Journal: Investigational New Drugs

Article Title: Structure-based design, synthesis, and evaluation of the biological activity of novel phosphoroorganic small molecule IAP antagonists

doi: 10.1007/s10637-020-00923-4

Figure Lengend Snippet: The interaction network between the N -terminal tetrapeptide (Ala-Val-Pro-Ile) of Smac and the XIAP BIR3 domain (based on the NMR data) ( a ) and representation of the peptide-binding groove of the BIR3 domain containing the bound N-terminal AVPI motif of Smac (based on 1G73.pdb) ( b )

Article Snippet: Phosphate buffer (pH 7.5) supplemented with γ-globulin (100 µg/mL) and NaN 3 (0.02% w/v ), fluorescent probe 4 and the XIAP BIR3 domain (Sino Biological, Beijing, China) were used for all measurements.

Techniques: Binding Assay

Journal: Investigational New Drugs

Article Title: Structure-based design, synthesis, and evaluation of the biological activity of novel phosphoroorganic small molecule IAP antagonists

doi: 10.1007/s10637-020-00923-4

Figure Lengend Snippet: Determination of the binding affinity of fluorescent probe 4 and the XIAP BIR3 domain. The assay was performed at a constant fluorescent probe concentration (2 nM) and serially diluted XIAP BIR3 domain (ranging from 2.5 µM to 0.0763 nM)

Article Snippet: Phosphate buffer (pH 7.5) supplemented with γ-globulin (100 µg/mL) and NaN 3 (0.02% w/v ), fluorescent probe 4 and the XIAP BIR3 domain (Sino Biological, Beijing, China) were used for all measurements.

Techniques: Binding Assay, Concentration Assay

Journal: Journal of Biological Chemistry

Article Title: Smac/DIABLO Selectively Reduces the Levels of c-IAP1 and c-IAP2 but Not That of XIAP and Livin in HeLa Cells

doi: 10.1074/jbc.m401253200

Figure Lengend Snippet: FIG. 6. Smac does not promote the auto-ubiquitination of XIAP in vitro. The purified XIAP (without tag) was incubated for 2 h at 30 °C with or without different concentrations of Smac or with Smac A in the reconstituted ubiquitination reaction system (Ub Mix) consisting of 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 2 mM Mg-ATP, 20 M mammalian ubiquitin, 100 nM rabbit E1, and 400 nM UbcH6 (E2). The reactions were stopped by adding equal volumes of 2 SDS sample loading buffer, and the products were subjected to SDS-PAGE followed by immunoblotting with anti-XIAP antibody.

Article Snippet: This paper is available on line at http://www.jbc.org 16963 at SO U T H E R N IL L IN O IS U N IV on M arch 5, 2015 http://w w w .jbc.org/ D ow nloaded from of human c-IAP2, and residues 244–263 of human XIAP, and the monoclonal antibody against human ubiquitin were purchased from R & D Systems.

Techniques: Ubiquitin Proteomics, In Vitro, Purification, Incubation, SDS Page, Western Blot

Journal: Oncotarget

Article Title: Protease nexin 1 induces apoptosis of prostate tumor cells through inhibition of X-chromosome-linked inhibitor of apoptosis protein

doi:

Figure Lengend Snippet: (A) In PC3 (1 × 10 5 ) cells, uPA activity is reduced following transfection with a PN1 expression vector (2 μg) ( N = 4, t -test, * P < 0.01). (B) Knock-down of PN1 via (10nM) siRNA increases uPA activity ( N = 4, one-way ANOVA, * P < 0.01). Mock treatment and scrambled siRNA (NEG) used as controls. (C) 1U or 5U of recombinant uPA proteins were added to the medium of PC3 cells (1 × 10 5 ) for 24 h and xiap mRNA transcripts were measured ( N = 4, one-way ANOVA, * P < 0.05). (D) Immunoblotting of proteins from PC3 conditioned media treated with 10nM control siRNA (NEG) or siRNA uPA (siuPA) and with or without transfection of PN1 (i) and quantitation of xiap transcripts (ii). (Two-way ANOVA; N = 3, * P < 0.01). (E) PC3 cells transfected with control or expression vectors (2 μg) for WT-PN1 or PN1-LRP binding mutant (mLRP) or RCL binding mutant (mRCL), and measurement of xiap expression by ELISA ( N = 4, one-way ANOVA, * P < 0.01). (F) PC3 cells transfected with control or expression vectors (2 μg) for WT-PN1 or treated with anti-LRP (50 μg/ml), anti-uPAR (50 μg/ml) blocking antibody for 24 h, and measurement of xiap expression by ELISA ( N = 4, one-way ANOVA with Tukey Test, * P < 0.01; ** refer to similarly significant comparisons between specific groups as denoted by the horizontal lines over the bar graph). (G) Immunoblotting of PC3 lysates transfected with PN1 vector or treated with anti-LRP or anti-uPAR blocking antibody for 24 h.

Article Snippet: All ELISA detection of XIAP protein was performed using the Human Total XIAP DuoSet IC (R&D Systems, DYC822) according to factory instructions.

