EMBO/soda-nel huggingface dataset:
USP19 modulates autophagy and antiviral immune responses by deubiquitinating Beclin-1 (2016)
Figure 1

Figure 1. USP19 positively regulates autophagy.

A Immunoblot analysis of the knockdown of exogenous USP19 in 293T cells expressing Flag-USP19 (top) or endogenous USP19 in HeLa cells (bottom) treated with USP19-specific siRNA or scrambled (Scr) siRNA.

B Representative images of GFP-LC3B puncta in HeLa-GFP-LC3B cells transfected with USP19-specific siRNA or scrambled siRNA during growth in normal medium (DMEM) or EBSS medium for 3 h. Arrows denote representative autophagosomes. Scale bar, 200 μm.

C Quantification of GFP-LC3B puncta in HeLa-GFP-LC3B cells transfected with USP19-specific siRNA or scrambled siRNA. Bars represented mean ± standard error of the mean (SEM) of triplicate samples (20 cells per sample). *p < 0.05, **p < 0.01 and ***p < 0.001.

D Immunoblot detection of the relative accumulation of LC3 I and LC3 II in HeLa cells transfected with USP19-specific siRNA or scrambled siRNA with rapamycin (250 nM) treatment for 12 h.

E HeLa cells were transfected with USP19-specific siRNA or scrambled siRNA and treated with indicated concentration of rapamycin for 12 h, p62 levels were detected by immunoblot.

F Human peripheral blood mononuclear cells (PBMCs) transfected with control or USP19-specific siRNA were treated with rapamycin (250 nM) for 18 h, the lysates were analyzed with each antibody.

G Flag-USP19 stably expressing 293T cells were incubated with DMEM or EBSS medium for 3 h in the absence or presence of 50 μM of chloroquine (CQ). Cells were then assayed for the relative accumulation of LC3-I and LC3-II by immunoblot analysis using anti-LC3 antibody.

H Flag-USP19 inducible HeLa cells were treated with 200 ng/ml doxycycline (Doxy) for overnight to induce the expression of Flag-USP19. The protein levels of p62 were analyzed after rapamycin treatment for 12 h at indicated concentrations.

I GFP-LC3B stably expressing HeLa cells were transfected with pcDNA3.1 empty vector or plasmid expressing the wild-type and the mutant forms of USP19 (CS or CS/HA). Pictures in (B) and (I) were taken using Leica DMI3000 B microscopy with a×100 oil-immersion objective. Scale bar, 200 μm.

J Average GFP-LC3B puncta per cell were calculated. Bars represented mean ± SEM of triplicate samples (20 cells per sample). *p < 0.05, **p < 0.01 and ***p < 0.001.
USP19 modulates autophagy and antiviral immune responses by deubiquitinating Beclin-1 (2016)
Figure 2

Figure 2. USP19 interacts with Beclin-1 complex member.

A 293T cells were transfected with plasmids encoding HA-USP19 and Flag-tagged Beclin-1 complex members (Flag-ATG14L, Flag-VPS15, Flag-Beclin-1, Flag-VPS34, Flag-UVRAG and Flag-AMBRA1), followed by immunoprecipitation (IP) with anti-Flag beads and immunoblot analysis with anti-HA. Throughout was the immunoblot analysis of whole-cell lysates (WCL) without immunoprecipitation.

B, C Extracts of A549 cells (B) or human PBMCs (C) incubated with EBSS for various time points (above lanes) were subjected to immunoprecipitation with anti-Beclin-1 and immunoblot analysis with indicated antibodies (shown on the left).

D HeLa cells were incubated in EBSS medium for 1 h after co-transfection eGFP-USP19 and dsRed-Beclin-1 for 48 h. Cells were imaged for eGFP and dsRed. Scale bar, 200 μm.

E Quantitative data are mean ± SEM of triplicate samples (20 cells per sample). *p < 0.05, **p < 0.01 and ***p < 0.001.

F Coimmunoprecipitation and immunoblot analysis of 293T cells transfected with deletion mutants of USP19 plasmid along with vector encoding Flag-Beclin-1.

G Coimmunoprecipitation and immunoblot analysis of 293T cells transfected with deletion mutants of Beclin-1 plasmid along with vector encoding HA-USP19.
USP19 modulates autophagy and antiviral immune responses by deubiquitinating Beclin-1 (2016)
Figure 3

Figure 3. USP19 stabilizes Beclin-1.

