RIP1 ubiquitination assays

Plasmids were constructed for RIP1 ubiquitination overexpression. Briefly, the coding regions of the genes were amplified from the Homo lung cDNA library via the PCR technique. Then the resulting fragments were cut with restriction enzymes, and inserted into pcDNA3.1-Myc and pcDNA3.1-HA-Ubiquitin (Invitrogen) to form pcDNA3.1-RIP1(WT)-myc and pcDNA3.1(+)-HA-Ub (RIP1 plasmid and HA-Ub plasmid) for overexpressing RIP1(WT) gene and ubiquitination, respectively. All constructs were sequenced for confirmation.

To determine the role of spermidine effects on RIP1 ubiquitination, the RIP1 plasmid and HA-Ub plasmid were co-transfected overexpression of RIP1 (WT) and ubiquitinated plasmid (2 μg/μL) for 48 h. The transfection efficiency was determined by using Western blot analysis.

To determine the role of spermidine effects on RIP1 deubiquitination by the activation of CLYD, H-FLS cells were divided into two groups: (i) shRNA-CYLD group and (ii) shRNA-SC group. Both groups were then co-transfected with RIP1 (WT) and ubiquitinated plasmid. After transfection of shRNA-CYLD lentivirus and RIP1 (WT) and HA-Ub plasmid, the transfected H-FLS cells were stimulated with 10 ng/mL of TNF-α for 2 min in the presence or absence of 9 μM spermidine for 1 h, then, cells were resuspended in 1 mL of cold PBS, and were subsequently directly lysed by RIPA lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 20 mM N-ethylmaleimide and 1% Triton X-100). The lysate was immediately incubated with anti-RIP1 and A/G Plus Agarose beads. Ubiquitinated RIP1 proteins were detected by immunoblotting with an HA antibody and were subsequently probed with anti-RIP1 as a loading control.