Anti mouse irdye 800cw secondary antibody

Whole cell lysates were isolated using RIPA buffer containing (mmol/l) 150 NaCl, 50 Tris pH 7.4, 1 EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and protease and phosphatase inhibitors. Twenty micrograms of protein was separated on 4–20% Tris-HCl gels (Bio-Rad, Hercules, CA, USA), transferred on nitrocellulose membranes, and blocked in 5% BSA for 1 h at room temperature. Blots were then incubated with the rabbit anti-HER-2/neu antibody (1:500 EMD; Millipore, Billerica, MA, USA), rabbit β-catenin antibody (1:500), rabbit T308 phospho-Akt or total Akt antibody (both from Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. Signals were detected with IRDye 680 secondary antibodies (1:5,000) for 1 h at room temperature and visualized using LI-COR Odyssey Imaging System (both from LI-COR, Lincoln, NE, USA). Blots were subsequently incubated with mouse GAPDH antibody (1:1,000; Sigma) and anti-mouse IRDye 800CW secondary antibody (1:5,000; LI-COR) to ensure equal protein loading. N=3.

Surface sensing of translation (SUnSET) was performed on dis3L2^wt and dis3L2¹² wing imaginal discs. Wing discs were dissected in batches of 30 and incubated in fully supplemented Shields and Sang M3 insect medium (Sigma-Aldrich, cat. no S8398) containing 2μg/ml Puromycin (Sigma-Aldrich, cat. no. P8833) for 1 hour at 25°C with rotation. Western blotting was performed to determine the levels of Puromycin incorporation with Tubulin as a loading control. Mouse anti-Tubulin primary antibody (Sigma-Aldrich, cat. no. T9026) was used at 1:2000 dilution. Mouse anti-Puromycin (clone 12D10) primary antibody (Merck Millipore, cat. no. MABE343) was used at 1:1000 dilution. Anti-mouse IRDye 800CW secondary antibody (LI-COR Biosciences, cat. no 926–32210) was used at 1:20,000 dilution to detect both primary antibodies. Each sample was run in parallel on two gels/membranes so the Tubulin band could be distinguished from Puromycin containing peptides. Quantification of Tubulin (46kDa) and Puromycin peptides (from smallest size visible to 240kDa) was achieved using LI-COR Biosciences Image Studio software. In each case Puromycin signal in dis3L2¹² tissues was compared to dis3L2^wt samples ran on the same gel. Uncropped blot is shown in S2 File.