Fig. 3.

Chemical inhibition of the LRS–RagD interaction. a Effect of alanine mutations at the indicated residues located in the aa 951–981 peptide region of LRS VC domain on the interaction of LRS and RagD was determined by GST pull-down assays. Purified GST or GST-LRS (aa 951–1176) proteins were incubated with Myc-RagD^WT-transfected SW620 cell lysates, precipitated with glutathione sepharose beads and the precipitation of Myc-RagD was analyzed by immunoblotting with anti-Myc antibody. b Effect of BC-LI-0186 on the interaction of LRS WT with RagD was determined by in vitro pull-down of GST-LRS (957–1176 aa) and Myc-RagD. c Relative band intensity of Myc-RagD in b was quantified and displayed as line graph. The inset represents the IC₅₀ value of BC-LI-0186. d Effect of BC-LI-0186 and BC-LI-0198 on the endogenous interaction of LRS with RagD. Cells were treated with 10 μM BC-LI-0186 for 1 h and cell lysates were subjected to immunoprecipitation with anti-LRS, anti-RagD, or anti-mTOR antibodies. Co-immunoprecipitation was confirmed by immunoblotting with the indicated antibodies. e Effect of BC-LI-0186 on the interaction of LRS WT and the indicated mutants with Myc-RagD was determined by co-immunoprecipitation. f SW620 cells were co-transfected with Myc-tagged LRS WT, S974A, or S953A mutant and HA-tagged RagD WT. Cells were treated with 10 μM BC-LI-0186 for 1 h and cell lysates were subjected to immunoprecipitation with anti-Myc antibody. Cellular levels of the indicated proteins were analyzed by immunoblotting with their specific antibodies