Anti prpf8

Dissected cerebella were rinsed with ice-cold PBS, chopped with a razor blade, and transferred into 1 ml of NET2 buffer (150 mM NaCl, 0.05% NP-40, and 50 mM Tris–HCl, pH 7.4, supplemented with 1:200 Protease Inhibitor Cocktail Set III [#539134; Merck Millipore]), where they were homogenized for 5 s on ice. The crude lysates were then sonicated with 30 consecutive pulses of 1 s (60% amplitude) and cleared by centrifugation at 15,000g for 5 min at 4°C. The cleared lysates were further incubated with 4 μg of Prpf8 antibody (#79237; Abcam) or with 4 μg of control IgG antibody (#I5381; Sigma-Aldrich) for 1 h at 4°C with continuous rotation. 30 μl of Protein G agarose beads (#sc-2002; Santa Cruz Biotechnology) was then added to the mixture, and the reaction was incubated for an additional 2 h at 4°C with continuous rotation. The beads were then washed five times with NET2 buffer and resuspended in 2× sample buffer (250 mM Tris–HCl, pH 6.8, 20% glycerol, 4% SDS, and 0.02% bromophenol blue), supplemented with 24 mM DTT (Sigma-Aldrich). Precipitated proteins were analyzed by Western blotting. Primary antibodies used for the immunoblotting were as follows: anti-GAPDH (#5174T, 1:1,000; Cell Signaling Biotechnology), anti-Prpf6 (#sc-166889, 1:500; Santa Cruz Biotechnology), anti-Prpf8 (#79237, 1:1,000; Abcam), and anti-Snrnp200 (#HPA029321, 1:250; Sigma-Aldrich).

For siRNA-mediated depletion, Cal51 cells were reverse transfected with 25 nM siRNA to the indicated genes using DharmaFECT1 (Dharmafect) transfection reagent. Transfected cells were harvested 54 hours later for RNA extraction, western blotting and fluorescence-activated cell sorting (FACS). Efficiency of depletion was either monitored by western blotting with the indicated antibodies or by qRT-PCR against the indicated genes. Antibodies used were anti-PRPF8 (Abcam), anti-Separase (Abcam), anti-CDC20 (Santa Cruz), anti-APC8 (Novus Biologicals), anti-Actin (Abcam).