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. 2007 Jan 31;81(8):4116–4129. doi: 10.1128/JVI.01924-06

FIG. 3.

FIG. 3.

E6large and E6small/E6* influence the cellular level of procaspase 8 in opposite directions. (A) Both E6large and E6small/E6* are expressed in cells. U2OS cells (2 × 106) were transiently transfected with pHAE6large (lane 1) or pHA-E6sense (lane 2). Forty-eight hours posttransfection, the cells were lysed in 100 μl of Laemmli buffer and subjected to separation by SDS-PAGE. Detection of expressed HA-E6 was performed using anti-HA rat polyclonal antibodies conjugated with HRP. (B) Cells expressing E6large display a decreased level of procaspase 8, while cells expressing E6small/E6* display an increased level of procaspase 8. U2OS cells stably transfected with HA-E6large (U2OSE6L12) or HA-E6small/E6* (U2OSE6S9), as well as U2OSE6tet24 cells grown in the absence or presence of 100 ng/ml doxycycline (dox) (1 × 106 per preparation), were lysed in 100 μl of Laemmli buffer, and 20 μg (total protein) of each cell lysate was separated by SDS-PAGE. Procaspase 8 was detected by immunoblotting using monoclonal anti-caspase 8 antibodies (top). The blot was reblotted with monoclonal antibodies directed against β-actin to normalize for loading (second blot). The level of HA-E6 expressed in each of these cell lines was also analyzed by immunoprecipitation (2 × 106 cells per preparation) using anti-HA monoclonal antibodies and then detected by using anti-HA rat polyclonal antibodies conjugated to HRP (third blot). (C) Stable clones transfected with E6large (U2OSE6L12) and E6small/E6* (U2OSE6S9) each expressed the expected version(s) of the E6 message, as determined by RT-PCR.