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: Acta Pharmaceutica Sinica. B

Article Title: A new perspective of triptolide-associated hepatotoxicity: the relevance of NF- κ B and NF- κ B-mediated cellular FLICE-inhibitory protein

doi: 10.1016/j.apsb.2020.02.009

Figure Lengend Snippet: Possible role of NF- κ B and NF- κ B-mediated pro-survival signals in TP/LPS-induced hepatotoxicity. (A) Experimental design to detect the time-dependent changes of NF- κ B and its related pro-survival factors. (B)–(G) Relative mRNA levels of NF- κ B target genes, including Ciap1 , Ciap2 , Flip L , Xiap , Tnfaip3 , and Nfkbia , were detected by qPCR with tubulin as the internal control ( n = 6). (H)–(L) Representative Western blots and relative intensity of protein bands of FLIP L , CIAP1, XIAP, and I κ B- α after the treatment of TP and LPS with tubulin as the loading control ( n = 4–6). (M) Representative photomicrographs of liver sections by IHC for P65 1 h after LPS application (200 × ). Scale bar = 50 μm. Results were expressed as mean ± SEM and statistical analysis was performed using Two-way ANOVA following by Tukey's multiple comparison test. *, # , $ P <0.05, **, ## , $$ P <0.01, ***, ### , $$$ P <0.001; ns, no statistical difference.

Article Snippet: Antibody against RIPK1 (17519-1-AP), mixed lineage kinase domain like pseudokinase (MLKL, 66675-1-Ig), myeloperoxidase (MPO, 22225-1-AP), and CIAP1 (10022-1-AP) were purchased from Proteintech (Chicago, IL, USA).

Techniques: Control, Western Blot, Comparison

Journal: Acta Pharmaceutica Sinica. B

Article Title: A new perspective of triptolide-associated hepatotoxicity: the relevance of NF- κ B and NF- κ B-mediated cellular FLICE-inhibitory protein

doi: 10.1016/j.apsb.2020.02.009

Figure Lengend Snippet: The primer sequences used for qPCR assay in mice.

Article Snippet: Antibody against RIPK1 (17519-1-AP), mixed lineage kinase domain like pseudokinase (MLKL, 66675-1-Ig), myeloperoxidase (MPO, 22225-1-AP), and CIAP1 (10022-1-AP) were purchased from Proteintech (Chicago, IL, USA).

Techniques:

Journal: PLoS ONE

Article Title: Induction of interferon-β and interferon signaling by TRAIL and Smac mimetics via caspase-8 in breast cancer cells

doi: 10.1371/journal.pone.0248175

Figure Lengend Snippet: MCF-7 cells were treated with LCL161 (10 μM) and/or TRAIL (100 ng/mL) for indicated time periods and total cell lysates were analyzed by immunoblotting for cIAP1 (A) the NFKB2 gene product p100 and p52 (B) or phosphorylated and total STAT1 (D). Intensity of bands in B and D were quantified (C and E) and normalized to LCL161+TRAIL (C) or TRAIL (E) treatment for 48 h. Protein levels of cIAP1 (F), p100 and p52 (G), or phosphorylated and total STAT1 (H) were measured in CAMA-1 cells that had been treated with LCL161 and/or TRAIL for 8 h in the absence or presence of zVAD-FMK (20 μM). Figures are representative of three independent experiments. Data in C and E indicate individual data points with bars representing the mean and error bars representing SEM, n = 3. * denotes p<0.05, ** p<0.01, *** p<0.001 using ANOVA followed by Tukey’s honest significance test.

Article Snippet: This was followed by incubation with primary antibodies against phosphorylated STAT1 (1:400, #9167), total STAT1 (1:400, #9172), FADD (1:400, #27825), caspase-8 (1:300, #9746), and FLIP (1:400, #56343) all from Cell Signaling Technology; p100/p52 (1:300, ab131539) and IFNAR1 (1:400, ab124764) from Abcam; cIAP1 (1:200, AF8181, R&D Systems); actin (1:2,000, #0869100, MP Biomedicals); and RIP1 (1:500, #610458, BD Biosciences).

Techniques: Western Blot

Journal: Oncotarget

Article Title: The BIRC6 gene as a novel target for therapy of prostate cancer: dual targeting of inhibitors of apoptosis

doi:

Figure Lengend Snippet: (A) Correlation of immunohistochemical staining intensity of BIRC6 and clinical (T) stages of prostate cancer (mean staining intensity ± S.E.M.). (B-D) Correlation of BIRC6 immunohistochemical staining intensity with the absence and presence of poor prognostic factors, such as recurrence of PSA, lymph node metastasis and prostatic capsule invasion. The statistical significance of positive trends was determined by the Chi square test for trend. (E) Representative images of correlated expressions between BIRC6 and survivin, XIAP and cIAP1. 20x magnification, scale bar, 100 μm.

Article Snippet: Immunohistochemical staining using rabbit polyclonal antibody against BIRC6 (NB110-40730, Novus Biological, 1:50), rabbit monoclonal antibody against Survivin (#2808, Cell Signaling, 1:50), monoclonal antibody against cIAP1 (MAB8181, R & D Systems, 1:200) and rabbit polyclonal antibody against XIAP (#sc-11426, Santa Cruz, 1:25) was conducted using a Ventana autostainer (model Discover XT; Ventana Medical System, Tucson, AZ) with an enzyme-labelled biotin-streptavidin system and a solvent-resistant DAB Map kit (Ventana).

Techniques: Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: The BIRC6 gene as a novel target for therapy of prostate cancer: dual targeting of inhibitors of apoptosis

doi:

Figure Lengend Snippet: (A-B) Western blotting showing protein levels of BIRC6, cIAP1 and survivin in two CRPC cell lines (A) PC-3 cells and (B) C4-2 cells transfected with Mock or increasing dosages of scrambled ASO (Scrb), dASOs 6w2 and 6w5 for 72 hr. (C) Comparison of dual IAP targeting and single IAP-targeting. Cell viability of PC-3 cells transfected with dASOs 6w2, 6w5 and siRNA-targeting BIRC6, cIAP1 or survivin, was determined by MTS assay at 72 hr after transfection. Cell viabilities of ASO- and siRNA-treated cells were normalized with corresponding Scrb ASO and siRNA controls. Error bars represent mean percentage cell viability ± S.D. Western blotting of 3 IAPs showing comparable amounts of reduced protein expression obtained with dASO and siRNA single IAP-targeting. (D) Proliferation of PC-3 cells transfected with mock, Scrb ASO, dASOs 6w2 and 6w5. Error bars represent mean cell number ± S.D. (E) MTS viability assay of C4-2 cells treated with dASOs.

Article Snippet: Immunohistochemical staining using rabbit polyclonal antibody against BIRC6 (NB110-40730, Novus Biological, 1:50), rabbit monoclonal antibody against Survivin (#2808, Cell Signaling, 1:50), monoclonal antibody against cIAP1 (MAB8181, R & D Systems, 1:200) and rabbit polyclonal antibody against XIAP (#sc-11426, Santa Cruz, 1:25) was conducted using a Ventana autostainer (model Discover XT; Ventana Medical System, Tucson, AZ) with an enzyme-labelled biotin-streptavidin system and a solvent-resistant DAB Map kit (Ventana).

Techniques: Western Blot, Transfection, Comparison, MTS Assay, Expressing, Viability Assay

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