Lamin A Rod Domain Mutants Target Heterochromatin Protein 1α and β for Proteasomal Degradation by Activation of F-Box Protein, FBXW10
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.