Journal: eLife

Article Title: PI(3,4,5)P3 allosteric regulation of repressor activator protein 1 controls antigenic variation in trypanosomes

doi: 10.7554/eLife.89331

Figure Lengend Snippet: ( A ) Diagram of procyclic forms PIP5Pase CN cell line. Constructs containing drug resistance markers flanked by ~500 bp of the 5’-untranslated region (UTR) and 3’-UTR of the PIP5Pase gene were used for homologous recombination and replacement of endogenous PIP5Pase alleles in T. brucei 29.13 procyclic cells. SaCas9 with guides targeting the PIP5Pase gene was used to cleave the PIP5Pase alleles and increase recombination rates. A tet-regulatable V5-tagged PIP5Pase allele (using pLEW100-3V5 vector) was inserted in the silent ribosomal spacer, for controlled PIP5Pase gene expression. Arrows indicated by 5’_Amp and 3’_Amp show hybridization sites of primes used to verify the constructs’ recombination. ( B ) Real-time PCR amplification from genomic DNA of T. brucei 29.13 (WT) or CN PIP5Pase cell line. Primers 5’_Amp and 3’_Amp, indicated in A, were used in amplification. The Tubulin gene was used as endogenous control. ( C ) Real-time PCR amplification of PIP5Pase from cDNA of CN PIP5Pase cell line growing in the presence of tet (tet +, 0.5 µg/mL for 24 hr) or after removal of tet from medium (Tet -) for several time points (indicated in graph x-axis). Tubulin was used as endogenous control. The data shown are the mean ± SDM of three biological replicates. ( D ) Subcellular localization of V5-tagged PIP5Pase in procyclic forms using T. brucei CN PIP5Pase (100 ng/mL of tet). PIP5Pase-V5 was detected with α-V5 monoclonal antibodies (mAb) followed by goat α-mouse IgG-AlexaFluor 488 (green). DAPI stains DNA (blue). DIC (Differential image contrast). N, nuclear DNA; K, kinetoplast DNA. ( E ) Cumulative growth curve of T. brucei CN PIP5Pase (0.5 µg/mL of tet). The data shown are the mean ± SDM of three biological replicates.

Article Snippet: Membranes were probed for 2 h at RT (or overnight at 4 °C) with mAb α-V5 (BioShop Canada Inc, catalog number TAG006.100) 1:2500 in 6% milk in PBS 0.05% Tween (PBS-T).

Techniques: Construct, Homologous Recombination, Plasmid Preparation, Gene Expression, Hybridization, Real-time Polymerase Chain Reaction, Amplification, Control, Bioprocessing