Immunoblotting of NCS-1 in Cell Lines

Cells were lysed as described previously [9 (link)], and immunoblotting was performed using the NuPAGE system (Invitrogen, Carlsbad, CA) and the Biorad wet transfer system (Bio-Rad Laboratories, Hercules, CA) according to the manufacturers’ protocols. For these studies three different human cell lines (SHSY5Y, HEK293, MB231) and C57BL/6 mouse tissues were used. Antibodies used were anti-NCS-1 FL-190, anti-HA (both from Santa Cruz Biotechnology, Santa Cruz, CA), and anti-FLAG (Sigma-Aldrich, USA). Protein expression was quantified by scanning densitometry by UN-SCAN-IT (Silk Scientific, Orem, UT) and normalized to β-actin loading controls. All Western blotting experiments were performed using three independent cultures.