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Knockdown, Recombinant, Western Blot, Control, Quantitation Assay, Binding Assay, Mutagenesis, Enzyme-linked Immunosorbent Assay, Blocking Assay

Journal:

Article Title: Chemically synthesized human survivin does not inhibit caspase-3

doi: 10.1110/ps.036145.108

Figure Lengend Snippet: (A) Ligation of (1–45)αCOSR and (46–142) at 1.5 h. The reaction was monitored by analytical HPLC on a Waters XBridge C18 column (4.6 × 150 mm, 3.5 μM) running a 30-min gradient of 25%–45% acetonitrile containing 0.1% TFA at a flow rate of 1 mL/min. (Insets) Mass spectra of (1–45)αCOSR and (46–142) determined by ESI-MS. (B) Ligated full-length survivin characterized by C18 RP-HPLC and ESI-MS. HPLC conditions: Waters symmetry 300 C18 column (4.6 × 150 mm, 5 μM) running a 30-min gradient of 5%–65% acetonitrile containing 0.1% TFA at a flow rate of 1 mL/min. (C) CD spectra of synthetic survivin at 5 μM in 5 mM phosphate buffer containing 0.1 mM TCEP, pH 7.5 (thin line), and at 25 μM in 5 mM phosphate buffer containing 0.1 mM TCEP and 50 μM Zn2+, pH 7.5 (thick line). (D) Representative size-exclusion chromatograms of synthetic survivin (3) and molecular mass standards (1, 2, 4, 5). Linear regression analysis of the correlation between logarithmic M r and retention time is illustrated in the inset. (E) Representative raw data from the hydrolysis of Ac-DEVD-AMC by caspase-3 in the absence and presence of different concentrations of XIAP and synthetic survivin. (F) Dose-dependent percent inhibition of caspase-3 by XIAP (filled circles), synthetic survivin without Zn2+ (empty squares), and synthetic survivin in the presence of Zn2+ (filled squares). Each curve is the mean of three independent experiments.

Article Snippet: EnzChek Caspase-3 assay kit #1 was purchased from Invitrogen; recombinant caspase-3 was obtained from Calbiochem and recombinant human XIAP, from R&D Systems.

Techniques: Ligation, Inhibition

Journal: Journal of Radiation Research

Article Title: Cell-permeable intrinsic cellular inhibitors of apoptosis protect and rescue intestinal epithelial cells from radiation-induced cell death

doi: 10.1093/jrr/rru094

Figure Lengend Snippet: Structures and purification of TAT-conjugated recombinant IAPs. ( A ) Schematic representation of TAT-conjugated recombinant IAPs. The recombinant proteins contain an HA tag at the N-terminal and a TAT sequence at the C-terminal. The locations of the BIR domains, RING domain and caspase recruitment domain (CARD) are shown in rectangles. Mutation sites of BIR3-RING (2R)-TAT are shown as K321R or K327. ( B ) Purification of TAT-conjugated recombinant IAPs. Purified proteins were subjected to SDS-PAGE (with 5–20% gradient gel) followed by Coomassie Blue staining. Lane 1, cIAP2-TAT; Lane 2, XIAP (FL)-TAT; Lane 3, BIR2-TAT; Lane 4, BIR3-RING-TAT, Lane 5, BIR3-RING (2R)-TAT; Lane M, molecular weight markers. The arrows indicate each purified protein.

Article Snippet: Preparation of cell extracts and activation of caspases in the cell extracts followed the protocol for the activity assay of recombinant human XIAP BIR3 domain (R&D Systems).

Techniques: Purification, Recombinant, Sequencing, Mutagenesis, SDS Page, Staining, Molecular Weight

Journal: Journal of Radiation Research

Article Title: Cell-permeable intrinsic cellular inhibitors of apoptosis protect and rescue intestinal epithelial cells from radiation-induced cell death

doi: 10.1093/jrr/rru094

Figure Lengend Snippet: Effects of TAT-conjugated IAPs on caspase activity. Inhibitory effect of TAT-conjugated IAPs on caspase activity was measured using Ac-DEVD-AMC substrate as described in the Materials and Methods. ( A–D ) Caspase-3 activity in cells transduced proteins. The reactions were performed using 50 ng of active caspase-3, and the indicated amounts of TAT-conjugated XIAP ( A ), GFP ( B ), BIR2 ( C ), or cIAP2 ( D ) are shown. After 30 min incubation at 37°C, caspase-3 activity was determined by measuring Ac-DEVD-AMC hydrolysis. Values are presented as mean and standard deviation of the experiments in triplicate. ( E ) Inhibition of caspase-7 activity by XIAP-TAT and BIR2-TAT. The reactions were performed using the indicated amounts of TAT-conjugated proteins and 20 ng of activated caspase-7. After 5 min incubation at room temperature, caspase-7 activity was determined by measuring Ac-DEVD-AMC hydrolysis. Values are shown as mean and standard deviation of experiments in triplicate. ( F ) Inhibition of caspase-9 activity by BIR3-RING-TAT and BIR3-RING (2R)-TAT. Caspases were activated by the addition of cytochrome c and dATP to IEC6 cell extracts. Caspase-9 inhibitor peptide (LEHD-CHO; 250 μM) or 2.5 μM of GFP-TAT, BIR3-RING-TAT or BIR3-RING (2R)-TAT was added. Values are presented as mean and standard deviation of experiments in triplicate.

