pebb ha ciap1  (Addgene inc)

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

Journal: Cell Death & Disease

Article Title: The LEF1/CYLD axis and cIAPs regulate RIP1 deubiquitination and trigger apoptosis in selenite-treated colorectal cancer cells

doi: 10.1038/cddis.2014.13

Figure Lengend Snippet: Selenite-induced degradation of cIAPs facilitated apoptosis by promoting DISC formation. ( a and b ) After cIAP silencing using an siRNA mixture containing sequences targeted to cIAP1 and cIAP2 for 24 h, HCT116 and SW480 CRC cells were treated with 10 μ M selenite and subjected to FACS and western blot analysis. ( c and d ) HCT116 and SW480 cells were transfected with a plasmid expressing cIAP1 before selenite treatment and were then subjected to FACS and western blot analysis. ( e ) Coimmunoprecipitation using a RIP1 antibody was performed after transfection of CRC cells with the cIAP1 plasmid, followed by incubation with K63 polyubiquitin chain-specific antibody, caspase-8 antibody and FADD antibody

Article Snippet: The cIAP1 plasmid was a gift from Colin Duckett (Addgene, plasmid #38232).

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Incubation

Journal: Cell Death & Disease

Article Title: The LEF1/CYLD axis and cIAPs regulate RIP1 deubiquitination and trigger apoptosis in selenite-treated colorectal cancer cells

doi: 10.1038/cddis.2014.13

Figure Lengend Snippet: Selenite demonstrated antitumour activity in a colon cancer xenograft model by triggering apoptosis via the LEF1/CYLD/cIAPs/caspase-8 signalling pathway. ( a ) Fourteen days after inoculation with HCT116 cells, nude mice (seven per group) were injected with PBS or selenite (2 mg/kg/d). Tumour volumes were calculated at the indicated intervals. The data are presented as the mean±S.D. ( b ) Representative images and quantitative analysis of in situ labelling of apoptosis cells using TUNEL assay; original magnification, 10 × . Scale bar, 100 μ m. * P <0.05. ( c ) Western blot analysis of proteins extracted from tumour tissues using antibodies against related molecules as indicated. ( d ) Immunohistochemistry results of the colon xenograft animal model using antibodies against cleaved caspase-8, cleaved caspase-3, CYLD and cIAPs (detecting both cIAP1 and cIAP2). Scale bar, 50 μ m

Article Snippet: The cIAP1 plasmid was a gift from Colin Duckett (Addgene, plasmid #38232).

Techniques: Activity Assay, Injection, In Situ, TUNEL Assay, Western Blot, Immunohistochemistry, Animal Model