Fig. 1: ABL kinase activities are potentiated following resistance to BRAF and BRAF/MEK inhibitors. | Nature Communications

Fig. 1: ABL kinase activities are potentiated following resistance to BRAF and BRAF/MEK inhibitors.

From: Combating acquired resistance to MAPK inhibitors in melanoma by targeting Abl1/2-mediated reactivation of MEK/ERK/MYC signaling

Fig. 1

a Parental (M14, Mel1617, 451-Lu), BRAFi (-BR), or BRAFi/MEKi-resistant (-BMR) cell lines were serum-starved and subjected to in vitro kinase assay (top two panels) or western blot (rest of the panels) for total protein expression. For kinase assays, ABL1 was immunoprecipitated with K12 antibody, whereas ABL2 was immunoprecipitated with ABL2-specific antibody19,62, and IPs incubated with substrate (GST-CRK) and radiolabeled (gamma-32P)-ATP (see “Methods”). Kinase quantitation for n = 3–5 biological replicates is shown in Supplementary Fig. 1d. b Resistant cell lines (a) were fractionated into nuclear (nuc) and cytoplasmic (cyto) fractions, equal percentages of cytoplasmic/nuclear fractions loaded for each line, and fractions blotted with the indicated antibodies. Lamin and tubulin blots were used to demonstrate the purity of nuclear and cytoplasmic fractions, respectively. ABL1 cytoplasmic:nuclear ratio is noted for each cell line, and quantitation for all lines is shown in Supplementary Fig. 1e. A representative of n = 2 independent experiments is shown. c Lysates from a were blotted with the indicated antibodies and quantified. Graph shown is mean ± SEM for three independent experiments. **p = 0.006 using single sample t-tests (two-sided) and Holm’s adjustment for multiple comparisons. d Primary human paired melanoma datasets (RNAseq-77940, 50535, 65185, EGAS00001000992; microarray-50509) were analyzed for ABL1 mRNA levels pre-/post-BRAFi, MEKi, or BRAFi/MEKi treatment. Numbers in each column indicate number of cases. All cases were included; repeated analyses using particular cut-off values are shown in Supplementary Fig. 1f). p = 0.008 for the combined set using a two-sided binomial (right). e Parental and resistant cells were serum-starved, treated with vehicle (DMSO) or SFK inhibitor, SU6656 (10 μM), for 16 h and lysates were subjected to in vitro kinase assay (top two panels) and western blot (all other panels) as in a.

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