Anti traf2 antibody

Hoechst 33342, Protein A/G agarose and a NuPAGE system were purchased from Invitrogen Life Technologies (Grand Island, NY, USA). AbuRPFK(5-Fam)-NH₂ peptide was obtained from Biomer Technologies (Pleasanton, CA, USA). Anti-cIAP1 antibody for western blot analysis was purchased from R&D Systems (Minneapolis, MN, USA). Anti-GAPDH, anti-caspase-8 and anti-PARP antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-RIPK1 antibody was obtained from BD Biosciences (Franklin Lakes, NJ, USA). Anti-TRAF2 antibody was obtained from Cell Signaling (Danvers, MA, USA). IAP antagonists were synthesized at TetraLogic Pharmaceuticals.

The fluorescence in situ hybridization (FISH) array assay was performed using a Fluorescence In Situ Hybridization Kit (C10910; RiboBio, China) according to its protocol. The 5’ FAM-labeled SNHG15 probe was designed and synthesized by GenePharma (Suzhou, China). The probe sequences are shown in Additional file 1: Table S2. After incubation with the SNHG15 probe, cells were treated with a mouse monoclonal anti-CD14 antibody (Abcam, Cambridge, UK) or anti-TRAF2 antibody (Cell Signaling Technology, MA, USA) for 2 h at 37 °C and were then washed three times with PBS. These cells were incubated with a fluorescently labeled secondary antibody (Proteintech, Chicago, USA) for 1 h at 37 °C and washed three times prior to nuclear staining with DAPI for 10 min. Finally, the microscopy sides were mounted with anti-fade mounting medium (Abcam, Cambridge, UK).

The expression plasmids HA-K48-UB (with all lysines except lysine 48 mutated to arginine) and HA-K63-UB (with all lysines except lysine 63 mutated to arginine) were purchased from Hanbio Technology (Shanghai, China). Ubiquitination assays were performed by immunoprecipitation and immunoblotting. Briefly, cell lysates were incubated with an anti-TRAF2 antibody (Cell Signaling Technology, MA, USA) overnight at 4 °C. Bound proteins were analyzed by immunoblotting with an antibody specific for ubiquitin (Invitrogen, MA, USA) after washing three times.