Smart system

The C-ICD and C-ICDmut peptides were synthesized by Fmoc chemistry using the PSSM-8 automated peptide synthesizer (Shimadzu, Kyoto, Japan) and purified by reverse-phase high-performance liquid chromatography on a C18 column with a linear gradient from 0 to 90 % CH₃CN in 0.1 % trifluoroacetic acid for 60 min at a flow rate of 1 ml/min. The purified peptides were digested with trypsin and separated using the Smart System (GE Healthcare, Little Chalfont, UK) on a RP300 column with a linear gradient from 0 to 90 % CH₃CN in 0.1 % trifluoroacetic acid for 50 min at a flow rate of 1 ml/min, and the fractions were collected. The individual fractions were analyzed by matrix-assisted laser desorption/ionization time-of-flight collision-induced dissociation tandem mass spectroscopy using the Axima Performance system (Shimadzu), and their amino acid sequences were confirmed by the 492HT protein sequencer (Applied Biosystems, Foster City, CA, USA). FITC-I (Dojindo) was used to label the peptides according to the manufacturer’s instructions. Briefly, the peptide and FITC-I were mixed at a weight ratio of 10:1 in phosphate-buffered saline (PBS) containing 2.5 mM carbonate and were reacted at 4 °C for 4 h. The FITC-labeled peptide was washed with PBS by five centrifugation cycles using an Amicon Ultra-10 K membrane filter (Millipore, Billerica, MA, USA).

Five-hundred mL of conditioned medium (C.M.) of FLAG-tagged Wnt3a (FLAG-Wnt3a)-expressing L cells in serum-free condition were prepared. HEPES buffer, pH7.4 (final 10 mM); EDTA, pH8.0 (final 1 mM); and protease inhibitors (final 1 mM PMSF, 5 μg mL⁻¹ leupeptin, 10 μg mL⁻¹  peptstatinA, and 5 μg mL⁻¹ aprotinin) were added to the C.M. Debris was removed by centrifugation for 30 min at 14,000 × g, and the supernatant containing FLAG-Wnt3a was applied to an anti-FLAG M2 affinity gel (Sigma) column that had been equilibrated with 15 mM HEPES buffer, pH7.4-150 mM NaCl. The column was washed with 10 column bed volumes of the same buffer containing 0.1 mM n-Dodecyl-β-D-maltoside. The bound proteins were eluted with the same buffer containing 125 μg mL⁻¹ FLAG peptide (Sigma). The eluate was concentrated 10–30 times with a Microcon centrifuge filter unit YM-100 (Millipore). Further purification was performed by using Superdex200 (PC3.2/30) gel filtration chromatography in a Smart-system (GE Healthcare). The elution of protein was profiled at 280 nm, and the eluate was sub-fractionated into 40-μL fractions.

For determining the stoichiometry of the complex, PDE6 concentration of ~2 µM with molar ratios of 1, 2 and 3 for PrBP/δ were subjected to gel filtration (50 µl sample volume, Superose 12 column, GE Healthcare, Germany) using SMART systems (GE Healthcare, Germany) in 20 mM BTP (pH 7.5), 130 mM NaCl, 1 mM MgCl₂, 1 mM TCEP at 10 °C. The column was previously calibrated using external protein standards (Thyroglobin (669 kDa), Ferritin (440 kDa), Catalase (232 kDa), Alcohol Dehydrogenase (158 kDa), Albumin (67 kDa), Ovalbumin (43 kDa), Chymotrypsine (25 kDa), RNase (13.7 kDa)).

The following procedures were performed at 4°C using the FPLC and SMART systems (GE Healthcare). Soluble materials from Escherichia coli BL21 (DE3) cells were dissolved in buffer A (20 mM sodium phosphate (pH 7.2), 0.3 M NaCl, 10% glycerol, and 10 mM β-mercaptoethanol), passed through Hitrap DEAE (GE Healthcare), and then loaded onto TALON resin (Clontech). After sequential washes with buffer A and buffer B (20 mM Tris-HCl (pH 8.0), 0.1 M NaCl, 10% glycerol, and 10 mM β-mercaptoethanol), the bound materials were eluted with 0.2 M imidazole in buffer B and loaded onto anti-FLAG M2 agarose. After a wash with buffer B, the bound materials were eluted with buffer B containing 0.1 mg/ml FLAG peptide (Sigma) and then loaded onto anti-HA-agarose (Sigma). After a further wash with buffer B, the bound materials were eluted with buffer B containing 0.1 mg/ml HA peptide (Sigma). The PCNA-enriched fractions detected by SDS-PAGE and Coomassie Brilliant Blue staining were loaded onto a MonoQ/PC1.6/5 column (GE Healthcare) and the proteins were eluted with a linear gradient of NaCl (0.1–0.5 M) in 20 mM sodium phosphate (pH 7.2), 0.1 mM EDTA, 10% glycerol, and 10 mM β-mercaptoethanol.