Fig. 2.
Reduced nuclear matrix association of Mof in Zmpste24−/− MEFs. (A and B) Colocalization of Mof and lamin A in nuclear matrix. (A) Nuclear matrices were prepared from wild-type MEFs and stained with anti-Mof and anti-lamin A/C antibodies. Staining with DAPI indicated efficient digestion of DNA in the isolated nuclear matrix. Control cells (labeled “TOTAL”) were processed directly without pre-extraction and stained with anti-lamin A/C antibody and DAPI. (B) Histogram:immunofluorescence signals collected from 488 channels (lamin A/C) and 594 channels (Mof) were overlaid to depict colocalization. (C) Zmpste24+/+ and Zmpste24−/− MEFs were subjected to subcellular fractionation to yield nucleoplasmic (S2), nuclear matrix + chromatin (P2), soluble chromatin + nucleoplasmic (S2′), nuclear matrix (P2′), and cytosolic (S1) fractions. Equal amounts of protein (20 μg) were resolved and Western-blotted with indicated antibodies. Coomassie brilliant blue (CBB) staining of the gel was performed to ensure equal loading of the fractions. (D) HEK293 cells were transiently transfected with Mof-myc together with FLAG-tagged lamin A or prelamin A for 48 h. As controls, cells were transfected with empty vector, FLAG-tagged lamin A, prelamin A or Mof-myc alone. Immunoprecipitation was performed with FLAG antibody-conjugated agarose beads, and immunoblots were probed with indicated antibodies. Three independent experiments were performed and identical results were obtained.