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Fig. 4. 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 
-galactosidase activity (SA -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 LAP2
(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 LAP2 antibody or hybridoma medium (DMEM) as a control, and immunoprecipitates (P) and supernatants (SN) were analyzed by immunoblotting using antibodies against LAP2 (upper panel) and ubiquitin (lower panel). Input (Inp) represents 1.5% of protein used for IPs. Black and white arrowheads point to ubiquitylated LAP2. 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-LAP2, ubiquitylated LAP2; pRb, retinoblastoma protein.
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