Western Blot Analysis of Protein Expression

Proteins were transferred to 0.45 μm Immobilon-P PVDF membrane (Millipore, Burlington, MA) in transfer buffer (25 mM Tris, 200 mM Glycine, 20% methanol) for 75 min at 500 mA at 4°C. The membranes were blocked with blotto (2.5% nonfat dry milk, 0.5% BSA, 0.5% NP-40, in TBST) for 30 min at room temperature and then incubated with rabbit anti-ZLD (1:750) (Harrison et al. 2010 (link)), or anti-Tubulin (DM1A, 1:5,000) (Sigma, St. Louis, MO), overnight at 4°C. The secondary incubation was performed with goat antirabbit IgG-HRP conjugate (1:3,000) (Bio-Rad, Hercules, CA) or antimouse IgG-HRP conjugate (1:3,000) (Bio-Rad) for 1 h at room temperature. Blots were treated with SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA) and visualized using the Azure Biosystems c600 or Kodak/Carestream BioMax Film (VWR, Radnor, PA).