Fig. 5

SNHG15 inhibits TRAF2 auto-ubiquitination. A Sliver-stained SDS-PAGE gels showing separated proteins in macrophages that were pulled down using a biotin-labeled SNHG15 probe. The bands in the highlighted regions were assessed by mass spectrometry, verifying TRAF2 as a protein specifically interacting with SNHG15. B The interaction of SNHG15 and TRAF2 was analyzed with the catRAPID database. C Western blot analysis of biotin-labeled SNHG15 probe-bound proteins in THP-1 and RAW264.7 cells using an anti-TRAF2 antibody. D RNA immunoprecipitation was performed with an anti-TRAF2 antibody to assess SNHG15 expression in macrophages by qRT-PCR. IgG was used as a control. E The interaction of TRAF2 with the full-length sequence or fragments of SNHG15 was assessed by RNA pull-down assays. F The effect of SNHG15 on the transcription level of TRAF2 was assessed by qRT-PCR. G The localization of SNHG15 in THP-1 and RAW264.7 cells was assessed by FISH. H Immunoblotting was performed to assess the ubiquitination of TRAF2 in THP-1 and RAW264.7 cells. I Immunoblot analysis of the ubiquitination of TRAF2 in HEK293T cells transfected to express TRAF2 and HA-tagged K48- or K63-linked ubiquitin. Right panel: The influence of different concentrations of SNHG15 on the ubiquitination of TRAF2. J Effects of forced expression or silencing of SNHG15 on MAPK and NF-κB in THP-1 and RAW264.7 cells were determined by western blotting. *** P < 0.001