A Immunoblot analysis of extracts of 293T cells transfected with expression vector for Flag-Beclin-1 and increasing doses of expression vector for HA-USP19 (wedge).

B HeLa cell line harboring inducible Flag-USP19 was induced with indicated concentration of Doxy for overnight, and the protein was harvested for immunoblot analysis.

C BECN1 mRNA levels of the same sample (B) were detected by real-time PCR. Bars represented mean ± SEM of triplicate samples.

D Human PBMCs were transfected with USP19-specific or scramble siRNA and treated with rapamycin (250 nM) for 18 h. The extracts were analyzed by immunoblot with indicated antibodies.

E Wild-type (WT) and USP19 KO A549 cells were cultured in EBSS for indicated time points, and the protein expression levels of Beclin-1 and ULK1 were detected by immunoblot.

F USP13 KO HEK293T cells were transfected with plasmids encoding Myc-USP5, Myc-USP19 and Myc-USP38 and the cellular extracts were analyzed by immunoblot.

G 293T cells transfected with plasmid expressing Flag-Beclin-1 and empty vector or Myc-tagged USP19 (WT, CS, CS/HA or ΔTMD), the lysates were analyzed by immunoblot.

H 293T cells were transfected with plasmid encoding Flag-Beclin-1 and treated with CHX (100 μg/ml) alone or with MG132 (10 μM), 3MA (10 mM), CQ (50 μM) as combination for 6 h or transfected with the HA-USP19 plasmid. The cell lysates were analyzed by immunoblot.
USP19 modulates autophagy and antiviral immune responses by deubiquitinating Beclin-1 (2016)
Figure 4

Figure 4. USP19 removes K11-linked ubiquitination of Beclin-1.

A HeLa cells were transfected with USP19 siRNA or scrambled siRNA and protein extracts were harvested after MG132 (10 μM) treatment for 3 h. Protein extracts were immunoprecipitated using anti-Beclin-1 antibody or with IgG as a negative control and analyzed by immunoblot using anti-ubiquitin and anti-Beclin-1 antibodies.

B Lysates of 293T cells transfected with plasmid expressing Flag-Beclin-1 and HA-tagged ubiquitin (HA-Ub (wild type), K6-linked-Ub, K11-linked-Ub, K27-linked-Ub, K29-linked-Ub, K33-linked-Ub, K48-linked-Ub or K63-linked-Ub), together with the empty vector or expression vector of Myc-USP19 and treated with MG132 (10 μM) for 3 h were immunoprecipitated with anti-Flag and immunoblotted with anti-HA.

C HeLa cells were transfected with USP19-specific siRNA and plasmid encoding Flag-Beclin-1, plus K11-linked-Ub. Protein was harvested after MG132 (10 μM) treatment for 3 h and immunoprecipitated with anti-Flag and immunoblotted with anti-HA.

D 293T cells were transfected with plasmid for Flag-Beclin-1 and HA-K11-Ub, together with the empty vector or expression vector for Myc-USP19 (WT, CS or CS/HA) and treated with MG132 (10 μM). Cell lysates were subjected to immunoprecipitation with anti-Flag and immunoblot analysis with anti-HA.

E Purified ubiquitinated Beclin-1 was incubated with immunopurified Flag-USP19 in vitro in deubiquitinating buffer. The immunoblot was probed with anti-HA.
USP19 modulates autophagy and antiviral immune responses by deubiquitinating Beclin-1 (2016)
Figure 5

Figure 5. The K437 site is critical for Beclin-1 degradation.

A Lysates of 293T cells transfected with plasmid for Flag-Beclin-1 and HA-K11-linked-Ub, together with empty vector or expression vector for Myc-USP5 or Myc-USP19 were immunoprecipitated with anti-Flag and immunoblotted with anti-HA.

B Immunoprecipitation and immunoblot analysis of 293T cells transfected with vectors expressing Flag-Beclin-1 and HA-K11-linked ubiquitin.

C 293T cell were transfected with Flag-Beclin-1 (WT or K437R) and treated with CHX (100 μg/ml) for indicated time points. The expression levels of WT and K437R Flag-Beclin-1 were analyzed by immunoblot.

D Quantification of the expression levels of WT and K437R Flag-Beclin-1.

