Figure 4.

Translocation of Lyn to the nuclei of 293T cells occurs only upon co-expression with MBNL1_42–43. (a) Representative confocal images of 293T cells co-transfected with plasmids carrying different MBNL1 isoforms (MBNL1_{40–41–42–43} in pEF1-Myc/HisA vector) and Lyn (pCMV6-XL4/Lyn). Nuclei were labelled with DAPI (blue), MBNL1 with anti-P9 (MBNL1_42–43) or anti-P11 (MBNL1_40–41) antibodies (red) coupled to immunodetection of Lyn (green). Scale bar 10 μm. (b) Representative WB analysis of whole-cell lysates from 293T cells transfected with expression vectors for Lyn, MBNL1₄₀, MBNL1₄₁, MBNL1₄₂ and MBNL1₄₃ probed with anti-Lyn antibody (top strip), anti-P11 antibody (middle strip) and anti-P9 antibody (bottom strip). (c) Representative co-immunoprecipitation of four-fifths of whole-cell lysates from 293T cells, overexpressing MBNL1₄₁ (pEF1/MBNL1₄₁ (left panel), lanes 1 and 2), or MBNL1₄₃ (pEF1/MBNL1₄₃ (right panel), lanes 3 and 4) and Lyn (pCMV6-XL4/Lyn, lanes 1–4). Thirty-six hours after transfection, whole-cell lysates were immunoprecipitated with anti-Lyn in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of the GST-Lyn SH3 domain. The immunocomplexes and one-fifth of the whole-cell lysates underwent WB analysis with anti-P11 or anti-P9 antibodies, respectively, and subsequently with anti-Lyn antibodies. The figure represents one of three independent experiments. The same set of experiments conducted with MBNL1₄₀ and MBNL1₄₂ isoforms demonstrated that these two proteins behave similarly to MBNL1₄₁ and MBNL1₄₃, respectively, again supporting the hypothesis that it is exon 5 that enables MBNL1s to interact with SFKs (data not shown). (d) Representative WB analysis of nuclear lysates from 293T cells transfected with expression vectors for Lyn (lanes 1–5), MBNL1₄₀ (lane 2), MBNL1₄₁ (lane 3), MBNL1₄₂ (lane 4) and MBNL1₄₃ (lane 5). The membranes were probed with anti-Lyn and with anti-Lamin B antibody as loading control. The resulting bands underwent densitometric analysis. The values (arbitrary units) represent the mean±S.D. of at least three experiments. Reference unit: lane 1 sample (only transfected with Lyn). **Significant difference for P<0.01 by Kruskal–Wallis analysis. (e) Representative WB analysis of nuclear lysates from 293T cells, transfected with expression vectors for Lyn (lanes 1–5), MBNL1₄₀ (lane 2), MBNL1₄₁ (lane 3), MBNL1₄₂ (lane 4) and MBNL1₄₃ (lane 5) and probed with anti-p-Tyr antibody. The membranes were reprobed with anti-Lamin B antibody as loading control