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. 2014 Dec 23;34(3):326–343. doi: 10.15252/embj.201489184

Figure 2.

Figure 2

USP45 interacts directly with ERCC1
  1. Extracts of HEK293 cells were analysed by gel filtration on a Superdex 200 10/300 GL preparative grade column (GE Healthcare) in buffer containing 0.15 M NaCl, and every second fraction denatured and analysed by immunoblotting with the indicated antibodies. The white dotted lines indicate that the samples were run on different polyacrylamide gels.
  2. Endogenous USP45 or ERCC1 was immunoprecipitated from HEK293 cell lysates and subjected to immunoblotting with the indicated antibodies.
  3. Yeast two-hybrid (Y2H) assays were performed with a GAL4 DNA-binding domain fusion and/or activation domain for each protein indicated in the table to detect interaction between these proteins. Cells grown on media lacking LEU, TRP and HIS (to select for bait and prey plasmids) or tested for lacZ reporter gene activity.
  4. Endogenous SLX4 and ERCC1 were immunoprecipitated from extracts of WT or ERCC1 KO MEFs (D), or WT or SLX4 KO MEFs (E) and subjected to immunoblotting with the indicated antibodies.
  5. Extracts of WT or SLX4 KO MEFs were analysed by gel filtration as described in (A). Similar results were obtained in at least two separate experiments.