Samples for gel electrophoresis were reduced with 100 mM dithiothreitol in 1x Laemmli buffer (0.0625 mM Tris pH 6.8, 2% (w/v) SDS stock, 10% (v/v) glycerol, 0.002% (w/v) bromophenol blue) and heat denatured on a thermoblock at 70°C for 10 minutes. Samples were run on a 4–12% Tris-Bis NuPage Novex “mini” gel (Invitrogen) at 130 V with 1X 2-(N-morpholino)ethanesulfonic acid (MES) running buffer (Invitrogen). Gels were stained with Lumitein™ (Biotium) according to the manufacturer’s protocol.
For Western blot analysis, gels were transferred onto PVDF (Millipore) membranes, previously equilibrated in 1x NuPage transfer buffer (Invitrogen) containing 12% (v/v) methanol, at 25 V for 45 minutes and at 35 V for another 45 minutes. Membranes were blocked with 1x PBS, 0.1% Tween-20 containing 5% nonfat dry milk for 30 minutes at room temperature and probed with horseradish peroxidase conjugated mouse monoclonal anti-FLAG M2 antibody (SIGMA) (1:1000), goat C20 anti-holo and C-terminus MIS antibody (Santa Cruz) (1:200), or rabbit MGH4 anti-holo and N-terminus MIS antibody (custom) (1:1000). Blots were washed two times, 5 minutes each at room temperature with 1x PBS, Tween-20 0.1%, and incubated with appropriate secondary antibody if necessary, and then washed three times 5 minutes again. Proteins bands were visualized with the ECL kit detection system (Perkin-Elmer) onto Kodak Biomax MR film. ImageJ (NIH, http://imagej.nih.gov/ij/) was used to perform densitometry to quantify the protein bands to compare cleavage of different constructs14 (link) which was averaged over at least three independent western blots.