Alcam

Cell extraction and western-blot were performed as previously described [33 (link)]. Incubation with primary antibodies was performed at 4°C overnight using 1:500 mAb ALCAM (MOG/07, Abcam, Cambridge, MA, USA); 1:2000 rAb a-Tubulin, 1:2000 rAb ERK1/2, 1:1000 rAb p-ERK1/2 (Cell Signaling Technology, Beverly, MA, USA), 1:2500 rAb Lamin (Santa Cruz, CA, USA), 1:5000 rAb mCherry ([57 (link)]; an aliquot of the antibody was kindly provided by Drs. Anna Santamaría and Erich A. Nigg). Nuclear, membrane and cytosolic fractionation of transfected Hec1A-ETV5 cells was performed by using a Subcellular Fractionation Kit (Pierce) as per the manufacturer's instructions.

Total proteins were extracted using an NE-PER kit (Nuclear and Cytoplasmic Extraction Reagents, Thermo Fisher Scientific Inc.) according to the manufacturer’s protocol. Protein levels were normalized using a BCA kit (Thermo Fisher Scientific Inc.). SDS/PAGE conditions were 10 μl of protein loaded to a 10% gel run at 120 V for 90 min. For Western blotting, proteins were transferred to PVDF membranes and immunoblotted with primary antibodies against DLG5 (1:600), ALCAM (1:800), and β-actin (1:4000) (Abcam, Cambridge, MA, U.S.A.). The secondary antibody was horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, U.S.A., 1:5000).