FIG. 2.

Lentiviral vector-mediated delivery of siRNA targeting PML-RARα results in specific knockdown of expression. (A) The indicated lentiviral siRNA expression constructs were cotransfected into 293T cells with an expression construct for the long isoform of PML-RARα. siPML-RARα/S and siPML-RARα/L target the breakpoint regions of the short and long PML-RARα isoforms, respectively. si GFP, irrelevant control siRNA. Immunoblotting of whole cell extracts was performed with anti-PML antibody to detect PML-RARα (right). An anti-α-tubulin antibody was used to confirm equal protein loading (bottom). (B) The short isoform of PML-RARα was cotransfected with the indicated lentiviral siRNA expression constructs, followed by Western blotting of whole cell extracts using anti-PML and anti-α-tubulin antibodies. NS, nonspecific band. (C) Control 293T cells or 293T cells stably expressing the short isoform of PML-RARα were left untransduced (lanes 1 and 4), or were transduced with a lentiviral vector expressing siRNA specific for the short PML-RARα isoform (lanes 2 and 5) or with control lentiviral vector expressing only GFP (lanes 3 and 6). Whole cell extracts were prepared 5 days posttransduction and Western blotting was performed with anti-RARα antibody to detect PML-RARα and anti-α-tubulin antibody. (D) NB4 cells were mock transduced or transduced with the indicated lentiviral vectors delivering siRNA against the breakpoint regions of the short or long PML-RARα isoform. Whole cell extracts were prepared 5 days posttransduction and immunoblotting was performed with anti-PML, anti-RARα, and anti-α-tubulin antibodies.