Fig. 4: Galectin-3 binds LPS to promote the activation of Rag GTPases and the targeting of mTOR to lysosomal surface.

a HEK-293T cells were stimulated with LPS (1 μg/ml) for 6 h followed by immunofluorescent staining for LPS (green) and Lamp2 (red) or galectin-3 (red). Scale bar: 10 μm. Each experiment was repeated three times with similar results. b In vitro purified RagA or RagC, p14 or p18, or galectin-3 was incubated with biotinylated LPS and mixed with streptavidin beads. The bound proteins were eluted for immunoblotting analysis with antibodies against the indicated proteins. Each experiment was repeated three times with similar results. c Prediction of the binding pose of LPS to galectin-3. d HEK-293T cells were transfected with FLAG-Gal3 or FLAG-metap2, starved of amino acids and glucose for 1 h, and stimulated with LPS (1 μg/ml) for 5 h. Cellular lysates were immunoprecipitated with anti-FLAG followed by immunoblotting with antibodies against the indicated proteins. Each experiment was repeated three times with similar results. e The Rag GTPases mutants with different nucleotide states. f HEK-293T cells were co-transfected with the indicated plasmids and treated with LPS (1 μg/ml) for 6 h. Cellular lysates were immunoprecipitated with anti-FLAG followed by immunoblotting with antibodies against the indicated proteins. Each experiment was repeated three times with similar results. g HEK-293T cells were co-transfected with the indicated plasmids and RagA or RagC with different nucleotide states treated with LPS (1 μg/ml) for 6 h. Cellular lysates were immunoprecipitated with anti-FLAG followed by immunoblotting with antibodies against the indicated proteins. Each experiment was repeated three times with similar results. h HEK-293T cells were infected with lentiviruses carrying shGFP or shGal3, starved of amino acids and glucose for 1 h, and stimulated with LPS (1 μg/ml) for 5 h followed by immunofluorescent staining for mTOR (green) and Lamp2 (red). Scale bar: 10 μm. Each experiment was repeated three times with similar results. i HEK-293T cells were treated with siRNAs targeting TSC2 or TSC2 plus galectin-3, starved of amino acid and glucose for 1 h, and stimulated with LPS (1 μg/ml) for 5 h for western blotting analysis of the level or phosphorylation of the indicated proteins. Each experiment was repeated three times with similar results.