Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - (SuppFigure1.tif; 607 KB): The effect of SAP155 Ab on the mobility of the CRCE 1:protein complex. Nuclear extracts were prepared from A549 cells and subjected to EMSA binding conditions by reacting with labeled CRCE 1. Following 20 min equilibration, no antibody, a control antibody (IgG), the antibody for SAP155, or the denatured antibody for SAP155 was introduced to the binding assay for an additional 30 min. Samples were then subject to electrophoretic separation using a 5% TBE-Polyacrylamide (27:1 acrylamide/bis-acrylamide). Positions of "supershifted" complexes are indicated by arrows. The data are representative of four determinations on four different occasions.
• Supplemental Figure 2 - (SuppFigure2.tif; 444 KB): A,B) The ability of individual SAP155 siRNA's to downregulate SAP155 and induce the activation of the Bcl-x(s) 5' splice site. A549 cells were transfected with 0.2 μM control siRNA or 0.2 μM of an individual SAP155 siRNA SMARTselection™ (SAP155-1, SAP155-2, SAP155-3, or SAP155-4) for 48 hr. A) Total protein lysates were produced and subjected to 7.5% SDS-PAGE analysis, transferred to polyvinylidene difluoride, and immunoblotted for SAP155 as described in "Experimental Procedures". B) Total RNA was extracted and analyzed by RT-PCR for the ability of each individual SAP155 siRNA to effect the alternative 5’ splice site selection of Bcl-x. The graph depicts the ratio of Bcl-x(L) mRNA to Bcl-x(s) mRNA for each selection as determined by densitometry of RT-PCR fragments stained with SYBR Gold (Invitrogen). C) The ability of SAP155 siRNA to activate Bcl-x(s) at low concentrations. A549 cells were transfected with 0.2 μM control siRNA or 0.01-0.025 μM of SAP155 SMARTpool® siRNA for 48 hr. Total RNA was extracted and analyzed by RT-PCR for the ability of SAP155 siRNA to effect the alternative 5' splice site selection of Bcl-x. The graph depicts the ratio of Bcl-x(L) mRNA to Bcl-x(s) mRNA as determined by densitometry of RT-PCR fragments stained with SYBR Gold (Invitrogen). Data are representative of two separate determinations on two separate occasions.