Fig. 3

SNHG17 interacts with PES1 and blocks ubiquitin/proteasome-dependent PES1 degradation in CRC cells. a RNA pull-down assays followed mass spectrometry analysis indicated that SNHG17 could bind to PES1. b RIP assays using an antibody against PES1 followed by RT-qPCR to determine the enrichment of SNHG17. c A series of truncated SNHG17 fragments were used for RNA pulldown assays to detect the binding domain of SNHG17 on PES1. d Deletion mapping for the domains of PES1 that bound to SNHG17. e Western blotting for the protein levels of PES1 after SNHG17 overexpression or knockdown. f SNHG17 regulated PES1 stability. HCT116 cells and LoVo cells were treated with CHX at 50 μg/ml and harvested for western blotting assays at the indicated time points. g Cells with SNHG17-overexpression or knockdown were treated with MG132 (20 μM) for 6 h, and then western blotting was performed to detect PES1 levels. h Ub-Flag and PES1-HA were transfected into HCT116 cells with SNHG17 overexpression. The cells were treated with MG132 for 6 h prior to lysis. Immunoprecipitation was performed with an anti-HA antibody, and western blotting analyses were performed with an anti-Ub antibody. i The effects of SNHG17 and/or Trim23 overexpression on the protein levels of PES1 in HCT116 cells. j SNHG17 knockdown increased the association between PES1 and Trim23. The PES1-HA plasmid or Trim23-Flag plasmid was co-transfected into si-NC- or si-SNHG17-transfected LoVo cells. k A series of truncated PES1 mutants were used for IP assays to detect the binding domain of PES1 on Trim23. The cells were treated with MG132 for 6 h prior to lysis.