Journal: Journal of autoimmunity
Article Title: Selective disruption of Traf1/cIAP2 interaction attenuates inflammatory responses and rheumatoid arthritis.
doi: 10.1016/j.jaut.2025.103377
Figure Lengend Snippet: Fig. 2. TRAF1 stabilizes cIAP2 and protects it from degradation (a) HEK 293T cells overexpressing cIAP2 with or without WT TRAF1 were treated with 5 μg/ml cycloheximide (CHX) to inhibit denovo protein synthesis for the indicated times and lysates were blotted for cIAP2, TRAF1 or as a loading control, β-actin (left panel). Densitometry of band intensity from 3 independent exper iments were quantified by ImageLab software (right panel). (b) shCtrl or shTRAF1 RAJI cells were treated with 5 μg/ml cycloheximide (CHX) for the indicated times, and lysates were blotted for cIAP2, TRAF1 or as a loading control, β-actin (left panel). Densitometry of band intensity from 3 independent experiments were quantified by ImageLab software (right panel). (c) HEK 293T cells overexpressing cIAP2 with or without WT, TRAF1 V203A were treated with cycloheximide (CHX) and blotted, as in panel a (left panel). Densitometry of band intensity from 3 independent experiments were quantified by ImageLab software (right panel). (d) HEK 293T cells overexpressing cIAP2 with or without WT were treated with or without 0.01 mM of the proteasome inhibitor, MG132 or 0.025 mM of the lysosomal inhibitor, chloroquine (CQ). (e) HEK 293T were transfected with various combinations of plasmids to overexpress cIAP2, WT TRAF1, cIAP1, cIAP2 H574A, or cIAP1 H588A as indicated and lysates were blotted for cIAP2, TRAF1 or as a loading control, β-actin. All blots are representative of three independent experiments. * non- specific band.
Article Snippet: The TRAF1 mutated plasmids were sub-cloned into c-Flag pcDNA3. c-Flag pcDNA3 was a gift from Stephen Smale (Addgene plasmid # 20011) [37]; Flag-cIAP2/pRK5 was a gift from Xiaolu Yang (Addgene plasmid # 27973) [38]; pEBB HA cIAP1 was a gift from Colin Duckett (Addgene plasmid # 38232) [39].
Techniques: Control, Software, Transfection
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