Figure 3

Knockdown effect of NH2-isofom specific ASOs on protein level compared to p73-shRNA. (A) Western blot analysis showing the levels of full-length TAp73 and amino-truncated p73 forms 24 h following cotransfection of 2 μg p73 expression plasmids and 500 nM antisense or nsc LNA-DNA gapmers. Actin was used as a loading control. Protein bands were quantitated in relative software units by the Bio-Imaging-Analyzer (Fuji) using the TINA program (shown as fold induction or reduction, respectively, normalized to actin control bands. (B) Protein levels of ΔEx2/3 and ΔNp73 in H1299 cells cotransfected with ΔEx2/3p73 and ΔNp73 expression plasmids along with ASO-116 (left) or ASO-185/451 (right) compared to control-ASO. Relative densitometric units analyzed as described in A are shown in the bottom panel. (C) QPCR indicating the endogenous expression levels of p73 isoform mRNAs normalized to RPS9 in HEK293 cells at 48 h after transfection with 1 μg p73-shRNA encoding plasmid relative to levels in control sh-sc treated cells (set as 1). Bar graphs show results from three independent experiments. Data are the mean ± SD. Immunoblot of H1299 cells with endogenous ΔN levels and transfected with 1 μg of expression plasmids for TAp73, ΔEx2, and ΔEx2/3. Cells were treated with Ad-shp73 or Ad-shGFP at moi 20. Forty-eight hours after infection, cells were lysed, and extracts were probed with anti-p73 (ER-15) antibody. β-actin was used for equal loading (bottom panel).