LINT-25 and LAP2α interact in vitro. (A) His-tagged, recombinant LINT-25 and LAP2α were incubated alone or together and mixed with immobilized LAP2α antibody. Immunoprecipitates (P) and supernatants (SN) were analyzed by immunoblotting detecting LAP2α (upper panel) and the His-Tag (lower panel). Input represents 10% of protein used for IPs. (B) In vitro translated, [³⁵S]methionine-labeled LINT-25 was incubated alone or together with in vitro translated and labeled LAP2α or LAP2β1-408 and mixed with immobilized LAP2α- and LAP2β antibodies (IP) or hybridoma medium (DMEM) as a control. Immunoprecipitates (P) and supernatants (SN) were analyzed by SDS-PAGE and autoradiography. Input represents 2% of protein used for IP. Note, that the signals of the different protein bands do not reflect the relative protein levels due to different numbers of methionines in the proteins' primary sequences. (C) Recombinant LINT-25 was transblotted to nitrocellulose, stained with Ponceau S (Ponc), or detected with αHis-tag reagent (αHis) or overlaid with recombinant LAP2α or buffer. Bound LAP2α was detected using LAP2α antibodies (αLAP2α). Numbers indicate molecular masses of marker proteins in kDa; mLINT-25, monomeric LINT-25; dLINT-25, dimeric LINT-25.

LINT-25 localizes to the nucleus and associates with chromatin during mitosis. (A) HeLa cells were lysed in hypotonic buffer with iodoacetamide (IAA) (left panel), and total lysates (Pre), nuclear (N) and cytoplasmic (C) fractions were analyzed by immunoblotting using antibodies against LINT-25, lamin A/C and LAP2 (right panel). HeLa cells were lysed in culture dishes in buffer with the indicated concentrations of Triton X-100 and salt and total lysates (Pre), insoluble pellet fractions (P) and supernatants (S) were analyzed by immunoblotting. Note that the LINT-25 double band is caused by additional alkyl-groups resulting from the IAA treatment (see also supplement). Numbers indicate molecular masses of marker proteins in kDa; mLINT-25, monomeric LINT-25; dLINT-25, dimeric LINT-25; La, lamin. (B,C) HeLa cells were fixed with formaldehyde and double-stained for immunofluorescence microscopy using LINT-25 antiserum and antibodies against the indicated antigens. DNA was stained with Hoechst dye. Confocal images of interphase cells (B) or various stages of mitosis (C) are shown. Arrowheads indicate outer core regions. Bars, 10 μm.

LINT-25 is upregulated and translocated to chromocenters in non-proliferating fibroblasts. BJ1 human foreskin fibroblasts (A,B), human primary skin fibroblasts (C) and mouse primary skin fibroblasts (D) were cultivated in normal medium (P), under low serum conditions for 6-9 days (Q6, Q7, Q9) followed by serum re-stimulation for 38 hours (Q9→P), or passaged in culture until reaching replicative senescence (S). Cells were processed for immunofluorescence microscopy (confocal images are shown. Bars, 10 μm) or immunoblotting of cell lysates (B, left panel). Samples were analyzed using antibodies against the indicated antigens or stained for senescence-associated β-galactosidase activity (SA β-gal). For testing mRNA levels (B, upper right panel), semiquantitative RT-PCR was performed using primers for indicated cDNAs. Ethidium bromide-stained agarose gels of PCR fragments are shown. In order to test proteasomal degradation of LAP2α (B, lower right panel), cells were starved in low serum medium for 4 days and incubated with or without proteasome inhibitor MG-132 during the last 12 hours. Proteins were precipitated from lysates using LAP2α antibody or hybridoma medium (DMEM) as a control, and immunoprecipitates (P) and supernatants (SN) were analyzed by immunoblotting using antibodies against LAP2α (upper panel) and ubiquitin (lower panel). Input (Inp) represents 1.5% of protein used for IPs. Black and white arrowheads point to ubiquitylated LAP2α. Note, that the ubiquitin blot is weak because of the large background detected in the region >110 kDa. Numbers indicate molecular masses of marker proteins in kDa; mLINT-25, monomeric LINT-25; dLINT-25, dimeric LINT-25; La, lamin; Ubi-LAP2α, ubiquitylated LAP2α; pRb, retinoblastoma protein.

LINT-25 is upregulated during differentiation. (A) E14.5 fetal liver-derived proliferating erythroid progenitors were switched to erythroid differentiation medium and processed for immunoblotting or immunofluorescence microscopy at the indicated time points, using antibodies as indicated. Numbers indicate molecular masses of marker proteins in kDa; mLINT-25, monomeric LINT-25; dLINT-25, dimeric LINT-25; La, lamin; pRb, retinoblastoma protein. (B) C2C12 myoblasts or myotubes differentiated in 1% FCS for 10 days were processed for immunofluorescence microscopy using antibodies against the indicated antigens. Confocal images are shown. Bars, 10 μm.

LINT-25 protein levels change during the cell cycle of proliferating cells. (A) BJ1 cells at different phases of the cell cycle were prepared by centrifugal elutriation and cell fractions were analyzed by DNA flow cytometry. Cell lysates were processed for immunoblotting using antibodies against LINT-25 and LAP2. FACS profiles from representative fractions and from an asynchronous culture (log) are shown. (B) WI-38 human lung fibroblasts were synchronized by an overnight aphidicolin block and released from the block for indicated time periods. Samples were processed for immunoblotting after the indicated time points and analyzed using antibodies against LINT-25, LAP2α and actin. The first lane (–) is a sample from untreated control cells; numbers on the right are molecular masses of marker proteins in kDa; mLINT-25, monomeric LINT-25; dLINT-25, dimeric LINT-25.