Lysate Preparation and Western Blotting

Larval lysates were prepared by crushing animals in lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% NP-40) with 1× protease inhibitor cocktail (Invitrogen) and clearing the lysate by centrifugation at 13,000 RPM for 10 min at 4°C. S2 cell lysates were prepared by suspending cells in lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% NP-40) with 10% glycerol and 1× protease inhibitor cocktail (Invitrogen) and disrupting cell membranes by pulling the suspension through a 25 gauge needle (Becton Dickinson). The lysate was then cleared by centrifugation at 13,000 RPM for 5 min at 4°C. Western blotting on lysates was performed using standard protocols. Rabbit anti-dSMN serum [28] (link) was affinity purified. For Western blotting, dilutions of 1 in 2,500 for the affinity purified anti-dSMN, 1 in 10,000 for anti-α tubulin (Sigma), 1 in 10,000 for monoclonal anti-FLAG (Sigma), 1 in 10,000 for polyclonal anti-Myc and 1 in 5000 for monoclonal anti-Myc (Santa Cruz) were used. Anti-FLAG antibody crosslinked to agarose beads (EZview Red Anti-FLAG M2 affinity gel, Sigma) or anti-Myc antibody crosslinked to agarose beads (Sigma) were used to immunoprecipitate FLAG and Myc tagged proteins from cells.