Figure 2

Activity of MBNL1 N and C-terminal domains in splicing repression and activation. A. Schematic of the regions of MBNL1 used in the experiments in this figure (top). Lower panel, anti-FLAG western blot showing expression of MBNL proteins. B. The Tpm1 ΔBP minigene was co-transfected with control (C2, lane 1) or MBNL1 targeting siRNAs (lanes 2–5). In addition, full-length MBNL1 (lane 5) or the MBNL1 N- or C-terminal domains (lanes 3–4) were cotransfected along with the MBNL1 siRNA. C. The Vldlr minigene was transfected alone (lane 1) or with full-length MBNL1 (lane 4) or the MBNL1 N- or C-terminal domains (lanes 2, 3). D. Anti-MS2 western blot of HeLa cells transfected with MS2 alone, lane 6, or MBNL1-MS2 fusion proteins: full length, lane 5; MBNL1 C-terminus, lane 4, MBNL1 N-terminus, lane 3; mock transfections lanes 1, 2. E. Schematic of the Tpm1 ΔBP DMS2 minigene in which the downstream MBNL site is replaced by a pair of MS2 hairpins (upper panel). Lower panel, RT-PCR of HeLa cells transfected with Tpm1 ΔBP (lane 1) or Tpm1 ΔBP DMS2 (lanes 2–6). Cotransfections with MS2 alone (lane 6) or the indicated MBNL1-MS2 fusion proteins (lanes 3–5). F. Schematic of the Vldlr-MS2 minigene in which the downstream MBNL site is replaced by an MS2 hairpin (upper panel). Lower panel, RT-PCR of HeLa cells transfected with Vldlr (lane 1) or Vldlr-MS2 (lanes 2–6). Cotransfections with MS2 alone (lane 6) or the indicated MBNL1-MS2 fusion proteins (lanes 3–5).