V7931

Drosophila brains were dissected in cold ringer solution, fixed using 4% paraformaldehyde for 20 min at room temperature (RT), and then washed in phosphate buffer with 0.3% Triton-X (PBT; 3× immediate washes followed by 3 × 20-min washes). Non-specific staining was blocked using 5% heat inactivated goat serum in PBT, and brains were then subjected to primary antibody staining overnight at 4 °C. Primary antibodies included chicken anti-GFP 1:500 (GFP-1020; AVES), mouse anti-FasII 1:25 (1D4; DSHB), mouse anti-EcRB1 1:25 (AD4.4; DSHB), rabbit anti-active-JNK (pJNK) 1:200 (V7931; Promega) and rabbit anti-Mamo 1:5000^{25 (link)}. Brains were rinsed (x3) then washed with PBT (3 × 20-min), stained with secondary antibodies for 2 h at RT, and washed again. Secondary antibodies included FITC donkey anti-chicken 1:300 (703-095-155; Jackson immunoresearch), Alexa fluor 647 goat anti-mouse 1:300 (A-21236; Invitrogen) and Alexa fluor 647 goat anti-rabbit 1:300 (A-21236; Invitrogen). When staining for Mamo, DAPI 1:1000 (D1306; Invitrogen) was added for 15 min and then rinsed three times prior to mounting. Brains were mounted on Slowfade (S-36936; Invitrogen) and imaged on Zeiss LSM 800 confocal microscope. Images were processed with ImageJ (NIH).

Mosquito tissues collected from individual mosquitoes were separately put into micro-centrifuge tubes containing 100μL of breaking buffer [50 mM Tris (pH 7.4), 1% IGEPAL, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethyl-sulfonylfluoride, 1X protease inhibitor mixture, and 1X phosphatase inhibitor mixture (Sigma-Aldrich, St. Louis, Missouri, USA)] and homogenized using a pellet pestle. The homogenates were centrifuged at 13,000 rpm for 5 min. The supernatants were transferred into a Qiashredder Column (Qiagen, Los Angeles, California, USA) and centrifuged again under the same conditions for 10 min. The flow-through was transferred to a clean micro-centrifuge tube to conduct a Western blot analysis using anti-phosphoric JNK antibody (V7931, Promega) and anti-JNK antibody (sc-571, Santa Cruz). The blot was developed by VisGlow Chemiluminescent Substrate and HRP (Visual Protein).

Dissections, fixations, and immunostainings were performed following standard procedures, except that for stainings to detect apoptosis in the pupal CNS, only pupae within the first 10 min of puparium formation were used in order to minimize biological variability in apoptosis levels over time. Primary antibodies used were rabbit anti-GFP (1:500 in larvae and pupae, 1:1,000 in embryos; A11122; Invitrogen), rabbit anti-DsRed (1:100; 632496; Takara Bio Inc.), rabbit anti-βgal (1:5,000; Cappel), mouse anti-Eve (1:5–1:10; 2B8; Developmental Studies Hybridoma Bank), mouse anti-Eve (1:20; 3C10; Developmental Studies Hybridoma bank), mouse anti-pERK (1:500 in retina and 1:100 in optic lobe; 9106; Cell Signaling Technology), mouse anti-Repo (1:250; 8D12; Developmental Studies Hybridoma Bank), rabbit anti-pJNK (1:200; V7931; Promega), rabbit anti-Myd88 (1:250; a gift from S. Wasserman), and rabbit anti-Dcp1 (cleaved Drosophila Dcp1 [Asp216]; 1:500; 9578S; Cell Signaling Technology). Secondary antibodies were directly conjugated Alexa Fluor 488, 546, and 647 (1:250, Molecular Probes) or biotinylated mouse or rabbit (1:300) followed by avidin amplification using the Vectastain ABC Elite kit (Vector Laboratories) or the Tyramide Signal Amplification kit (T20922; Thermo Fisher Scientific), using the manufacturer’s instructions. For sample sizes, see Table S2.