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The PH domain of TBC1D23 directly interacts with LKB1 a Schematic diagram of full-length (FL) and various truncations of TBC1D23, and a summary of LKB1 binding determined by GST pull-down. b, c HEK293T cells were transfected with indicated constructs for 24 h. Cell lysates were subjected to precipitation with GST beads, and the bound proteins were immunoblotted with indicated antibodies. The graph showed ratios of bound Flag to GST quantified by densitometry using Image J software. d HEK293T cells were co-transfected with mCherry-TBC1D23 WT or 3 K and Flag-LKB1 for 24 h, and control cells were co-transfected with mCherry-TBC1D23 WT or 3 K and an empty vector. Cells were harvested and subjected to immunoprecipitation with anti-Flag beads followed by immunoblotting with indicated antibodies. The ratios of bound mCherry to Flag derived from a densitometric analysis of the blot were shown. e A schematic diagram of full-length (FL) and truncated mutants of LKB1, and a summary of TBC1D23 binding determined by immunoprecipitation. f HEK293T cells co-transfected with LKB1 FL or its truncations (vector as control) and GST-TBC1D23 PH were lysed and incubated with anti-Flag beads, the immunoprecipitates were then examined by immunoblotting. The graph showed ratios of bound GST to Flag quantified by densitometry using Image J software. g GST-FAM21-R11, GST-LKB1-LFa WT, mutants, or GST pull-down of purified TBC1D23 PH domain. h HEK293T cells were transfected with indicated constructs. 24 h later, cell lysates were precipitated with GST beads and immunoblotted with indicated antibodies. The graph showed ratios of bound GFP to GST quantified by densitometry using Image J software. i Isothermal titration calorimetry of an LKB1-LFa motif peptide (GADEDEDLFDIEDDIIYTQ) titrated into TBC1D23 PH domain. Top and bottom panels show raw and integrated heat from injections, respectively. Experiments in (a–i) were performed in triplicate. Results are presented as mean ± SD. P values were determined by one-way ANOVA, followed by followed by Dunnett’s test (b, f, h), or by unpaired two-tailed t test (c, d). Source data are provided as a Source data file.
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