4), 5 mM MgCl2, 5 mM KCl, 1 mM DTT and 1× protease inhibitor cocktail (Invitrogen, Carlsbad, CA, USA). Cells were mechanically lysed with a glass GSK2126458 homogenizer and centrifuged at 2,000 rpm. The supernatant was centrifuged at 15,000 rpm and the pellet was washed and resuspended in 100 μl of the hypotonic buffer. Total proteins were quantified by the Bradford assay (BioRad, Hercules, CA, USA). Identical masses of membrane fractions were seeded on a PVDF membrane (Hybond-P; GE Healthcare, Chalfont St. Giles, Buckinghamshire, England) previously activated with methanol and washed with TBS buffer with the aid of the BIO-DOT SP apparatus (Bio-Rad, Hercules, CA, USA). Once seeded,

membranes were blocked with a 5% low-fat milk in TBS solution and washed Vistusertib in vivo with TBS. Incubation with the anti-NeuGc-GM3 antibody 14F7 (10 μg/ml) was performed at room temperature for 1 h. After washing them with TBS-T buffer, membranes were incubated with

the biotinylated anti-mouse antibody (Vector Laboratories, Burlingame, CA, USA) and then incubated with a streptavidin linked to peroxidase solution (Vector Laboratories, Burlingame, CA, USA). Bands were detected by the ECL method (GE Heathcare, Chalfont St. Giles, Buckinghamshire, England) following the manufacturer’s instructions. Membranes were analyzed with the ImageJ analysis software (National Institute of Health) and the intensity of each band was recorded and expressed as arbitrary units. Indirect immunoperoxidase staining Tumor cells were cultured for 24 h in chamber-slides

(Nalge-Nunc, Rochester, NY, USA) in serum-free DMEM-F12 medium containing 250 μg/ml of BSM (Sigma, St. Louis, MO, USA), and later 7-Cl-O-Nec1 solubility dmso formalin-fixed. Subsequently, monolayers Beta adrenergic receptor kinase were stained by the Vectastain kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions. 14F7 mAb was used as primary antibody at a concentration of 10 μg/ml. Cells were counterstained with hematoxylin. Adhesion assay B16 or F3II cells were seeded (40,000 cells/well) in 96-well plates in D-MEM supplemented with 2 or 5% FBS, in the presence or absence of 50-100 μg/ml of purified NeuGc (Sigma, St. Louis, MO, USA). Cells were incubated at 37°C in a CO2 incubator for 60 min. After incubation, cells were washed twice with 1× PBS buffer and fixed with methanol (100 μl/well). After a 10-min incubation, cells were stained with a 0.1% crystal violet solution (100 μl/well) for 10 min. After washing thoroughly with distilled water, 60 μl/well of a 10% methanol-5% acetic acid solution were added and the plate was shook for a few minutes. Absorbance at 595 nm was measured. Proliferation assay B16 or F3II cells were seeded (2,500 cells/well) in 96-well plates in D-MEM supplemented with 1, 5 or 10% FBS, in the presence or absence of 50-100 μg/ml of purified NeuGc. Plates were incubated at 37°C in a CO2 incubator for 72 h. After incubation, cells were treated with MTT (0.