Article Snippet: Preparation of cell extracts and activation of caspases in the cell extracts followed the protocol for the activity assay of recombinant human XIAP BIR3 domain (R&D Systems).

Techniques: Activity Assay, Incubation, Standard Deviation, Inhibition

Journal: Journal of Radiation Research

Article Title: Cell-permeable intrinsic cellular inhibitors of apoptosis protect and rescue intestinal epithelial cells from radiation-induced cell death

doi: 10.1093/jrr/rru094

Figure Lengend Snippet: Transduction of TAT conjugated–recombinant IAPs into IEC6 cells. ( A ) Transduction of TAT conjugated-GFP proteins. IEC6 cells were treated with 0.5 μM of GFP or GFP-TAT at 37°C for 1 h. As control, cells were treated with protein-storage buffer. Localization of the recombinant proteins was visualized by fluorescence microscopy. Merged GFP fluorescence (green) and differential interference contrast (DIC) images are shown. ( B ) Transduction of TAT conjugated–recombinant IAPs. IEC6 cells were treated with either 0.5 μM of XIAP(FL)-TAT, BIR2-TAT, BIR3-RING-TAT, BIR3-RING(2R)-TAT or cIAP-TAT for 1 h. As a control, cells were treated with protein-storage buffer. Localization of the recombinant proteins was visualized by immunofluorescence analysis using anti-HA antibody (green). Nuclear DNA was counterstained with Hoechst 33258 (blue). ( C ) Transduction levels of Tat-conjugated IAPs in cells. IEC6 cells were treated with 0.5 μM of each of the TAT-conjugated IAPs for 1 or 2 h. Cell lysates were subjected to immunoblot analysis with the indicated antibodies.

Article Snippet: Preparation of cell extracts and activation of caspases in the cell extracts followed the protocol for the activity assay of recombinant human XIAP BIR3 domain (R&D Systems).

Techniques: Transduction, Recombinant, Control, Fluorescence, Microscopy, Immunofluorescence, Western Blot

Journal: Journal of neuropathology and experimental neurology

Article Title: Apoptosis and proliferation markers in diffusely infiltrating astrocytomas: profiling of 17 molecules.

doi: 10.1097/01.jnen.0000235857.79502.c3

Figure Lengend Snippet: FIGURE 3. (A) Western blot anal- ysis of caspases and inhibitor of apoptosis proteins in normal brain tissue (NBT, lanes 1Y7), diffuse astrocytoma (lanes 9Y15), anaplas- tic astrocytoma (lanes 17Y21), and glioblastoma multiforme (lanes 24Y31). Controls included glioma cell line U251 (lanes 8 and 22), melanoma cell line A875 (lane 16), and a carcinoma tissue sample (lane 23), which were known to express BIRC5 and caspase 3. GAPDH was used as internal con- trol. (B) Western blot analysis of BIRC5 in one original blot. Simul- taneous probing of BIRC5 and GAPDH illustrate the specificity of the antibodies. Absence of BIRC5 (first 5 lanes from left) in 5 astro- cytoma samples and presence of BIRC5 in glioma cell U251 (lane 6) and a carcinoma sample (lane 7). A prestained broad-range (11Y170 kDa) protein ladder was used for SDS-PAGE and aligned to the blot on the right.

Article Snippet: The following primary antibodies at indicated dilutions were used for IHC: CASP3 (rabbit polyclonal, 1:250; Santa Cruz Biotechnology, Santa Cruz, CA), CASP6 (goat polyclonal, 1:100; Santa Cruz), CASP7 (goat polyclonal, 1:100; Santa Cruz), CASP8 (goat polyclonal, 1:100; Santa Cruz), CASP9 (rabbit polyclonal, 1:100; Santa Cruz), CASP10 (goat polyclonal, 1:100; Santa Cruz), CASP14 (goat polyclonal, 1:300; Santa Cruz), CIAP1 (rabbit polyclonal, 1:100; Santa Cruz), CIAP2 (rabbit polyclonal, 1:100; Santa Cruz), Xlinked inhibitor of apoptosis protein (XIAP; rabbit polyclonal, 1:300; ProteinTech Group, Inc., Chicago, IL), BIRC5 (Survivin) (rabbit polyclonal, 1:500; R and D Systems, Inc., Minneapolis, MN), LIVIN (goat polyclonal, 1:500; R and D), APAF1 (rabbit polyclonal, 1:100; Santa Cruz), SMAC (goat polyclonal, 1:300; Santa Cruz), BCL2 (mouse monoclonal, 1:200; Zymed Laboratories Inc., San Francisco, CA), PHH3 (rabbit polyclonal, 1:500; Upstate Group LLC, Charlottesville, VA), and Ki67 (MIB1) (mouse monoclonal, 1:100; DakoCytomation, Glostrup, Denmark).

Techniques: Western Blot, SDS Page