Imagequant las4000 reader

S2 cells were lysed in 50 mM Tris-HCl (pH 7.8), 150 mM NaCl, 1 mM EDTA (pH 8.0), 1% Triton X-100, 0.01% Igepal, and protease inhibitors (Roche Applied Science). Protein concentration was quantified using Pierce 660 reagent (22660, Thermo Scientific) according to manufacturer’s instructions. Following SDS-PAGE, proteins were detected by immunoblotting with anti-Flag M2 (1:1000, Sigma Aldrich), anti-GFP (1:5000, TP401, Acris) and anti-α-Spectrin (1:1000, RRID: AB_528473) antibodies, followed by incubation with corresponding HRP-conjugated secondary antibodies (Jackson Immuno Research). Chemiluminescent signal was captured using ImageQuant LAS4000 reader (GE Healthcare).

For each sample, 1 mg of S2 cell protein lysate was incubated with GFP-Trap beads (gtma-20, Chromotek, RRID:AB_2631406) overnight. Following five washes with Lysis buffer, bound proteins were eluted with Glycine-HCl on 37°C, followed by neutralization. Proteins resolved on 10% SDS-PAGE were detected by immunoblotting with anti-Flag M2 (mouse, 1:1000, F1804, Sigma Aldrich, RRID:AB_262044), anti-GFP (rabbit, 1:5000, TP401, Acris, RRID:AB_2313770) and anti-α-Tubulin (mouse, 1:1000, DSHB, AA4.3, RRID:AB_579593) antibodies, followed by incubation with corresponding HRP-conjugated secondary antibodies (Jackson Immuno Research). Chemiluminescent signal was captured using ImageQuant LAS4000 reader (GE Healthcare, RRID:SCR_014246).
Hemolymph was collected from twenty third instar larvae per replicate in 100 μL PBS. After centrifugation, 5000 rpm for 10 min, the supernatant was precipitated with 100 μL of ice cold acetone for 1 hr at −20°C. Afterwards, the proteins were pelleted and resuspended in 50 μL in Lysis buffer (see above), and equal volumes were resolved on 10% SDS-PAGE. Immunoblotting was carried out with anti-Endostatin (rat, 1:1000, Harpaz et al., 2013 (link)) and anti-PPO1 (rabbit, 1:750, Jiang et al., 1997 (link)) antibodies.