Sc 32790

HeLa and Caco-2 cells expressing or not eGFP-galectin-3 were lysed for 30 min in RIPA-buffer (30 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycolate, 0.1% SDS, 1 mM EDTA), supplemented with a protease inhibitor cocktail (Sigma-Aldrich). After removal of the insoluble material by centrifugation at 12,000 g, the lysates were either mixed with a concentrated Laemmli buffer or incubated for 3 h at 4 °C with GFP-Trap beads (Chromotek) to immunoprecipitate eGFP-galectin-3. The proteins in the lysates and the immunoprecipitates were separated by SDS-PAGE followed by a transfer to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked in Tris-buffered saline buffer containing 5% bovine serum albumin and 0.1% Tween 20 for 1 h at room temperature, before incubation with appropriate combinations of primary and secondary antibodies and visualization using the Li-cor Odyssey Imaging System. The primary antibodies used were a mouse anti-galectin-3 mAb (1:1000, sc-32790, Santa Cruz Biotechnology), a rabbit polyclonal anti-GFP (1:2000, Proteintech) and a mouse anti-GAPDH mAb (1:2000, Proteintech). Uncropped and unprocessed scans of the blot are included in the Source Data file.