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. 2011 Jan 21;12(2):142–148. doi: 10.1038/embor.2010.206

Figure 2.

Figure 2

Preservation of SUMO conjugates in nuclear and cytoplasmic fractionation. (A) HeLa cells in suspension were heat-stressed at 43°C for 1 h or control-treated at 37°C. In all, 10% of cells were lysed directly into Laemmli's sample buffer (lanes 1 and 4), with the remainder fractionated into cytoplasmic and nuclear extracts (lanes 2, 3, 5 and 6). The efficiency of fractionation and SUMO conjugates preservation was analysed by using tubulin, lamin A/C and SUMO2 antibodies (lanes 1–6). Nuclear lysates were used for purification with RNF4wt or RNF4mut crosslinked to beads. Eluates were analysed by immunoblot (lanes 7–10). Nuclear lysates represent 20% of the input for the pull-down experiments. (B) Silver-stained SDS–PAGE of the RNF4-dependent pull-downs from nuclear lysates from control and heat-stressed HeLa cells. Cyt., cytoplasmic; Nuc., nuclear; RNF4, RING-finger 4; SDS–PAGE, SDS–polyacrylamide gel electrophoresis; SUMO, small ubiquitin-like modifier.