HP1/H3K9Me3 interaction is unaffected by HP1 phosphorylation following BXB-ER activation.

<p>(A) Western blot for HP1β of untreated and 15 hours estrogen treated (*) 3T3BXB-ER cells. Lysates in the “H3K9Me3 IP” (right hand) panel were immunoprecipitated using an antibody specific for H3K9Me3 and Western blotted for HP1β. (B) Autoradiograph of ultra high resolution 2D SDS PAGE gels (pH 4.5–5.5, region shown around pH 4.9, MWr ∼22 kD) of lysates from cells labeled metabolically with ³²P <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013322#pone.0013322-Lovri1" target="_blank">[35]</a>. Cells were either untreated, activated with estrogen for 20 min (*) or activated and treated with PD98059 at the same time (* + inhibitor). The spot in the middle of the panels showed reproducible changes in phosphorylation intensity in 3 experiments, the one shown is a representative. Bars show relative intensities of the phosphorylation intensity of this spot, relative to all spots on the gel. This spot was later identified as HP1α (see C/D). (C/D) Spots shown in (B) were identified by Peptide Mass Fingerprinting. For this the spots were cut out from the gel, digested with trypsin and processed for identification by MALDI ToF-MS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013322#pone.0013322-Lovri1" target="_blank">[35]</a>. Panel C shows the raw data from the MALDI ToF MS. Peaks were manually picked and submitted to a MASCOT (Matrix Science, UK) search of the non-redundant MSDB database. (D) Murine HP1α was identified as the best match to the sequences in the database with a MASCOT score of 91 (scores >70 denoted statistical significance at the 0.05% level). Mass accuracy was set to 100 ppm and three missed tryptic cleavages were allowed, as HP1α contains a higher than normal amount of Arg (R) and Lys (K). Panel D also shows the 59% sequence coverage of the matching peptides in red. Sequence coverage went down to 46% with more typical setting of one missed tryptic cleavage allowed (not shown). (E) Western blot for HP1β from high resolution 2D SDS PAGE gels (pH 3–5.6, region shown around pH 3.8, apparent MW ∼22 kDa). The spots shown are the only signals generated from a much larger Western blot. Spot 1 is the least acidic spot recognized by the HP1β antibody, with the smallest apparent MW. The unnumbered spot is an artifacts, unique to this experiment. 5 experiments with similar results were performed.