Plasmodium palmitoylation machinery engineered in E. coli for high‐throughput screening of palmitoyl acyl‐transferase inhibitors
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...Source data are available online for this figure. ª 2020 The Authors The EMBO Journal 39: e104168 | 2020 3 of 22 isp3::6HA, by tagging endogenous ISP1 with a triple V5 epitope (3V5) in the isp3::6HA parasite (Appendix Fig S1) and observed similar co-localization of ISP1/ISP3 at the periphery of zygote protrusion (Fig 2B)....
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...18 of 22 The EMBO Journal 39: e104168 | 2020 ª 2020 The Authors...
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...Indeed, the Cter75 fused with ª 2020 The Authors The EMBO Journal 39: e104168 | 2020 7 of 22 a BFP::Myc peptide could be palmitoylated by co-transfected WT DHHC2-HA in HEK293 cells, while the Cter75 containing C4A mutations (Cter75*) failed to be modified (Fig 5M)....
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...We further generated a doubly tagged parasite (DTS), isp1::3V5/ 2 of 22 The EMBO Journal 39: e104168 | 2020 ª 2020 The Authors B C D E F in vitro 2 4 6 8 10 12 0 20 40 60 80 100 Time post fertilization (h) Δisp1/3 wildtype *** ****** P ar as ite o f s ta ge I an d II (% ) Δisp1/3 wildtype H J wildtype Δisp1/3 wild typ e ** 0 2 4 6 8 10 12 S al iv ar y gl an d sp or oz oi te s p er m os qu ito (× 10 3 ) Δis p1/ 3 wild typ e G wildtype Δisp1 Δisp3 Δisp1/3 Py/Pf.isp1 ISP3 (HA) Hoechst Py/Pf.isp3 6HA 4 5 6 7 O oc ys ts p er m os qu ito (D ay 7 ) 0 50 100 150 200 250 300 350 **** n=55 n=59 A Δis p3 wild typ e Δis p1 Δis p1/ 3 ** *** * ns C on ve rs io n ra te to m at ur e oo ki ne te (% ) 0 20 40 60 80 3V5 ISP1 (V5) Hoechst Pyisp1 Pfisp1 Pyisp3 Pfisp3 I Δis p1/ 3 0 20 40 60 80 C on ve rs io n ra te to m at ur e oo ki ne te (% ) *** *** *** ** wi ldt yp e Δis p1 /3 Δisp1/3 rescued with Py isp 1 Pf isp 1 Py isp 3 Pf isp 3 ** P ar as ite o f s ta ge I an d II (% ) 0 20 40 60 80 100 Δis p1/ 3 wild typ e in vivo 25 25 15 Δisp1/3 rescued with HA ISP3 V5 ISP1 P28 D TS Δ is p1 /3 P yi sp 1:...
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...16 of 22 The EMBO Journal 39: e104168 | 2020 ª 2020 The Authors required to fully elucidate the mechanisms by which the IMC recruits the palmitoylated DHHC2....
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