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. 2021 Aug 12;118(33):e2108963118. doi: 10.1073/pnas.2108963118

Fig. 4.

Fig. 4.

Expression of WT or mutant FUT1s in E. coli or within Leishmania. (A) Expression and purification of recombinant GST-FUT1 fusion proteins from E. coli visualized following SDS-PAGE. Lanes 1–6 are GST-FUT1 (WT). Lane 1: before-induction whole-cell lysates; lane 2: postinduction whole-cell lysates; lane 3: soluble fraction; lane 4: insoluble fraction; lane 5: elution; lane 6: concentrated eluted protein; lane 7, purified GST-CAT-MUT; lane 8, purified GST-MTP-MUT; and lane 9, purified HIS-BLOCK-MUT. Molecular weight markers are shown on the left. The images from lanes 1–4 and 5–9 are from separate experiments. (B) Western blot analysis of C-terminal HA-tagged FUT1s expressed in Leishmania. Lysates from parasites expressing the indicated HA-tagged FUT1s in a ∆fut1−/+pXNGPHLEO-FUT1 background are shown (the presence of untagged WT FUT1 was required as none of the mutants were viable in its absence; Fig. 6). Western blots were performed with anti-HA to visualize the tagged FUT1 expression, and anti–L. major H2A as a loading control.