.

a In vitro kinase assay based on endogenous CDK12 immunoprecipitated from HEK293 cells. Cells were serum-starved overnight, and treated with BVD-523 (2 µM) or PD184352 (10 µM) for 30 min before PMA stimulation (100 ng/ml) for another 30 min. Total cell lysates were immunoblotted with specific antibodies. For kinase reactions, recombinant GST-RPB1 was detected by Coomassie staining and samples were immunoblotted with specific RPB1 phospho-Ser2/Ser5 antibodies. CDK12 kinase activity was quantified using Ser2 phosphorylation as output. b Proliferation assay was performed using melanocytes and BRAF-mutated melanoma cell lines with increasing doses of THZ531 for 72 h. Respectively, the IC₅₀ for melanocytes, >1 µM; A375, 222 nM; Colo829, 183 nM; WM164, 63 nM; WM983A, WM983B and SK-MEL-5, 55 nM. c Same as a, except that endogenous CDK12 was immunoprecipitated from A375 cells. d A375 cells were serum-starved overnight, and treated with BVD-523 (2 µM), PD184352 (10 µM) or THZ531 (500 nM), for 6 h. e A375 cells stably expressing wild-type CDK12 (WT) were serum-starved overnight, and treated with BVD-523 (2 µM) or PD184352 (10 µM), for 1 h. Immunoprecipitated Myc-CDK12 was assayed for phosphorylation by immunoblotting with CDK12 phosphospecific antibody targeted against Thr548. f Same as a, except that CDK12 was immunoprecipitated from A375 cells stably expressing wild-type CDK12 (WT) or the CDK12 T548A mutant. Data are represented as mean ± SD of independent experiments, n = 3 (a, c), n = 6 (f) and independent replicates n = 6 (b). d Representative data of independents n  =  3. (a, c, f) Significance was determined using unpaired two-tailed Student’s t-tests. Source data are provided as a Source Data file.