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. 2007 Aug 3;27(3):420–434. doi: 10.1016/j.molcel.2007.06.016

Figure 4.

Figure 4

PTB/nPTB Knockdown Switches Splicing of α-Actinin

(A) Schematic representation of pA α-actinin minigene reporter containing α-actinin EF1a, NM, SM, and EF2 exons and complete introns (thin black lines). PTB represses SM exon inclusion.

(B) RT-PCR analysis of pA alternative splicing in HeLa cells after RNAi with negative control C2 siRNA (lane 1) or with siRNAs against PTB (lanes 2 and 3, P1 and P2), nPTB (lanes 4 and 5, N1 and N3), or PTB+nPTB (lanes 6–9). Lane 10, PCR negative control. Numbers to the left indicate size markers (nt) in lane M. The four amplicons shown schematically to the left are 289, 223, 208, and 142 nt in size. The percentage of each the four products is shown below each lane. For ease of presentation, values are rounded to whole numbers and SD are not shown.

(C) RT-PCR analysis of pA alternative splicing in HeLa cells after RNAi with control C2 siRNA (lane 1) or with siRNAs against PTB and nPTB (lanes 2–19). Increasing amounts (1, 10, 100, and 800 ng) of expression plasmids for PTB1 (lanes 4–7), PTB4 (lanes 8–11), nPTB (lanes 12–15), or smPTB (lanes 16–20) were cotransfected. pGEM4Z (lanes 1 and 2) and pCMV-β-gal (lane 3) were negative controls. Lane 20, PCR negative control. Only lanes 1–3 were carried out as triplicate repeats. Note that lane 2 in (C) is equivalent to lane 7 in (B). The lower effects of siRNAs P1 and N3 in (B) were associated with a lower degree of nPTB knockdown than in (C) (data not shown).