FIGURE 9.

ΔNp63α affects SNAIL1 protein level and YB-1 binding to SNAIL1 and YB-1 mRNAs in MDA-MB-231 cells. a, MDA-MB-231 cells were transfected with an empty vector (mock) or a fixed amount (1 μg in 60-mm dishes) of ΔNp63α or ΔNp63γ expression vector. 24 h after transfection, cells were harvested, and total extracts were prepared. 20 μg of each extract were loaded on SDS-PAGE and subjected to immunoblot with the indicated antibodies. b, MB-MDA-231 cells were transfected with an empty vector or ΔNp63α encoding plasmid. 24 h after transfection, total RNA was purified and retrotranscribed as described under “Experimental Procedures.” The expression level of ΔNp63α was checked by immunoblot (right panel). WB, Western blot. PCR was performed with primers designed to specifically amplify SNAIL1, TWIST, or HPRT transcripts and analyzed by agarose gel electrophoresis (left panel). Plot showing the level of SNAIL1 and TWIST transcripts was normalized respect to HPRT. Values are the mean of three independent experiments (lower panel). Nuclear extracts from ΔNp63α transfected MB-MDA-231 cells were immunoprecipitated with anti-YB-1 antibody. After reverse cross-linking, the YB-1-bound RNA was purified, retrotranscribed, and subjected to quantitative RT-PCR analysis using oligonucleotides designed to specifically amplify SNAIL1 transcript (c) or YB-1 transcript (d). Plots represent the % of enrichment of SNAIL1/YB-1 transcript normalized as indicated under “Experimental Procedures.” 1:50 of the input extract was loaded as control. The values are the means ± S.D. of three biological replicates.