Amersham hybond p 0.45 pvdf

Twenty embryos for each condition were manually dechorionated. Proteins were extracted by homogenizing embryos in buffer (200 mM Tris/HCl, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40, 10% Glycerol, 1 mM NaFl and sodium orthovanadate) on ice and quantified by a standard bicinchoninic acid method. A total of 50 μg of total protein extract were then separated on 10% polyacrylamide gels and transferred onto membranes (GE AmershamTM HybondTM P 0.45 PVDF). Membranes were cut at the level of the molecular weight of interest, blocked with 2% skim milk in TBST, and incubated with the primary antibody diluted in 2% skim milk in TBST (Abcam ab128298 polyclonal rabbit anti-PANK2 antibody, 1:1000, Origene TA890010 monoclonal mouse anti-actin antibody, 1:1000) and then with the appropriate secondary antibody also prepared in 2% skim milk (1:2000, from Pierce). Images were acquired with Li-Cor Odyssey Image station, and band intensity was quantified by ImageJ software without any modification of the original data. Original images were partially cropped to fit the final figure.

Lysates from wild-type or per⁰¹ larval brains were sonicated in 2x Laemmli, separated by SDS-PAGE and transferred to PVDF membranes (Amersham^TM Hybond^TM P 0.45 PVDF, GE Healthcare Life science, Chicago, IL, United States). Blots were incubated overnight at 4°C with the following antibodies: rabbit anti-phospho-Akt (Ser473) (1:1000; clone D9E, Cell Signaling, Danvers, MA, United States), rabbit anti-Akt (1:1000, Cell Signaling), rabbit anti-phospho-Drosophila-p70 S6k (1:1000; Cell Signaling), mouse anti-α-Tubulin (1:5000, clone NDM1A, Merck, Darmstadt, Germany). After incubation with HRP-coupled secondary antibodies, signal detection was done with the ECL Plus detection reagents (GE Healthcare Life Science, Buckinghamshire, United Kingdom) and a ChemoCam ECL Imager equipped with a 16 bit camera (Intas, Göttingen, Germany). Exposure times were adjusted to allow for quantification of signal intensities within the dynamic range of the camera system.

Western blot analysis of membrane‐bound TREM2 was performed as previously described (Kleinberger et al, 2014). For Western blot analysis of Aβ, lysates were prepared with 8 M urea lysis buffer (8 M Urea, 50 mM Tris–HCL pH 7.4) freshly supplemented with a protease inhibitor cocktail (Sigma‐Aldrich) and incubated on ice for 30 min. The lysate was mixed with Laemmli sample buffer supplemented with beta mercaptoethanol, and equal amounts of protein were separated by SDS–PAGE. After transfer onto polyvinylidene difluoride membranes (Amersham Hybond P 0.45 PVDF, GE Healthcare Life Science) or nitrocellulose membranes (GE Healthcare Life Science), blots were blocked for 1 h with I‐Block^™ (Thermo Fisher Scientific) and exposed to 5F4 for TREM2 and sTREM2 or 2D8 (Shirotani et al, 2007) for Aβ. Signals were visualized with HRP‐conjugated secondary antibodies using ECL kit (Thermo Fisher Scientific).