Figure 3. Expression of mature miR-155 does not inhibit efficient Env expression.

(A) Un-transfected HeLa cells (lane 1) or HeLa cells transfected with 2 µg of either pCMV-Empty vector backbone (lane 2), pMIR155HG (lane 3) or Env expression vectors pAD8-140 miR-155S (lane 4), pAD8-140 miR-155 (lane 5) or pAD8-140 (lane 6). Forty-eight hours post-transfection, total RNA was isolated and resolved using denaturing urea PAGE. Membranes were subsequently probed with an [³²P]-ATP radio-labelled oligonucleotide complementary to the mature guide strand of miR-155. Equal loading of RNA was confirmed by probing with a [³²P]-CTP labelled, DNA probe specific for U6 snRNA. (B) Protein lystates were isolated from similarly transfected HeLa and resolved using SDS-PAGE. Expression levels of Env were analysed by immunoblotting with anti-gp120 D7324 and anti-β-actin antibodies.