Mouse anti perk

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.

In total, 1 mL of cell suspension (1 × 10⁵ cells) was loaded onto a standard scWest chip for 5 min. Optical visualization was performed in order to confirm and mark single-cell capture sites, followed by gentle washing in 1× suspension buffer (ProteinSimple, San Jose, CA, USA). The scWest chip was then submitted to the Milo system (ProteinSimple) for lysis (10 s), electrophoretic separation (60 s, 240 V), and protein immobilization (240 s). Protein targets were probed on-chip for 2 h at RT with primary antibodies, including goat anti-neuroblastoma RAS viral (v-ras) oncogene homolog (NRAS) (Abcam), mouse anti-p-ERK (Cell Signaling Technology, Beverly, MA, USA), rabbit anti-p-AKT (R&D Systems, Minneapolis, MN, USA), rabbit anti-histone H3 (Cell Signaling Technology), rabbit anti-NRASQ61R (Spring Bioscience), and mouse anti-vimentin (Dako), and then for 1 h at RT with secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA), including donkey anti-goat IgG, Alexa Fluor 488; donkey anti-mouse IgG, Alexa Fluor 555; and donkey anti-rabbit IgG, Alexa Fluor 647. The probed chip was washed, air-dried, and analyzed using GenePix 4400A Scanners (Molecular Devices, San Jose, CA, USA).