Ponceau s solution

Cell lysates were harvested in RIPA buffer (Sigma) containing Complete Protease Inhibitor (Roche) and Phospho Stop (Roche) on ice and run on SDS-PAGE gels. PVDF membranes were stained for galectin-3 (Abcam, ab53082, 1:500, or BioLegend, M3/38, 1:1000), galectin-8 (Abcam, ab69631, 1:500), and α-tubulin (Cell Signaling, 2125, 1:1000). Lectin blotting for glycans was performed using PNA-Biotin (Sigma) followed by detection using the ABC Elite Kit (Vector Labs, PK-6100) or PNA-Peroxidase conjugates (Sigma). Ponceau S solution (Thermo) was used for total protein detection following transfer to PVDF membranes.
For liver homogenate western blots, mice bearing 393M1 flank tumors or no tumors were perfused by intracardiac injection of 20mL of phosphate buffered saline. Livers were then excised and 50mg portions were added to gentleMACS M tubes (Miltenyi Biotech) in 4.5mL of RIPA (Sigma) with Complete Protease Inhibitors (Roche). Tissue was dissociated using the gentleMACS Octo Dissociator (Miltenyi Biotech) and run on polyacrylamide gels as described above.

GSH-beads coupled to GST-PAK1-PBD (PAK02, Cytoskeleton) which were three times washed with 50 mM HEPES pH 7.5, 100 mM NaCl, 5 mM MgCl₂ and 5 mM β-mercaptoethanol. Experiments were performed with 60 μg (60 μl) GST-PAK1-PBD bound to GSH beads and 30 μg RHO^WT, RHOA^L57V or RAC1^WT bound either to GppNHp or GDP in 50 mM HEPES pH 7.5, 100 mM NaCl, 5 mM MgCl₂ , 5 mM β-mercaptoethanol and 100 μM GppNHp or GDP for one hour at 4°C with head-over-head rotation. Beads were washed three times with 50 mM HEPES pH 7.5, 100 mM NaCl, 5 mM MgCl₂ and 5 mM β-mercaptoethanol and boiled in 30 μl 4x Laemmli sample buffer (Bio-Rad Laboratories) supplemented with β-mercaptoethanol. Samples were analyzed utilizing immunoblotting on nitrocellulose membranes (Millipore) followed by staining with Ponceau S solution (Thermo Fisher Scientific).