FIGURE 2:

Expression of E6 reporters. TfR RNAi cell lines containing RNAi^R HA:E6 or HA:E6ΔG were cultured without or with tetracycline. (A) Cell density was enumerated by hemocytometer and cultures were adjusted to starting density daily. Data are presented for three biological replicates (mean ± SD). All subsequent analyses were performed after 24 h of silencing. (B) Wild-type RNAi-sensitive E6 (E6^wt) and RNAi^R HA:E6 or HA:E6ΔG (E6^R) transcript levels were assessed by qRT-PCR. Results are normalized to uninduced controls and are presented as fold changes for three biological replicates (mean ± SD). (C) Cell extracts were subjected to immunoblotting with rabbit anti-TfR (αTfR), mAb anti-HA (αHA), rabbit anti-Hsp70 (αH70), or anti-BiP (αBiP). All lanes have 10⁷ cell equivalents. Mobilities of molecular mass markers are indicated. Star indicates a nonspecific background polypeptide consistently observed with anti-TfR. (D) Receptor-mediated uptake of fluorescent transferrin (Tf) and tomato lectin (TL) was measured by flow cytometry. Data are presented as median fluorescent intensity (MFI ± SD) for three biological replicates and are normalized to unsilenced control cells.