Anti prpf6

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).