Figure 4: dDsk2 does not mediate proteolytic degradation of the dHP1c complex.
From: dDsk2 regulates H2Bub1 and RNA polymerase II pausing at dHP1c complex target genes
(a) The levels of ROW, dHP1c and dDsk2 are determined using western blot analysis in extracts prepared from control S2 cells treated with dsRNA against LacZ (lanes 1–4: increasing amounts of extract are analysed) and on depletion of dDsk2 (lane 5). Tubulin levels are also presented as loading control. Antibodies used were rabbit polyclonal αROW and αDsk2, rat polyclonal αdHP1c and mouse αTubulin. The position of markers of known molecular weight (kDa) is indicated. (b) On the top, wing imaginal discs obtained from dsk2RNAi; ptc-GAL4 larvae were immunostained with rabbit polyclonal αdDsk2 (green) and rat polyclonal αHP1c (top), αWOC (centre) and αROW (bottom) antibodies (red). DNA was stained with 4,6-diamidino-2-phenylindole (DAPI, blue). The arrows indicate the A/P boundary where the ptc promoter is specifically active. On the bottom, wing imaginal discs obtained from control wild-type larvae were stained with rabbit polyclonal αdDsk2 and rat polyclonal αHP1c, αWOC and αROW antibodies (in red). Scale bar, 50 μm. (c) p54/Rpn10 does not co-immunoprecipitate with the dHP1c complex. IPs were performed with rabbit polyclonal αROW (lane 3), αdHP1c (lane 4) and control αDDP1 (lane 2) antibodies. IP materials were analysed by western blot using mouse polyclonal αp54/Rpn10 (top) and rat polyclonal αdHP1c (bottom). Lane 1 corresponds to 2.5% of the input material. The position of markers of known molecular weight (kDa) is indicated. (d) Nuclear extracts obtained from S2 cells expressing the indicated V5-tagged dDsk2 constructs (lanes 2–4) were subjected to immunoprecipitation with rabbit polyclonal αHP1c (centre) and αROW (right) antibodies, and analysed by western blot using mouse monoclonal αV5 antibodies. The panel on the left corresponds to 1% of the input material used in each case for the immunoprecipitation. Lane 1 corresponds to a mock extract prepared from S2 cells expressing no V5-tagged construct. The position of markers of known molecular weight (kDa) is indicated.
