PTBP1 binds specifically to TP63 premRNA sequences. A,TP63 exon 12 genomic locus and positions of the RNAs tested in RNA/protein shift assays. Top lane, RBPmap predicted PTBP1 binding sites. Middle, Positions and names of the RNA fragments tested along the TP63 exon 12 genomic sequence. Bottom lane, nucleotidic conservation among vertebrates. B, Immunopurification of PTBP1/RNA complexes or control experiments (Ig and beads only) from HaCaT cell extracts. Immunopurification was controlled by PTBP1 and PCNA Western blot analysis as indicated in input (10%) or immunoprecipited (IP) samples (top). Protein MW are indicated on the side of the membranes. The asterisk denotes the light chain of the IgG. Bottom, RT-PCR detection of TP63 pre-mRNA with primers located in intron 11 and exon 12. C, Same as B from SCC9 cell extracts. D, qRT-PCR detection in HaCaT cells of different mRNA or pre-mRNA portions of TP63 from IP or control samples relative to input signal. Statistical assessment between PTBP1 and control IP was measured by a Student t test (n = 3). E, PTBP1/RNA-binding curves [bound/(bound + free)] obtained for the indicated RNAs (n ≥ 4). RNA mutated to remove PTBP1 binding sites are shown in gray in A. The calculated Kd and their SD is shown for each plot.