Fig. 1.
siRNA-mediated silencing of P-protein expression in HEK293T cells. HEK293T cells stably transected with doxycycline-inducible P2 RNAi were treated with 0.1µg/ml doxycycline for 96 h to knockdown expression of P2 protein. Whole cell lysates, purified ribosomes and cytoplasmic fractions were prepared post-treatment and separated by SDS-PAGE. Proteins were transferred to nitrocellulose, and probed with monoclonal antibody against the conserved C-termini of P-proteins (3BH5). P1 and P2 protein levels are expressed as a percentage relative to levels in undepleted cells. Levels were calculated from the band intensities, which were normalized to either P0 (whole cell lysates and purified ribosomes) or ß-actin (cytoplasmic fraction). Representative blots from 2–3 independent experiments are shown.