Amicon centricon

Purification of neuronal IP₃R1 reconstituted was performed as previously described^{20 (link),67 (link)}. Briefly, rat cerebellar membranes were solubilized in 2 mM Lauryl Maltose Neopentyl Glycol (LMNG, Anatrace) and 0.1% (w/v) l-α-phosphatidylcholine (PC, Sigma) for 2 h at 4 °C. Non-solubilized material were cleared by ultracentrifugation (100,000 x g) and the supernatant was applied to an immunoaffinity matrix pre-coupled with monoclonal antibodies (10 mg; T433 epitope of IP₃R1) in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.02 mM LMNG. Reconstitution of IP₃Rs into lipid-nanodiscs occurred “on-column” as we described in ref. 20 (link): 1.2 mM POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine, Avanti Polar Lipids) prepared in 3% DDM (n-dodecyl-β-d-maltoside, Avanti Polar Lipids, Inc), 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM DTT, 1 mM EDTA was added to the IP₃R1-bound immunoaffinity column matrix followed by the addition of 0.6 mg/ml MSP1E3D1 (Cube Biotech). Detergent removal was achieved by the addition of 0.25 mg/ml activated Bio-Beads-SM2 (BioRad) for 16 h at 4 °C. IP₃R1 was eluted with 500 μM of T433 peptide and concentrated using a 100 kDa Amicon centricon (Millipore).

Twelve mL of overnight culture of S. aureus CGMCC 1.89 grown in broth medium at 37 °C were collected and centrifuged; the supernatant was then filter-sterilized by passing through a 0.22-µm-pore-size sterile syringe filter. The sterilized supernatant was concentrated 15-folds by ultrafiltration [CBU(15x)] in a 3-kDa cut-off Amicon Centricon filter (MWCO 3 kD, Millipore) to collect the molecules larger than 3 kDa. The CBU(15x) was then injected into flies (DvWT and DvdelARP) with a dose of 69 nL each fly with the Nanoliter 2010 microinjection system (WPI, USA). Injected flies were subsequently maintained at 26 °C for experiments. Dead flies were daily counted over a period of seven days. Seventy flies per condition were used in this study. We also prepared a 15x concentrated sample through lyophilization by vacuum freeze-drying and resuspended with water but found that it lost some components relative to the original supernatant, as identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and thus discarded it in our study.

IGF-1 and IGF-2 were assayed with a previously validated RIA
[26 (link)]. Briefly, the IGFs were separated from their binding proteins by acidification (25 μL of samples in 2 ml of chlorhydric acid, 0.01 N). After, samples were ultrafiltered through an Amicon® Centricon® centrifugal filter with a molecular weight cut-off of 30 kDa (Millipore Corporation, Bedford; MA, USA) with two centrifugation steps. After lyophilisation, samples were dissolved in 500 μl binding buffer (0.03 M NaH₂PO₄, pH 7.4, 0.02 M EDTA, 0.2 g/L protamine sulphate, 0.2 g/L sodium azide, 0.05% Tween 20). A classical RIA was performed with these recovered samples using an antibody against human IGF-1 or IGF-2 (Amano Pharmaceutical Co., Nagoya, Japan) with human IGF-1 or 2 as a standard. Thereafter, free and bound IGFs were separated with a buffer containing active charcoal and centrifugation.