α ciap1  (R&D Systems)

Journal: bioRxiv

Article Title: Inhibition of IAPs induces programmed cell death and inflammatory signaling in patient-derived metastatic breast cancer organoids

doi: 10.1101/2024.08.28.610103

Figure Lengend Snippet: (A) hMOs were grown for three to seven days in growth medium followed by 24 h treatment with 10 µM of BV6, Birinapant (Bi), ASTX-660 (ASTX) and LCL-161 (LCL), respectively, or DMSO as vehicle control. Subsequently, CellTiter-Glo® viability assays were performed and the values were normalized to non-treated controls. Error bars represent standard deviation. * p < 0.05, ** p< 0.01, *** p < 0.005. (B) hMOs, treated as in (A) were harvested and Western blotting was performed with antibodies recognizing cIAP1, cIAP2, XIAP as well as total and cleaved caspase-3. Vinculin served as loading control. Representative blots of three independent experiments are shown.

Article Snippet: The following antibodies were used in this study: α-Vinculin (V9131, Sigma), α-RIPK1 (610459, BD Biosciences), α-phospho-RIPK1 S166 (657465, Cell Signaling Technologies), α-RIPK3 (13526, Cell Signaling Technologies), α-phoshpo-RIPK3 S227 (ab209384, Abcam), α-MLKL (14993, Cell Signaling Technologies), α-phospho-MLKL S358 (91689, Cell Signaling Technologies), α-caspase-3 (9662S, Cell Signaling Technologies), α-cleaved-caspase-3 (9661, Cell Signaling Technologies), α-cIAP1 (AF8181, R&D Systems), α-cIAP2 (3130, Cell Signaling Technologies), α-XIAP (610716, BD Biosciences).

Techniques: Control, Standard Deviation, Western Blot

Journal: Cellular and Molecular Life Sciences

Article Title: A20 undermines alternative NF-κB activity and expression of anti-apoptotic genes in Helicobacter pylori infection

doi: 10.1007/s00018-022-04139-y

Figure Lengend Snippet: Inhibition of H. pylori -mediated alternative NF-κB signaling pathway by A20 requires TIFA. a WT and A20 KO_1 AGS cells were infected with H. pylori . Total cell lysates were harvested and IP was performed using an antibody against TRAF2 or isotype-matched IgG (IgG). The numbers indicate the band intensities of cIAP1 (normalized to the respective band intensities of TRAF2) of H. pylori -infected samples relative to uninfected control. b WT and TIFA-knockout (TIFA KO ) AGS cells were infected with H. pylori or treated with 30 ng/ml LTα 1 β 2 . Total cell lysates were subjected to IP as in ( a ). c AGS cells were transfected with non-target-specific siRNA (scr) or siRNAs targeting TRAF2 (TRAF2 si ) or cIAP1 (cIAP1 si_8 ) for 48 h prior to infection with H. pylori . Total cell lysates were used for IP with an antibody against TIFA or isotype-matched IgG. d Following incubation of recombinant human A20 and TIFA proteins in vitro, IP was performed using an antibody against TIFA. e as in ( d ) but IP was performed using an antibody against A20 or isotype-matched IgG. f A20 KO_1 AGS cells were infected with H. pylori and IP was performed as in ( c ). IB analyses were performed with TIFA-immunoprecipitates without and with one hour incubation with 100 ng recombinant A20 protein prior to elution of immunoprecipitates. A representative blot of at least two experiments was shown

Article Snippet: Antibodies used in this work were as follows: α-A20 (5630, used for detection in IB after TRAF2 IP); α-cIAP1 (7065); α-IκBα (4812); α-NF-κB2 p100/p52 (4882); α-NIK (4994); α-TIFA (61,358); α-TRAF3 (61095); α-phospho-IκBα (9246); α-phospho-p100 (4810); and α-phospho-RelA (3031) from Cell Signaling Technology; α-A20 (sc-166692), α-HDAC1 (sc-7872), α-RelA (sc-81334), α-RelB (sc-226) and α-TRAF2 (sc-136999) from Santa Cruz Biotechnology; α-TRAF3 (Invitrogen, 700121); and α-GAPDH (Merck Millipore, MAB374).

Techniques: Inhibition, Infection, Control, Knock-Out, Transfection, Incubation, Recombinant, In Vitro

Journal: Cellular and Molecular Life Sciences

Article Title: A20 undermines alternative NF-κB activity and expression of anti-apoptotic genes in Helicobacter pylori infection

doi: 10.1007/s00018-022-04139-y

Figure Lengend Snippet: Alternative NF-κB signaling contributes to cell survival in H. pylori infection. a AGS cells were transfected with non-target-specific siRNA (scr) or siRNA targeting RelB (RelB si_E1 ) for 48 h. After H. pylori infection for 24 h, cells were harvested and stained with annexin V-FITC/PI, and analyzed by flow cytometry. A graph showing data from three independent experiments is depicted (means ± SD). ## p < 0.005, * p < 0.01 and ** p < 0.001. Representative dot plots are shown for each treatment. b A proposed model for A20’s role in the negative regulation of alternative NF-κB signaling pathway in H. pylori infection. H. pylori -induced activation of the classical NF-κB pathway results in the up-regulation of A20 expression. A20 binds to TIFA in the TIFA/NIK regulatory complex, which in turn destabilizes the association of TIFA with the complex. This restores the stability of cIAP1 in the complex and this TIFA-free NIK regulatory complex recovers the ability to mediate the proteasomal degradation of NIK, leading to the shutdown of alternative NF-κB signaling

Article Snippet: Antibodies used in this work were as follows: α-A20 (5630, used for detection in IB after TRAF2 IP); α-cIAP1 (7065); α-IκBα (4812); α-NF-κB2 p100/p52 (4882); α-NIK (4994); α-TIFA (61,358); α-TRAF3 (61095); α-phospho-IκBα (9246); α-phospho-p100 (4810); and α-phospho-RelA (3031) from Cell Signaling Technology; α-A20 (sc-166692), α-HDAC1 (sc-7872), α-RelA (sc-81334), α-RelB (sc-226) and α-TRAF2 (sc-136999) from Santa Cruz Biotechnology; α-TRAF3 (Invitrogen, 700121); and α-GAPDH (Merck Millipore, MAB374).

Techniques: Infection, Transfection, Staining, Flow Cytometry, Activation Assay, Expressing