Peroxidase conjugated anti goat or anti rabbit secondary antibody

Proteins extracts were obtained by washing the cells once with PBS and resuspending in lysis buffer (50 mM Tris pH6.8, 2% SDS, 10% glycerol, bromophenol blue, β-mercaptoethanol). Samples were then boiled 5 min at 95°C, subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes. Membranes were blocked overnight in TBS-T (Tris-Buffered Saline (TBS), 0.05% Tween-20) containing 5% skim milk, and incubated with the appropriate antibody. Membranes were washed four times in TBS–T, then incubated for 1 hour with the appropriate peroxidase-conjugated anti-goat or anti-rabbit secondary antibody at 1/5,000 (EMD Millipore). Membranes were washed again as described, incubated with Western Lighting Plus ECL reagents (PerkinElmer) according to manufacturer’s instructions, and exposed to film. Poly(A)⁺ RNA or genomic DNA was spotted onto Hybond N+ membranes (GE Healthcare, cat. #RPN119B), and subjected to dot blotting with the indicated antibodies as described^{33 (link)}. Original western blot and dot blot scans are provided in Supplementary Figure.