Values are expressed as the
percentage of the GFP level ± SE, with the P-value following each, calculated using Student’s t test. For Western Geneticin order blotting, there were three biological replicates, each run in triplicate sets of serial dilutions (1:2, 1:4, and 1:8), with the exception of the HM1:IMSS nontransfected samples having one biological replicate rather than three. Protein levels were not statistically different between the Igl1 (272–300), Igl (1198–1226), and Igl (2777–2805) PDGFR inhibitor samples (tested with ANOVA, one-tailed, α = 0.05, 0.1 < P < 0.25) or the GFP, HM1:IMSS, and Igl (2412–2440) samples (tested with ANOVA, one-tailed, α = 0.05, P > 0.25). A representative Western blot is shown in Figure 2. Figure 2 Western blot for Igl shRNA transfectants. A representative Western blot is shown with one biological replicate each for the GFP control shRNA transfectant, the Igl1-specific (272–300), the selleck products Igl (1198–1226), the Igl (2412–2440), and the Igl (2777–2805) shRNA transfectants. HM1:IMSS samples are not shown. Results shown 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 performed for each sample. Each membrane was probed with anti-actin antibody as a loading control, or with anti-Igl1 antibody. Igl1 protein levels for the Igl shRNA and GFP shRNA transfectants and HM1:IMSS nontransfected amebae are summarized in Table 4. Knockdown of Igl mRNA Short sections of Igl were amplified via qRT-PCR using template cDNAs synthesized from the Igl and control GFP shRNA transfectant mRNAs. Four oligo pairs were used
to amplify Igl. Two sets of oligos targeted both Igl1 and Igl2 simultaneously, with one pair amplifying a 5′ section and the other a 3′ section conserved in both Igl1 and Igl2. The two others were specific for Igl1 or Igl2, targeting selleck screening library a non-conserved region. The oligo sequences and regions of Igl transcript amplification are shown in Table 3, and summarized qRT-PCR data for Igl is shown in Table 5. All samples were compared to the GFP control shRNA transfectants. Three of the four Igl shRNA transfectants showed knockdown of Igl transcripts for all sets of oligo pairs, ranging between ~60 and ~80% of the Igl level in the GFP shRNA control (Table 5). Igl (2412–2440) shRNA transfectants did not show any knockdown, and the HM1:IMSS nontransfected trophozoites were not statistically different from the GFP shRNA control (Table 5). Table 5 Summary of Igl mRNA levels in Igl shRNA transfectants shRNA transfectant or control sample Igl 5′ oligo pair P-value Igl 3′ oligo pair P-value Igl1 oligo pair P-value Igl2 oligo pair P-value GFP6 100.0 ± 4.1 — 100.0 ± 4.9 — 100.0 ± 3.0 — 100.0 ± 4.0 — HM1:IMSS 101.4 ± 4.3 0.7741 96.1 ± 3.5 0.3239 105.5 ± 3.1 0.1382 103.9 ± 6.1 0.5713 Igl (2412–2440) 100.6 ± 5.0 0.9172 103.4 ± 9.1 0.7717 91.1 ± 6.9 0.
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