Immun star ap detection system

To prepare embryonic extracts, 0-2-hour old embryos were dechorionated, frozen and stored at -80°C. Ovaries (400 pairs) or embryos (the volume of dechorionized embryos ~70 μl) were homogenized in a Dounce homogenizer in 9 volumes of cold IP Buffer (20 mM HEPES pH 7.0, 150 mM NaCl, 2,5 mM MgCl₂, 0.1% Triton X-100, Complete Mini protease inhibitor cocktail (Roche), 0,2 mM NaVO₄, 20 mM NaF, RiboLock 1U/μl (ThermoScientific)). The extracts were cleared by centrifugation at 16,000 g at 4°C for 10 min. Supernatants were incubated with anti-HA magnetic beads (20 μl, Pierce) for 30 min at room temperature on a rotator. After 3 washes in IP buffer the bound proteins were eluted from the beads by boiling in 100 μl of Laemmli protein loading buffer (31,25 mM Tris-HCl, pH 6.8, 12,5% glycerol, 1% SDS, 0.005% Bromophenol Blue, 2,5% β-mercaptoethanol) for 5 min. Beads were pelleted and the supernatant was saved for Western blot analysis. Samples were resolved on 8% SDS-PAGE gel and transferred onto Immobilon-P PVDF membrane (Millipore). Blots were developed using the Immun-Star AP detection system (Bio-Rad Laboratories), in accordance with the manufacturer’s recommendations.

Proteins were extracted with 8 M urea, 0.1 M Tris-HCl, pH 7.0, 1% SDS, fractionated by SDS-PAGE (12% acrylamide gel) and transferred to a PVDF membrane (Immobilon-P, Millipore). Blots were developed using alkaline phosphatase-conjugated secondary antibodies (Sigma) and the Immun-Star AP detection system (Bio-Rad). The following antibodies were used for detection: murine monoclonal anti-lamin Dm0 (1:2000; ADL67^{49 (link)}), rabbit polyclonal anti-lamin C²⁵ (1:10000), murine monoclonal anti-beta Actin (1:3000; ab8224, Abcam).

Transient transfections of S2 cells were performed with the help of FuGENE^® HD Transfection Reagent (Promega# E2311) according to the manufacturer’s instructions. 3–4 days after the transfection the cells were harvested and subjected to immunostaining. For immunoprecipitation, anti-HA Magnetic Beads (#88836 Thermo Scientific) or anti-FLAG M2 Magnetic Beads (M8823 Sigma) were used. Protein samples (S2 cells or fly extracts) were applied to SDS-PAGE, transferred onto PVDF membrane according to standard protocols. The blots were analyzed using antibodies in a dilution 1:1000 against germinal NACβ, monoclonal mouse anti-FLAG M2 (Sigma F3165) and monoclonal mouse anti HA-Tag antibodies (mAB#2367, Cell Signaling Tech). Alkaline-phosphatase-conjugated anti-rabbit or anti-mouse antibodies (Sigma) were used as secondary reagent at a dilution of 1:20,000. Blots were developed using the Immun-Star AP detection system (Bio-Rad Laboratories) in accordance with the recommendations of the manufacturer, the signal was detected using the BioRad Chemi Doc MP Imaging System.