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. 2013 Feb 15;32(6):805–815. doi: 10.1038/emboj.2013.24

Figure 5.

Figure 5

Accumulation of SUMOylated Rap1 molecules in uls1-Δ cells. Strains 210-2d (WT) and 210-5b (uls1-Δ) transformed with empty vector pRS316 and strains 210-2d (WT), 210-5b (uls1-Δ), 210-3b (rap1-2R) and 210-13c (uls1-Δ rap1-2R) transformed with YEp195-CUP-His-Smt3 were grown exponentially (upper panel) or allowed to reach stationary phase (lower panel). Protein extraction and pull-down were done under denaturing conditions. Input sample is 1/2000th of the total extract subjected to the pull down. Rap1 was detected with a polyclonal antibody (left and central panels). The membrane was then rehybridized with a second polyclonal antibody against SUMO (right panel; the antibody sensitivity did not allow us to detect SUMO conjugates in the input samples (data not shown)). Position of mono-SUMOylated Rap1 is marked with a filled circle. Positions of poly-SUMOylated Rap1 are marked with stars. A background of unmodified Rap1 is detected in all pull-down samples and its position is marked with an empty circle. Total protein extraction from stationary cells was less efficient. Non-specific signals are marked with minus signs.