Knockdown of Ptbp1/PTBP1 affects splicing of PKM in MLL-transformed cells. (A) Schematic depiction of alternative splicing options for PKM RNA. Concentrations of available Ptbp1/Hnrnpa1 dimers altered exon retention in PKM RNA. High levels of Ptbp1/Hnrnpa1 favored alternative inclusion of exon 10, thus preferentially producing isoform PKM2. (B) Analysis of PKM splice isoforms. RNA was isolated from selected shRNA (LNGFR)-positive and -negative murine and human cells. After reverse transcription, the relevant PKM region was amplified by PCR and digested with the PstI enzyme, which exclusively cuts the cDNA of the PKM2 isoform. Digested and undigested PCR products are shown. Please note that an additional murine shRNA (shRNA2275) was included in this experiment. (C) PKM protein isoform analysis. Proteins were extracted from cells as in panel B and subjected to western blot analysis with antibodies specific for PKM1, PKM2, or total PKM, as indicated. (D) MLL-ENL activity correlated with Ptbp1 expression and Pkm2/Pkm1 ratio. Left: RNA was isolated from primary cells transformed by a tamoxifen-inducible version of MLL-ENL at the indicated time points (0 days [0d], TAM present and MLL-ENL active). Pkm1/2 splice analysis was performed by detection of the Pkm2-specific RFLP through digestion with PstI. Ptbp1 transcript levels in the same RNA samples were determined by quantitative reverse transcription-PCR.