Visualizing smORF Peptide Localization

S2 cells were cultured in 10% FBS (v/v) Schneider’s media at 25°C to a confluent stage. Transfections and staining protocols were carried out as described in Pueyo et al. (2016b) (link). We used primary rabbit and mouse anti-Flag antibody (1:500; Sigma) and secondary Donkey anti-mouse FITC (1:1000; Jackson ImmunoResearch), anti-mouse Rhodamine (1:400;Jackson ImmunoResearch) and anti-rabbit Cy5 (1:400; Jackson ImmunoResearch) for detection of tagged-smORF peptides. For detection of mitochondria S2 cells were incubated in 500 nM Mitotracker Red CMXRos (Life Technologies) for 45 min and then fixed for immunohistochemistry. For Lysosome colocalization experiments, cells were incubated in Lysotracker-DND99 (1:1000; Molecular Probes) for 15 min, then mounted and observed in vivo. Images of 5–10 cells per transfection culture were captured on the LSM510 Axioskop 2 or on the Leica TCS SP8 confocal microscopes. For colocalization analysis Z-stack images were taken and analysed with the ImageJ plugin “Mander’s Coefficients” which was used to calculate Pearson’s correlation coefficient of tag to tag signal in 2 different channels.