Extended Data Fig. 3: Generation and validation of an ESB1 conditional knockout.

A) Western blot validation of the cKO cell line and the BSF pDex577 tagged ESB1 exogenous expression cell line (the intermediate in cKO generation), both induced with 10 ng/ml doxycycline. Predicted molecular weights for ESB1 are: 108 kDa (untagged), 137 kDa (Ty::mNG tag) and 145 kDa (6×Ty::mNG tag). The uncropped Ponceau-stained membrane and anti-mNG blot are shown. B) Western blot validation of the BSF pDex577 tagged ESB1 exogenous expression cell line induced with 100 ng/ml doxycycline for overexpression. C) Validation of genetic modifications of the ESB1 conditional knockout (cKO). Schematics represent the deleted and tagged loci and primer binding sites and orientations, uncropped DNA gels shows the resulting PCR products from extracted genomic DNA. D) mRNA abundance in the BSF pDex577 tagged ESB1 exogenous expression cell line, plotting RPKM of uniquely mapped RNAseq reads. The exogenous expression prior to addition of doxycycline (0 h) is plotted relative to the parental pJ1339 cell line. Other plots are 12, 24 and 48 h after addition of 10 ng/ml doxycycline relative to the cell line grown without doxycycline. Each shows n = 1 induction replicate. E) mRNA abundance in the cKO, plotting RPKM of uniquely mapped RNAseq reads. The cKO prior to doxycycline washout (0 h) is plotted relative to the BSF pDex577 tagged ESB1 exogenous expression cell line induced with 10 ng/ml doxycycline. Other plots are 12, 24 and 48 h after doxycycline washout relative to the cell line grown with 10 ng/ml doxycycline. Each shows n = 1 induction replicate. F) mRNA abundance in the procyclic form KO, plotting RPKM of uniquely mapped RNAseq reads for three clonal KO cell lines relative to the parental cell line.