Fig. 1: Galectin-3 interacts with the components of the mTORC1 signaling machinery on lysosomal surface. | Nature Communications

Fig. 1: Galectin-3 interacts with the components of the mTORC1 signaling machinery on lysosomal surface.

From: Intracellular galectin-3 is a lipopolysaccharide sensor that promotes glycolysis through mTORC1 activation

Fig. 1

a Immunopurification and mass spectrometry analysis of Gal3-, RagA-, or RagC-associated proteins in HEK-293T cells. Cellular extracts from HEK-293T cells expressing FLAG-Gal3, FLAG-RagA, or FLAG-RagC were subjected to affinity purification with an anti-FLAG affinity column and eluted with excess FLAG peptides. The eluates were resolved by SDS-PAGE and silver-stained. The protein bands were retrieved and analyzed by mass spectrometry. b Galectin-3 is co-immunoprecipitated with RagB and RagC. The schematics were from the BioPlex, STRING, and CF-MS explorer databases. c HEK-293T cells were transfected with FLAG-Gal3 or FLAG-metap2. 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. d Co-immunoprecipitation in HEK-293T cells with anti-galectin-3 followed by immunoblotting with antibodies against the indicated proteins, or immunoprecipitation with antibodies against the indicated proteins followed by immunoblotting with antibodies against galectin-3 or against the components of the mTORC1 signaling. Each experiment was repeated three times with similar results. e HEK-293T cells were transfected with GFP-Gal3 followed by immunofluorescent staining for Lamp2 (red), p18 (red), or RagC (red). Scale bar: 10 μm. Each experiment was repeated three times with similar results. f FPLC analysis of FLAG affinity eluates in HEK-293T cells stably expressing FLAG-Gal3. Chromatographic elution profiles and immunoblotting analysis of the chromatographic fractions are shown. Equal volume from each fraction was analyzed, and the elution position of calibration proteins with known molecular masses (kDa) are indicated. Western blotting of galectin-3-containing complex fractionated by Superose 6 gel filtration is shown. g GST pull-down assays with GST-fused galectin-3, RagA, RagC, p14, p18, SLC38A9, or ATP6V1B2 and in vitro transcribed/translated proteins as indicated. Each experiment was repeated three times with similar results. h Co-immunoprecipitation in HEK-293T cells transfected with FLAG-Gal2 or FLAG-Gal4 with anti-FLAG followed by immunoblotting with antibodies against the indicated proteins. Each experiment was repeated three times with similar results.

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