Bioruptor 300

Cells harvested with RIPA buffer containing phosphatase and protease inhibitors (Thermo Fisher Scientific) were sonicated (Bioruptor 300, Diagenode) for 5 minutes and protein concentration was measured using Direct Detect® Spectrometer (Merck Millipore). Boiled samples (20 μg protein, 4 μl LDS, and 1.4 μl reducing agent) were separated on Bis-Tris protein gel (Thermo Fisher Scientific) and transferred to nitrocellulose membrane (LI-COR Biosciences). The membrane was blocked with 5% nonfat skim milk (Bio-Rad) for one hour and incubated with primary antibodies (ICAM1 0.03 μg/ml, VCAM1 0.22 μg/ml, IL-1β 2 μg/ml and, TNF-α 2 μg/ml) overnight at 4 °C. Proteins were detected using appropriate HRP-conjugated antibodies (rabbit anti goat 0.75 μg/ml, goat anti rabbit 0.67 μg/ml) and SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific) to obtain images with ImageQuant LAS 4000 Imager (GE Healthcare Life Sciences).

Total protein lysates were collected in RIPA buffer supplemented with protease (cOmplete™ ULTRA Tablets, Mini, EDTA-free, EASYpack Protease Inhibitor Cocktail, Sigma) and phosphatase (PhosSTOP™, EASYpack, Sigma) inhibitors. Lysate were sonicated (5 cycles, 10 seconds on, 15 seconds off) using a Bioruptor® 300 (Diagenode). Protein concentrations were quantified using the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific) and 40-100μg of protein was loaded for blot analysis after heat denaturation in 1X sample buffer (60mM Tris-HCl, pH 6.8, glycerol, 2% (w/m) SDS, 0.005% β-ME). Fractionation was carried out by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) or nitrocellulose membrane (0.2 μm) by overnight wet transfer. Membranes were blocked in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) plus 5% nonfat milk for 1 hour at room temperature. Primary and secondary antibody incubations were performed overnight at 4°C and 1 hour at room temperature, respectively, at the recommended dilutions. Blots were developed according to the manufacturer’s recommendations with Clarity Max™ Western ECL Substrate (Biorad) for HRP-conjugated secondary detection, or with the Odyssey (Li-Cor Biosciences) system. ImageJ⁷⁵ (link) was used to quantify band intensities.

All steps were carried out on ice/at 4 °C and all used buffers contained in addition Protease inhibitor cocktail tablets (cOmplete Tablets; Roche) and 0.5 mM PMSF. Cells were washed twice with PBS before lysis. For whole cell extracts pelleted cells were resuspended in buffer L (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM MgCl₂,) and sonicated 10× with an interval of 30″ on medium setting (Bioruptor300; Diagenode). Protein concentrations were measured using Quick Start^TM Bradford 1× Dye Reagent from BIO RAD according to the manufacturers manual and equal amounts loaded for each lane. For nuclear extracts, pelleted cells were incubated for 10′ in lysis buffer (10 mM Hepes-KOH, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT). Nuclei were pelleted and washed once with lysis buffer, resuspended in extraction buffer (20 mM Hepes, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and 0.5 mM DTT) plus Benzonase (Novagen; 100 U/ml) and incubated on ice for 20′. Supernatant after centrifugation served as the nuclear extract.

Ovaries were dissected from NASP¹/ Df(3R), NASP²/Df(3R) or OrR females fattened for two days on wet yeast in Ephrussi Beadle Ringers (EBR). Stage 12 egg chambers were isolated, re-suspended in LB3 [63 (link)] and sonicated using a Bioruptor 300 (Diagenode) for five cycles of 30s on and 30s off at maximal power. Lysates were treated with RNase and Proteinase K and genomic DNA was isolated via phenol-chloroform extraction. qPCR was performed using primers previously described [64 (link)].

Cells were harvested with RIPA buffer (Thermo Fisher Scientific, Oslo, Norway) containing phosphatase and protease inhibitors (No. 78420 and 78430, respectively, Thermo Fisher Scientific, Oslo, Norway) and sonicated (Bioruptor 300, Diagenode, Belgium) for 5 min. The protein concentration was measured by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Oslo, Norway) according to the manufacturer’s instructions. Immunoblotting was performed as described previously [32 (link)]. Primary antibodies anti-HER2 (rabbit-anti-human, 0.66 μg/mL), β-Actin (rabbit-anti-human, 0.67 μg/mL) and HRP-conjugated antibody (goat-anti-rabbit, 0.33 μg/mL) were used. Blots were visualized by ChemiDoc MP imaging system (Bio-Rad, Oslo, Norway).