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. 2016 Dec 30;18(2):241–263. doi: 10.15252/embr.201642386

Figure 5. SIM is the minimum region in Nup358‐IR required for binding to AGO2.

Figure 5

  1. Schematic representation of human Nup358‐IR region and the constructs used in this study. IR, internal repeats; SIM, SUMO‐interacting motif; 1, 2, SIM1 and SIM2. Amino acids substituted in Ubc9 mutant are indicated with red asterisks.
  2. HEK293T cells were transfected with indicated constructs, and immunoprecipitation (IP) was performed using GFP‐specific antibodies and probed for HA‐AGO2 by Western blotting (WB) using HA antibodies. Endogenous RanGAP and Ubc9 were probed with specific antibodies.
  3. Lysates prepared from cells expressing the indicated constructs were subjected to IP and WB using indicated antibodies. The presence of endogenous RanGAP and Ubc9 was determined by WB.
  4. GFP‐control, GFP‐IR1 + 2 wild type, or mutants were co‐expressed with HA‐AGO and IP and WB analyses were performed to detect the interaction using indicated antibodies.
  5. Lysates prepared from cells expressing the indicated proteins were immunoprecipitated with GFP‐specific antibodies, and the presence of specific proteins in the immunoprecipitates was detected by WB with indicated antibodies.