Fig. 4: Pro-BA facilitates EML4-ALK degradation through the ubiquitin-proteasome system.

A H3122 cells were treated with Pro-BA alone or in combination with MG132 (10 µM) or CQ (25 µM), for 6 h. The protein levels of EML4-ALK and GAPDH were subsequently assessed by immunoblotting (upper panel). The bar graph presents a quantitative analysis of EML4-ALK protein levels derived from three independent replicates shown in Fig. 4A and Supplementary Fig. 4A (lower panel). B H3122 cells were treated with cycloheximide (CHX) alone, or in combination with Pro-BA, or with both Pro-BA (100 nM) and MG132 (10 µM) for different time intervals, followed by immunoblotting to measure EML4-ALK and GAPDH expression (upper panel). Quantitative analysis of the Western blot results for EML4-ALK from three separate repeats is presented in Fig. 4B and Supplementary Fig. 4B (lower panel). C Immunoblot analysis was conducted on H3122 cells pre-treated with BA (10 μM) for 2 h and then incubated with Pro-BA (100 nM) for 12 h to assess the expression of specific proteins (upper panel). The bar graph presents a quantitative analysis of EML4-ALK protein levels derived from three independent experiments shown in Fig. 4C and Supplementary Fig. 4C (lower panel). D H3122 cells with stable expression of the indicated sgRNA were exposed to either DMSO or Pro-BA for 24 h, and the levels of the indicated protein were assessed by immunoblotting. E HEK293T cells co-expressing HA-GID4 and Flag-EML4-ALK were treated with DMSO, Pro-BA (500 nM), or Brigatinib (500 nM) for 24 h. The cells were then subjected to immunoprecipitation using anti-FLAG® M2 Magnetic Beads, followed by immunoblotting with the specified antibodies. F HEK293T cells coexpressing HA-GID4, Flag-EML4-ALK, and myc-Ub were incubated with DMSO, Pro-BA (500 nM), or Brigatinib (500 nM) for 24 h, then treated with MG132 (10 μM) for 4 h. Ubiquitylation of Flag-EML4-ALK was analyzed by denaturing immunoprecipitation (IP) with an anti-myc-tag antibody. G ITC measurement of the affinity of Pro-BA (left) and Pro-PEG3-BA (right) with ALK (1094-1400 aa). H HEK293T cells co-transfected with pHTN-GID4 and pNLF1-N-ALK were exposed to Brigatinib, Pro-BA, or Pro-PEG3-BA at the indicated concentration for 6 h. Data represented as normalized NanoBRET ratio. Data are presented as the mean ± SD (n = 3 independent experiments). Statistical analysis was performed using one-way ANOVA followed by Fisher’s LSD test (two-tailed) for pairwise comparisons. I HEK293T cells coexpressing HA-GID4 and Flag-EML4-ALK were incubated with DMSO, Pro-BA (500 nM), Pro-PEG3-BA (500 nM), or Brigatinib (500 nM) for 24 h, respectively. After immunoprecipitation with anti-FLAG® M2 Magnetic Beads, followed by immunoblotting with various antibodies indicated. J HEK293T cells coexpressing HA-GID4, Flag-EML4-ALK, and myc-Ub were treated with DMSO, Pro-BA (500 nM), Pro-PEG-BA (500 nM), or Brigatinib (500 nM) for 24 h, followed by the addition of MG132 (10 μM) for 4 h. Ubiquitination of Flag-EML4-ALK was examined by denaturing immunoprecipitation (IP) with anti-myc-tag antibody. For (A–C), data are shown as the mean ± SD (n = 3 independent experiments), two-tailed Student’s t-test. Source data are provided as a Source Data file.