Fig. 4

RBM4 disrupts LKB1/STRAD/MO25 heterotrimeric complex by competitively binding to LKB1. a, b Protein levels of MO25, STRAD, and RBM4 were examined in KYSE150 and KYSE450 cells with stable depletion (a) or overexpression (b) of RBM4. c Immunoprecipitation assay was performed in KYSE150 cells expressing Flag-RBM4, HA-LKB1 or Flag-LKB1, HA-RBM4 respectively, and the protein complex precipitated by Flag-M2 agarose beads were analyzed through western blotting. d, e Immunoprecipitation assay was carried out in KYSE150 cells expressing a fixed amount of Flag-MO25 and increased amount of RBM4 (d), or elevated amount of siRBM4 (e, left), or a fixed amount of Flag-LKB1 with increased amount of siRBM4 (e, right) respectively after 8-h PS341 (10 μM) treatment. The protein complexes were precipitated with anti-Flag followed by western blotting analysis. f Immunoprecipitation assay was performed in KYSE150 cells expressing HA-LKB1 and Flag-RBM4, or Flag-RBM4(1-177), Flag-RBM4(78-364), Flag-RBM4(178-364) in the presence of PS341 (10 μM). The Flag-tagged precipitated-complexes were analyzed with a specific HA or Flag antibody. Asterisks indicate non-specific bands. g Immunoprecipitation assay was carried out in KYSE150 cells expressing Flag-RBM4 and HA-LKB1, or HA-LKB1(1-243), HA-LKB1(1-317), HA-LKB1(Δ146-186), HA-LKB1(88-243), HA-LKB1(88-433) in the presence of PS341 (10 μM). The Flag-tagged precipitated-complexes were analyzed using western blotting. h The levels of LKB1 and RBM4 were examined in the cytoplasm and nucleus of KYSE150 cells expressing HA-RBM4 following PS341 (10 μM) treatment for 8 h. SE, short exposure; LE, long exposure. i Confocal immunofluorescence microscopy was utilized to examine the localization of Flag-RBM4 and HA-LKB1 in Cos7 cells. Scale bar = 10 μm