Fig. 5 | Signal Transduction and Targeted Therapy

Fig. 5

From: RBM4 dictates ESCC cell fate switch from cellular senescence to glutamine-addiction survival through inhibiting LKB1-AMPK-axis

Fig. 5The alternative text for this image may have been generated using AI.

RBM4 recruits E3 ligase TRIM26 to promote LKB1 degradation in nucleus. a KYSE150 cells with RBM4 stably knockdown were treated with 50 μg/mL cycloheximide (CHX) for the indicated time course. The protein levels of LKB1 and RBM4 were detected by a western blot assay. b Immunoprecipitation assay was performed in RBM4-depleted KYSE150 cells co-expressing Flag-LKB1 following PS341 (10 μM) treatment for 8 h to examine the ubiquitination of LKB1. The protein complexes were precipitated by anti-Flag M2 resin and analyzed with the indicated antibodies. Asterisks indicate non-specific bands. c Immunoprecipitation assay was carried out in cells expressing Flag-LKB1 and GFP-RBM4, with different HA-Ub vectors containing distinct lysine mutations in the presence of PS341 (10 μM). The protein complexes were precipitated by anti-Flag M2 resin and analyzed by a western blot assay. d KYSE150 cells co-transfected with the indicated vectors expressing Flag-LKB1, GFP-RBM4 or HA-Ub in the presence of PS341 (10 μM) were subjected to nucleocytoplasmic fractionation followed by a co-immunoprecipitation assay with anti-Flag M2 resin to analyze the ubiquitination of LKB1 in the cytoplasm and nucleus respectively. SE, short exposure; LE, long exposure. e The interaction between LKB1 and TRIM26 was determined by an immunoprecipitation assay with anti-Flag antibody following PS341 (10 μM) treatment for 8 h. f Immunoprecipitation assay was carried out to determine the Flag-LKB1-bound poly-ubiquitin with or without HA-TRIM26 overexpression following PS341 (10 μM) treatment for 8 h. g Immunoprecipitation assay was performed in RBM4-depleted or control cells after PS341 (10 μM) treatment for 8 h to assess the interaction between LKB1 and TRIM26. h Immunoprecipitation assay was carried out to investigate the effect of RBM4 and TRIM26 on the ubiquitination of LKB1 in the presence of PS341 (10 μM). i Confocal immunofluorescence microscopy was used to examine the localization of Flag-RBM4 or Flag-LKB1 with HA-TRIM26 in HeLa cells. Scale bar = 10 μm. j Immunoprecipitation assay was performed in KYSE150 cells expressing Flag-TRIM26 and HA-LKB1, or HA-LKB1(1-243), HA-LKB1(1-317), HA-LKB1(Δ146-186), HA-LKB1(88-317), HA-LKB1(88-433) in the presence of PS341 (10 μM). The Flag-tagged precipitated-complexes were analyzed by western blotting. Asterisks indicate non-specific bands

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