Figure 3

Steady state PfUT protein levels in the pfut mutants and the parental 3D7 strain. (a) Western blot analysis. Total protein lysates were prepared from late trophozoites cultured for 120 h in the presence or absence of 5 mM GlcN. Proteins were fractionated by SDS-PAGE and transferred to a PVDF membrane. The following antisera were used: anti-PfUT (raised against residues 473 to 712 of the N-terminal domain; rabbit; 1:1000); anti-α-tubulin (mouse, 1:1000); and anti-HA-tag (mouse, 1:1000). Representative Western blots are shown. Blots were cropped at the top and bottom to show the bands of interest. The uncropped, full-length blots are presented in Supplementary Fig. 9a (first and second panel) and 9b (third and fourth panel). A size marker is indicated in kDa. (b) The amount of steady state PfUT was quantified, on the basis of the anti-PfUT hybridization signal intensities, and normalized to the α-tubulin followed by normalization to steady state PfUT protein levels in untreated 3D7. Each symbol represents an independent biological replicate. A box plot analysis is overlaid over the individual data points, with the median (thin grey line), mean (thick black line) and the 25% and 75% quartile ranges being shown. Statistical significance between the data sets was calculated using Holm-Sidak one way ANOVA. n.s. – not significant. (c) As in (b) with the exception that the anti-HA signal intensities were analyzed and normalized to the corresponding α-tubulin signal followed by normalization to steady state PfUT-HA protein levels in untreated pfut mutants. This figure was reproduced from the PhD thesis by Jankowska-Döllken³².