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The interaction between TBC1D23 and LKB1 is dynamically regulated by AMPK activation. a, b HEK293T cells were transfected with Flag-LKB1 (a) or GFP-LKB1 CRD (b) and GST-TBC1D23 PH, co-transfection of GST-vector and Flag-LKB1 (a) or GFP-LKB1 CRD (b) as the negative control. 24 h post-transfection, cell lysates were subjected to glucose starvation for indicated time and precipitation with GST beads, followed by immunoblotting with indicated antibodies. The graph showed ratios of bound Flag to GST (a) or GFP to GST (b) quantified by densitometry using Image J software. Values for each time point were shown relative to the ratio of control cells without glucose starvation. c, d HEK293T cells were transfected with indicated constructs. Twenty-four hours later, cells were treated with (d) or without (c) culture medium deprived of glucose for 2 h before harvest and precipitation with GST beads. Precipitates were resolved by SDS-PAGE and analyzed by western blotting. The graph showed ratios of bound Flag to GST quantified by densitometry using Image J software. e, f HEK293T cells were co-transfected with indicated constructs. Twenty-four hours later, cells were collected and subjected to pulldown with GST beads (e) or glucose-starvation for 2 h before harvest and pulldown with GST beads. Precipitates were resolved by SDS-PAGE and analyzed by western blotting with indicated antibodies. The graph showed ratios of bound GFP to GST quantified by densitometry using Image J software. n = 3 independent experiments (a–f). Results are presented as mean ± SD. P values were determined one-way ANOVA, followed by Dunnett’s test (a, b), or by unpaired two-tailed t test (c–f). Con indicates co-transfection of Flag-LKB1 (c) or GFP-CRD (e) and GST-TBC1D23 FL. AMPK O/E indicates co-transfection of Flag-AMPKα1, Flag-LKB1 (d) or GFP-CRD (f), and GST-TBC1D23 FL. g A simplified model depicts the mechanism underlying the dynamic interaction between TBC1D23 and LKB1. Energy stress stimulates the TBC1D23/LKB1 interaction, which promotes the recruitment of LKB1 to the Golgi, which in turn promotes phosphorylation and activation of AMPK (①), and activated AMPK further facilitates the interaction between TBC1D23 and LKB1 (②). Source data are provided as a Source data file.
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