Figure 3

Overexpression of PTB enhances IRES activity: A) IRES activity of Di-cis constructs (see Fig. 1B) transiently transfected into N2A cells either in the presence of endogenous PTB or overexpressed human PTB (huPTB) by co-transfection with the pC1-PTB construct. IRES activity is represented as the ratio of Firefly to Rennila luciferase (FLuc/RLuc), with the activity of control vector pRF set to "1". Each construct was tested 4 times and each experiment was done in triplicates. Data are expressed as mean ± SEM. B) Western blot of Di-cis constructs as summarized in Fig. 2A. All constructs were transiently transfected into N2A cells in the presence of either endogenous PTB (-) or over-expressed huPTB (+). 30 μg of total cytosolic proteins were resolved by 10% SDS-PAGE and immunodetection was done using anti-GFP (1:2,000) as primary antibody and peroxidase labeled anti-mouse IgG (1:7,500) as secondary antibody. C) Western blot of endogenous PTB and over-expressed huPTB from transiently transfected N2A cells. Immunodetection was done by using anti-PTB (1:1,000; Invitrogen) as primary antibody and peroxidase labeled anti-mouse IgG (1:7,500, Jackson ImmunoResearch) as secondary antibody. Note: endogenous and over-expressed huPTB is detected as a doublet band. D) Western blots applying β-actin served as loading control.