The pathogenic basis of malaria. Figure S3. PfGRP170 is Expressed Throughout the Asexual Life CycleTMP was removed from tightly synchronized ring stage PfGRP170\GFP\DDD parasites and protein was isolated throughout the asexual Rabbit Polyclonal to NOM1 life cycle. Lysates were separated on a Western blot and probed with anti\GFP to visualize PfGRP170\GFP\DDD and anti\PfEF1 as a loading control. CMI-21-na-s003.tif (772K) GUID:?06141D86-42AB-43D5-B779-AD91E76AAA40 Figure S4. Conditional mutants of PfGRP170 localize to the ERSynchronized PfGRP170\GFP\DDD ring stage parasites were incubated with and without TMP for 24?hours. Parasites were then fixed with paraformaldehyde and stained with either DAPI, anti\GFP, and anti\BiP (ER) (A) or DAPI, anti\GFP, and anti\ERD2 (Golgi) (B). Images were TRC051384 taken as a Z\stack using super resolution microscopy and SIM processing was performed on the Z\stacks. Images are displayed as a maximum intensity projection. The scale bar is 2?m. CMI-21-na-s004.tif (1.1M) GUID:?2B7909FD-4F11-4E77-AF2A-D9824C562DF6 Figure S5. PfGRP170\BirA localizes to the parasite ER and biotinylates proteins. (A). Western blot of 3D7 (parental) and TRC051384 PfGRP170\BirA expressing parasites probed with anti\HA and anti\EF1. (B). Paraformaldehyde fixed PfGRP170\BirA parasites stained with anti\HA (PfGRP170\BirA), anti\PfPMV (ER), and DAPI. The images were taken with Delta Vision II, deconvolved and are displayed as a maximum intensity projection. The scale bar is 5?m. (C). A western blot analysis of 3D7 (parental) and PfGRP170\BirA parasites following a 24\hour incubation with biotin is shown. A fluorophore\labeled streptavidin secondary antibody was used to visualize biotinylated proteins. A control with PfGRP170\BirA parasites incubated without biotin is also shown. Anti\EF1 is used as a loading control. CMI-21-na-s005.tif (1.6M) GUID:?82582157-5E2D-4AF4-AA7F-65EA7A0590D5 Figure S6. Relative transcript abundance of proteins identified in both the anti\GFP co\immunoprecipitation and BioID mass spectroscopy approachesThe relative transcript abundance of the 11 PfGRP170 interacting proteins identified in Figure 4. The data are plotted using previously published genome\wide real\time transcription data46. CMI-21-na-s006.tif (2.3M) GUID:?35106F87-3514-42E6-8D13-5020BD8F1C2A Figure S7. PfGRP170 is not Required for Trafficking to the Host RBC.Tightly synchronized ring stage PfGRP170\GFP\DDD parasites were incubated with and without TMP for 24?hours. Following this incubation, parasites were fixed with acetone and stained with DAPI, anti\GFP (PfGRP170) and either anti\PfFIKK4.2 (A), anti\PfMAHRP1C (B), or TRC051384 anti\PfHSP70X (C). The images were taken with Delta Vision II, deconvolved, and are displayed as a maximum intensity projection. The scale bar is 5?M. Mean Fluorescent Intensity (M.F.I) was calculated for the exported fraction (PfFIKK4.2, PfMAHRP1C, and PfHSP70x) from individual cells. Data are from two independent experiments and is displayed as box\and\whiskers plots (whiskers represent the maximum and minimum M.F.I). The significance was calculated using an unpaired t test (NS?=?not significant). CMI-21-na-s007.tif (2.9M) GUID:?E7DE5DBA-F9C0-4F7D-9293-B99FBC32FD5A Figure S8. Overexpression of PfGRP170 does not Confer Artemisinin ResistanceTightly synchronized ring stage 3D7 and PfGRP170\BirA parasites were incubated with either 1% DMSO (Control) or Dihydroartemsinin (DHA) for 6?hours. After 6?hours the drug is removed by washing the culture with complete RPMI. Parasitemia was calculated using Giemsa stained thin blood smears at 0?hours (to calculate starting parasitemia) and 72?hours after either DMSO or DHA exposure. Four independent replicates of the experiment were completed for 3D7 and three for PfGRP170\BirA. The growth rate of the 3D7 and PfGRP170\BirA parasites, incubated only with DMSO, was calculated after 72?hours (A). The percent survival of parasites was calculated for 3D7 and PfGRP170\BirA after DHA exposure was calculated after 72?hours (B). CMI-21-na-s008.tif (680K) GUID:?48BB4C9E-18B2-4F79-B0AB-866271005F1A Figure S9. EIF2\ levels do not change in PM1 parasites in the presence or absence of TMP or a PK4 inhibitor. (Top) Synchronized ring stage PM1 parasites were incubated with and without TMP TRC051384 and in the presence and absence of 2?M PK4 inhibitor TRC051384 GSK2606414 for 24?hours. Protein was isolated from these.