Figure 1.
Localization of HP1α, β and γ in HeLa cells expressing lamin mutants.
(A–C) HeLa cells transiently transfected with the indicated GFP-tagged lamin constructs were stained with antibodies to HP1α, β or γ respectively and counterstained with DAP1 (insets). Arrows indicate transfected cells depleted of HP1α or β. Bar, 10 µm.
Table 1.
Effect of proteasomal inhibitors on HP1α and HP1β protein levels.
Figure 2.
Localization of methylated histones in cells expressing lamin mutants.
(A,B) HeLa cells were transiently transfected with the indicated GFP-tagged lamin constructs and stained with antibodies to H3K9me3 or H4K20me3 respectively. Bar, 10 µm.
Figure 3.
Expression of chromatin markers in HeLa cells expressing lamin mutants.
Lysates of sorted, transfected cells and untransfected cells (∼50,000 cells per sample) were analyzed by western blotting with antibodies to the indicated proteins. The results shown are representative of three independent experiments.
Figure 4.
Restoration of HP1β after treatment with proteasomal inhibitors.
HeLa cells transiently transfected with lamin constructs were treated with 6 µM MG132 or 10 µM lactacystin and stained with antibodies to HP1β. Arrows indicate transfected cells showing restoration of HP1β. Bar, 10 µM.
Table 2.
Effect of proteasomal inhibitors on localization of GFP-lamin A constructs.
Table 3.
Real-time PCR assays with stably transfected cells.
Figure 5.
Restoration of emerin after treatment with proteasomal inhibitors.
HeLa cells transfected with lamin constructs were treated with MG132 or lactacystin and stained with an antibody to emerin. Arrows indicate dispersed emerin staining; arrowheads point to restored emerin localization. Bar, 10 µM.
Figure 6.
Effects of MG132 on lamin A processing.
(A) Localization of LAΔ50. HeLa cells transfected with GFP-tagged LAΔ50 were treated with MG132 and stained with antibodies to HP1α, β or γ. Arrows indicate untreated transfected cells showing retention of HP1α or β. (B) Localization of pre-lamin A. HeLa cells transfected with lamin constructs were treated with MG132 and stained with an antibody to pre-lamin A. Arrowheads point to weak staining of untransfected cells. (C) Lamin profile after treatment with MG132. HeLa cells transfected with lamin constructs and untransfected cells were treated with MG132 and analyzed by western blotting with antibodies to GFP (upper blot) and lamin A/C (lower blot); U, untreated and T, treated with MG132. (D) Localization of GFP-HP1β. HeLa cells were co-transfected with GFP-HP1β and RFP-tagged lamin constructs. Bar, 10 µm in panels A and B, 5 µm in panel D.
Figure 7.
Stability and solubility of HP1 isoforms.
(A) Protein turnover in the presence of cycloheximide. HeLa cells were treated with cycloheximide (CHX) for 0-28 h and levels of indicated proteins were determined by western blot analysis followed by a semi-quantitative analysis by scanning of blots. Graphical data is representative of two separate experiments. (B) Basal levels of ubiquitination of HP1 isoforms. Individual HP1 isoforms were immunoprecipitated from HeLa cell lysates and analyzed by western blotting with antibodies to ubiquitin as well as to HP1α, β or γ. Vertical arrows point to multi-ubiquitinated forms of HP1α or β; arrowheads, HP1; ns, non-specific bands; asterisk, IgG light/heavy chain; molecular mass markers: phosphorylase b, 94 kD; albumin, 67 kD; ovalbumin, 45 kD; carbonic anhydrase, 30 kD; trypsin inhibitor, 21 kD. (C) Matrix association of HP1 isoforms. HeLa cell monolayers were extracted with Triton X-100, Tween-20 and sodium deoxycholate (strong) or with Triton X-100 only (mild) followed by nuclease digestion and salt extraction. The extracted pellet, E and unextracted cell lysates, U were analyzed by western blotting with antibodies to HP1 isoforms and lamin A/C.
Figure 8.
Effects of FBXW10 on HP1 isoforms and nuclear envelope proteins.
HeLa cells transfected with RFP-tagged FBXW10 were stained with antibodies to HP1 isoforms, emerin or lamin A. Arrows indicate transfected cells depleted of HP1α or β; arrowheads point to dispersed emerin localization. Values represent mean ± SD, calculated from three independent experiments in which n = 75 transfected cells were analyzed in each experiment. Bar, 10 µM.