Overexpression of Drosophila Ph Proteins

Drosophila S2 cells were procured from Expression Systems and were grown at 27°C in ESF921 media. Stable S2 cell lines overexpressing either PH-WT (BLRP-2XFLAG-Ph) or PH-ML (BLRP-2XFLAG-Ph-L1547/H1552R) under inducible metallothionein promoter were grown as described previously by Wani et al (32 (link)). Expression of PH in Drosophila S2 cells or cell lines expressing PH-WT or PH-ML was checked by immunoblotting using anti-FLAG and anti-PH antibodies (Figs 1 and S1). Given the duplicated nature of ph genes (ph-p and ph-d) and dominant negative nature of PH-ML mutation, overexpression strategy instead of CRISPR-Cas9 strategy was taken.