Anti galectin 3

Cells and exosomes were lysed with RIPA lysis buffer containing protease and phosphatase inhibitors. Nuclear proteins were extracted with a kit (CWBIO, Beijing, China). Protein samples were separated by SDS-PAGE, and then transferred to PVDF membranes (Millipore, Burlington, MA, USA). After blocking for 1 h, the PVDF membranes were incubated with primary antibodies overnight at 4℃. The primary antibodies used were anti-CD81 (1：1,000; Cell Signaling Technology, Danvers, MA, USA), anti-CD63 (1：1,000; Cell Signaling Technology), anti-TSG-101 (1：1,000; Cell Signaling Technology), anti-calnexin (1：1,000; Cell Signaling Technology)，anti-Galectin-3 (1：1,500; Abcam, Cambridge, UK), anti-α-SMA (1：1,000; Bioworld Technology, Bloomington, MN, USA), anti-Collagen Ⅰ (1：1,000; Bioworld Technology), anti-Pe-riostin (1：1,000; Proteintech, Wuhan, China), anti-IL-1β (1：1,000; Bioworld Technology), anti-TNF-α (1：1,000; Bioworld Technology), anti-TGF-β (1：1,000; Abcam), anti-β-catenin (1：1,000; Cell Signaling Technology), anti-GAPDH (1：3,000; CWBIO, Beijing, China), and anti-Histone (1：1,000; Cell Signaling Technology). The next day, membranes were incubated at 37℃ for 1 h with hor-seradish peroxidase-linked goat anti-rabbit or anti-mouse antibodies (1：3,000; ABM, Richmond, Canada).

The reaction mixture was prepared by mixing 2 μg of recombinant OGT, 3 μg of recombinant galectin 3(Abcam, ab89487), 1 μl of 2 M UDP-GlcNAz and making the volume up to 50 μl with OGT assay buffer (50 mM Tris–HCl, pH 7.5, 1 mM dithiothreitol, 12.5 mM MgCl₂). Negative controls were prepared by removing either OGT, galectin 3 or UDP-GlcNAz from the reaction mixture. A positive control was prepared by substituting casein kinase II (NEB, P6010S) for galectin 3 in the reaction mixture.
The OGT assay mixtures were incubated for ∼90 min in a 37 °C water bath. The reaction mixtures were then added to 10K filters and spun at 14,000 rpm for 30 min. The filters were washed with 100 μl of PBS and spun down at 14,000g for 10 min twice. To recover the protein, the membranes were flipped and spun down at 1000g for 3 min. One microliter of biotin phosphine was added and incubated for 1 h at 40 °C. The samples were then boiled for 10 min in 1× sample-loading buffer, and proteins were separated by 10 to 20% SDS-PAGE, transferred to nitrocellulose paper, blocked in odyssey blocking buffer for 1 h, and probed with streptavidin IR800 and anti-galectin 3 (Abcam). Membranes were imaged using a LI-COR Odyssey IR imager.

After a 48-h transfection with GFP-tagged galectin 3, mutants cells were rinsed with ice-cold PBS and lysed in 1 ml of ice-cold RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) with protease inhibitor mixture (Sigma-Aldrich). The conditioned media was also collected in some experiments and spun down at 3000g for 10 min to remove any cell debris; supernatants were transferred to fresh eppendorf tubes. Cell lysates were centrifuged at 13,300 rpm for 10 min. Supernatants were transferred to fresh eppendorf tubes. The samples were immunoprecipitated using 25 μl bead slurry of GFP-Trap Agarose (Chromotek) (GFP-Trap consists of an anti-GFP Nanobody/V_HH coupled to agarose beads to immunoprecipitate GFP-fusion proteins). The beads were preblocked for 1 h on ice with 3% BSA (Sigma-Aldrich) in RIPA buffer. After immunoprecipitation, beads were rinsed three times with wash buffer (10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.05 % Nonidet P40 Substitute, 0.5 mM EDTA) and boiled for 10 min in 1× sample-loading buffer, and proteins (2% of input supernatant and 100% of the immunoprecipitate) were separated by 10 to 20% SDS-PAGE, transferred to nitrocellulose paper, and probed with the following antibodies: anti-Ezrin (Cell Signaling), anti-galectin 3 (Abcam), and anti-RL-2 (Abcam). Membranes were imaged using a LI-COR Odyssey IR imager.