All samples were normalized to actin, and compared to the GFP control using Student’s t test. URE3-BP Doramapimod in vitro protein levels are GSK690693 nmr not statistically different between the URE3-BP (350–378) and (580–608) samples (two-tailed Student’s t test for comparing two sample averages, P = 0.3262) or between the GFP and HM1:IMSS nontransfected samples (two-tailed Student’s t test for comparing two sample averages, P = 0.2346). A representative Western blot is shown in Figure 3. Figure 3 Western blot for URE3-BP shRNA transfectants. A representative Western

blot is shown with three biological replicates each (one dilution shown) for GFP control, URE3-BP (350–378), and URE3-BP (580–608) shRNA transfectants. HM1:IMSS samples are not shown. Results are representative of three biological replicates per shRNA transfectant with each sample run in triplicate. Serial dilutions of the crude lysates (1:2, 1:4, and 1:8) were also done for each

sample. Each membrane was probed with anti-actin antibody as a loading control, or with anti-URE3-BP antibody. URE3-BP protein levels are PF-6463922 mouse summarized in Table 6. Knockdown of URE3-BP mRNA Three different oligo pairs, one amplifying the 5′ end of URE3-BP, one the 3′ end, and one a section in the middle, were used in qRT-PCR to amplify URE3-BP in cDNA from GFP shRNA control transfectants, URE3-BP (350–378) and URE3-BP (580–608) shRNA transfectants, and HM1:IMSS nontransfected trophozoites. Oligo sequences are shown in Table 3. Actin was used as the normalization control. The URE3-BP (350–378) shRNA transfectant had an average of about 69% of the GFP control URE3-BP transcript level, and the URE3-BP (580–608) shRNA transfectant had about 13% of the of the GFP shRNA

control URE3-BP level (Table 7). Table 7 Summary of mRNA levels in GFP shRNA control transfectants, URE3-BP shRNA transfectants, and nontransfected HM1:IMSS trophozoites shRNA transfectant or control sample URE3-BP 5′ oligo pair P-value URE3-BP middle oligo pair P-value URE3-BP 3′ oligo pair P-value GFP 100.0 ± 2.9 — 100 ± 2.8 — 100 ± 4.3 — HM1:IMSS 106.4 ± 5.8 0.2928 108.9 ± 5.6 0.1008 102.8 ± 5.0 0.5792 URE3-BP (350–378) 67.0 ± 2.5 <0.0001 67.4 ± 2.0 <0.0001 72.2 ± 2.8 <0.0001 URE3-BP (580–608) 12.4 ± 0.8 <0.0001 13.5 ± 3.3 <0.0001 12.5 ± 3.8 <0.0001 IMP dehydrogenase The average URE3-BP transcript level as measured by qRT-PCR and normalized to actin was defined as being 100% in the GFP shRNA control transfectants. HM1:IMSS nontransfected amebae were also included. Three different oligo pairs amplifying the 5′, middle, and 3′ sections of URE3-BP were used (sequences and locations are shown in Table 3). Student’s t test was used for statistical analysis. Three biological replicates were each assayed in quadruplicate with each oligo pair, with the exception of the HM1:IMSS samples, which had one biological replicate. Values are expressed as the percentage of URE3-BP mRNA of the GFP control shRNA transfectant level ± SE, with the P-value following each.