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: Human Molecular Genetics

Article Title: XIAP and cIAP1 amplifications induce Beclin 1-dependent autophagy through NFκB activation

doi: 10.1093/hmg/ddv052

Figure Lengend Snippet: cIAP1 overexpression induces autophagy. ( A ) HeLa cells previously transfected with empty vector (C-), wild-type cIAP1 or cIAP1 H588A expression constructs for 48 h were treated with DMSO or 400 n m bafilomycin A1 (Baf A1) during the last 4 h. Blots were probed with the indicated antibodies and the HA indicates the cIAP1 constructs. Densitometric measurements of LC3-II bands were normalized to the corresponding actin bands and are shown in the histogram on the right. ( B ) HeLa cells stably expressing mRFP-GFP-LC3 transfected with empty vector (C-), wild-type cIAP1 or cIAP1 H588A expression constructs for 48 h were fixed and subjected to automatic counting of LC3 vesicles. The histogram shows the percentage relative to C- of the number/cell of autophagosomes (mRFP+/GFP+) (AP), autolysosomes (mRFP+/GFP-) (AL) and both of them (total). ( C ) HeLa cells previously transfected with empty vector (C-), wild-type cIAP1 or cIAP1 H588A expression constructs for 48 h were subjected to western blotting. Densitometric measurements of p62 bands were normalized to the corresponding actin bands and are shown in the histogram on the right. ( D ) HeLa cells were co-transfected with the GFP-HttQ74 expression construct plus empty vector (C-), wild-type cIAP1 or cIAP1 H588A expression constructs for 48 h. The cells were then fixed and the percentage of transfected cells with aggregates was calculated as shown in the histogram. At least 150 cells were counted per sample. The values shown in all the histograms represent the mean ± standard deviation from at least three independent experiments performed in triplicate samples/condition. The P -values were determined using Student's t -test. See also Supplementary Material, Figure S8 .

Article Snippet: Ashwell (Addgene plasmid 11559) , pEBB-HA-cIAP1 and pEBB-HA-cIAP1 H588A were provided by C.S.

Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing, Construct, Stable Transfection, Western Blot, Standard Deviation

Journal: Human Molecular Genetics

Article Title: XIAP and cIAP1 amplifications induce Beclin 1-dependent autophagy through NFκB activation

doi: 10.1093/hmg/ddv052

Figure Lengend Snippet: cIAP1 induces Beclin 1 transcription via p65/NFκB activation. ( A ) HeLa cells were transfected with empty vector (C-), wild-type cIAP1 or cIAP1 H588A expression constructs for 48 h. Densitometric measurements of Beclin 1 bands were normalized to the corresponding actin bands and are shown in the histograms on the right. ( B ) mRNA from HeLa cells previously transfected with empty vector (C-), wild-type cIAP1 or cIAP1 H588A expression constructs for 48 h was analysed by qRT-PCR for Beclin 1-actin mRNA. The levels of Beclin 1 mRNA were normalized to Actin mRNA levels. ( C ) HeLa cells previously transfected with empty vector (C-), wild-type cIAP1 or cIAP1 H588A expression constructs for 48 h were subjected to western blotting to detect P-IκB and IκB levels. The blots shown are from the same set of experiments. Densitometric measurements of phospho-IκB (P-IκB) bands were normalized to the corresponding bands of actin and are shown in the histogram below. ( D ) HeLa cells previously transfected with empty vector (C-), wild-type cIAP1 or cIAP1 H588A expression constructs for 48 h were subjected to a ChiP assay. The amount of in vivo binding of endogenous p65 to Beclin 1 and actin (as a negative control) promoters was quantified by real-time PCR. Data are representative of three independent experiments. The values shown in all the histograms represent the mean ± standard deviation from at least three independent experiments performed in triplicate samples/condition. The P -values were determined using Student's t -test.

Article Snippet: Ashwell (Addgene plasmid 11559) , pEBB-HA-cIAP1 and pEBB-HA-cIAP1 H588A were provided by C.S.

Techniques: Activation Assay, Transfection, Plasmid Preparation, Expressing, Construct, Quantitative RT-PCR, Western Blot, In Vivo, Binding Assay, Negative Control, Real-time Polymerase Chain Reaction, Standard Deviation