A shRNA directed against green fluorescent protein (GFP) [30], with a sequence matching nothing in the E. histolytica genome, was utilized as a control for transfection and hygromycin selection for the Igl and URE3-BP transfectants. GFP shRNA transfectants were selected with the same level of hygromycin as other shRNA transfectants. For EhC2A, a scrambled control matching nothing in the E. histolytica genome was created, containing the same nucleotides as the EhC2A (363–391) shRNA,

but in a different order. Sequences of the shRNA sense strands are shown in Table 1. Non-transfected HM1-IMSS amebae were also included, with the results for Western blotting and qRT-PCR being statistically the same as the GFP controls. Three biological replicates were grown per shRNA transfectant, and one for the nontransfected HM1:IMSS amebae. All sample trophozoites were grown in 25 cm2 tissue culture flasks, and were harvested for crude lysate ARRY-438162 cost and for RNA isolation on the same day from the same flask. For protein and mRNA comparison, actin was used as the “”housekeeping”" control gene, as a loading and normalization control. Knockdown of Igl protein Four Igl shRNA constructs 4EGI-1 purchase targeted Igl. One construct, Igl1 (272–300), specifically targeted Igl1. Three

constructs, Igl (1198–1226), Igl (2412–2440), and Igl (2777–2805), were targeted to sequences conserved in both Igl1 and 2 (Table 1). The GFP shRNA transfectants were click here used as controls. Transfected trophozoites were selected with 100 μg/ml hygromycin for 48 hours before harvesting. Blots were probed with anti-Igl1 antibody, and with anti-actin antibody as a loading and normalization control. The level of Igl1 in the GFP shRNA transfectants was defined to be 100% (Figure 2, Table 4). The Igl1-specific (272–300) shRNA transfectant had a decreased amount of Igl1 protein, 27.8 ± 3.9%, as compared to the GFP shRNA control (Figure 2, Table 4). Igl (1198–1226) had 42.3 ± 6.2% and Igl (2777–2805) had 38.1 ± 9.4% of the GFP control Igl1 level. The Igl (2412–2440) Methane monooxygenase shRNA construct had no effect on Igl1 levels (95.3 ± 9.7% of the level in the GFP shRNA transfectants)

(Figure 2, Table 4). HM1:IMSS nontransfected amebae were not statistically different from the GFP shRNA control (Table 4). The Igl (1198–1226) and Igl (2777–2805) transfectants, when selected with 30 μg/ml hygromycin rather than 100 μg/ml, yielded less knockdown, having ~70% and ~65% of the control level of Igl1 (data not shown). Table 4 Summary of Igl1 protein levels in Igl shRNA transfectants shRNA Transfectant or Control Sample % of Igl1 protein level (± SE) P-value GFP 100.0 ± 3.6   HM1:IMSS 115.5 ± 11.8 0.1449 Igl (2412–2440) 95.3 ± 3.2 0.2078 Igl1 (272–300) 27.8 ± 1.3 < 0.0001 Igl (1198–1226) 42.3 ± 2.1 < 0.0001 Igl (2777–2805) 38.1 ± 3.1 < 0.0001 The average level of Igl1 protein in the GFP control shRNA transfectants was defined as 100% expression of Igl1 protein for computational purposes.