Mouse anti puromycin clone 12d10 primary antibody

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.

Surface sensing of translation (SUnSET) was performed on wild-type (pcm^WT1) and pacman null (pcm¹⁴) wing imaginal discs. Wing discs were dissected in batches of 30 and incubated in Shields and Sang M3 insect medium (Sigma-Aldrich, cat. no S8398) containing 2μg/ml puromycin (Sigma-Aldrich, cat. no. P8833) for 2 h at 25°C. 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 (∼50 kDa) and puromycin peptides (from smallest size visible to 245 kDa) was achieved using LI-COR Biosciences Image Studio software.