C2 P1. PTB. p50. BAG-1. p46. p36. Actin. 1.2. 1. 0.8. p1. 0.6. Relative luciferase activity. c2. 0.4. 0.2. 0. pRF. pRBagF. Supplementary data 1. HeLa cells were transfected with siRNAs directed against PTB P1
Relative luciferase activity
Supplementary data 1.
HeLa cells were transfected with siRNAs directed against PTB P1
(AAC UUC CAU CAU UCC AGA GAA) or a control sequence and C2 (AAG GUC CGG CUC CCC CAA AUG)
as described previously (Mitchell et al 2005). Cells were then transfected with the di-cistronic plasmid pRBagF (as
described; Coldwell et al 2000) or the control plasmid pRF. Lysates were prepared from transfected cells
and luciferase activities were measured and calculated as previously described (Mitchell et al., 2003) using
a Dual-Luciferase reporter assay system (Promega). Western analysis was performed on these samples to
detect expression of PTB, and BAG-1 whilst actin was used as a loading control. It can be seen that when PTB levels are reduced there is a specific reduction in the level of the p36 isoform of BAG-1 and a corresponding reduction in BAG-1 IRES activity.