Fig. 1

Actin is almost undetectable as soon as 72 h after rapamycin induction of act1 excision. a Quantitative immunofluorescence assay (IFA) of actin. Vacuoles were stained with α-ACT1 (Soldati) at 0, 24, 48, 72 and 96 h post rapamycin induction. Fluorescence intensity was analysed using CellProfiler software. The internal background was calculated for each vacuole and was then subtracted from the calculated intensity and plotted with mean and 95% confidence intervals. Dotted line shows the host cell background calculated using a YFP ⁺ parasite strain without antibodies. n = 60 vacuoles analysed per time point. The datasets were compared with a two-tailed Student’s t-test. **** P < 0.0001, ** P < 0.01. b Representative images of the labelled vacuoles used to analyse the ACT1 levels over time. Images shown were processed together under the same conditions to highlight the ACT1 signal intensity in the vacuoles. After 72 h actin was undetectable in YFP(+) vacuoles by IFA. Scale bar: 10 μm. c Western blot analysis of actin protein level. Immunoblot made with parasite lysates taken at 0, 24, 48 and 72 h after rapamycin induction. Aldolase was used as a loading control. Expression of YFP upon act1 excision was checked using α-GFP. d Relative levels of actin were analysed with LiCor Odyssey Image Studio 5.0 and normalised first using aldolase loading control and then compared against the LoxPAct1 control. The dataset was compared with a two-tailed Student’s t-test. Error bars represent standard deviation. * P < 0.05, n = 4