Fig. 7. Association of Rb with LAP2 and disruption of Rb-LAP2 complexes by mutant lamin A. The expression and localization of Rb and LAP2 were investigated by double immunofluorescence and confocal microscopy by co-staining undifferentiated (a) and differentiated myoblasts (b) with anti-LAP2 antibodies (left) and anti-RbS780 (centre). Micrographs are displayed as individual black and white or two-colour merged images (right). The formation of multiprotein complexes containing Rb, LAP2 and lamin C was investigated by immunoprecipitation (c-e) from cultures containing undifferentiated myoblasts (left), differentiated myoblasts (centre) and myotubes (right). Cell extracts were immunoprecipitated with anti-LAP2 antibodies (c,e) or anti-lamin C antibodies (d) and blotted with anti-RbS780 (c,d) or anti-LAP2 antibodies (e). sup, supernatant; Ppt, precipitate; ^*, hyperphosphorylated Rb; -, hypophosphorylated Rb (c,d); arrow in e, LAP2. Expression of mutant (mut) lamin A disrupts LAP2 and leads to loss of expression of Rb (f-i). C2C12 myoblasts were transfected with wild-type (f,h) or mutant (g,i) lamin A constructs and fixed and stained with antibodies against LAP2 (f,g) or RbS780 (h,i) before (top) or after (bottom) cultures were induced to differentiate. Each montage shows the exogenous lamin (left), the endogenous LAP2 or Rb (middle) and two-colour merged images (right). Arrowheads (g, bottom) show transfected cells in which LAP2 is no longer detected. Arrows (i, bottom) show transfected cells in which RbS780 is no longer detected. Bars, 10 µm.