E Immunoblot analysis of protein extracts of 293T cells transfected with empty vector or vector for HA-USP19, together with plasmid expressing WT or K427R, K430R or K437R mutant of Flag-Beclin-1.

F HeLa cells transfected with BECN1 sgRNA and restored with WT and K437R Flag-Beclin-1 were stained by anti-LC3 antibody. Scale bar, 200 μm.

G Quantification of LC3 puncta shown in (F). Bars represented mean ± SEM of triplicate samples. *p < 0.05, **p < 0.01 and ***p < 0.001.
USP19 modulates autophagy and antiviral immune responses by deubiquitinating Beclin-1 (2016)
Figure 6

Figure 6. USP19 suppresses type I IFN signaling in a Beclin-1-dependent manner.

A Luciferase activity in 293T cells transfected with an ISRE (left) or IFN-β (right) promoter-driven luciferase reporter and together with plasmid encoding RIG-I (N) or cGAS and STING.

B Protein lysates of WT and USP19 KO A549 cells infected with SeV at indicated time points were immunoblotted with indicated antibodies.

C RT-PCR analysis of gene transcription in WT and USP19 KO A549 cells after infection with SeV at indicated time points.

D Phase-contrast (PH) and fluorescence microscopy images of WT and USP19 KO A549 cells infected with VSV-eGFP at an MOI of 0.01 for the indicated time length. Scale bar, 50 μm.

E Human PBMCs were transfected with control or USP19-specific siRNA, followed by treatment with influenza A/Puerto Rico/8/34 (H1N1) (PR8) (MOI = 5) at different time points, the lysates were analyzed with each antibody.

F Human PBMCs were transfected with control or USP19-specific siRNA were infected with H1N1 virus (MOI = 5) at different time points. Relative expression levels of IFNB, ISG15, ISG54, ISG56 mRNA and H1N1 nucleoprotein (NP) RNA were measured by real-time PCR.

G Relative expression levels of IFNB mRNA and interferon-stimulated genes, including ISG54 and ISG56 in WT, BECN1, ATG5 and SQSTM1 KO cells with or without USP19.

H, I Luciferase activity in WT and BECN1 KO 293T cells transfected with an ISRE (H) or IFN-β (I) promoter-driven luciferase reporter after infection with SeV for indicated time points. Bars in (A), (C), (F)-(I) represented mean ± SEM of triplicate samples. *p < 0.05, **p < 0.01, ***p < 0.001; NS, not significant.
USP19 modulates autophagy and antiviral immune responses by deubiquitinating Beclin-1 (2016)
Figure 7

Figure 7. Beclin-1 inhibits the interaction between RIG-I and MAVS.

A Luciferase activity in WT and BECN1 KO 293T cells transfected with an ISRE (left) or IFN-β (right) promoter-driven luciferase reporter after infection with SeV for 12 h.

B WT and BECN1 KO 293T cells were infected with SeV for indicated time points, then protein extracts were analyzed by immunoblot using the indicated antibodies.

C Protein extracts of THP-1 cells infected with SeV for various time points (upper lanes) were subjected to immunoprecipitation with anti-Beclin-1 and immunoblot analysis with antibodies shown on the left.

D Coimmunoprecipitation and immunoblot analysis of protein lysates of A549 cells infected with SeV.

E 293T cells were transfected with vectors encoding HA-Beclin-1 and Flag-MAVS or Flag-MAVSΔCARD, followed by IP with anti-Flag beads and immunoblot analysis with anti-HA.

F Coimmunoprecipitation and immunoblot analysis of WT and BECN1 KO 293T cells transfected with HA-MAVS plasmid along with vector encoding Flag-RIG-I (N) in the absence or presence of USP19.

G Protein extracts of scramble or USP19-specific siRNA treated 293T cells transfected with HA-MAVS plasmid along with vector encoding for Flag-RIG-I (N) in the absence or presence of Beclin-1 were subjected to immunoprecipitation and immunoblot analysis.

H Luciferase activity in 293T cells transfected with scramble or USP19-specific siRNA and ISRE luciferase reporter after SeV infection for indicated time points with or without Beclin-1.

I Luciferase activity in 293T cells transfected with scramble or USP19-specific siRNA and IFN-β luciferase reporter after SeV infection for indicated time points with or without Beclin-1.

J A proposed working model to illustrate how the USP19-Beclin-1 axis positively regulates autophagy and negatively regulates type I IFN signaling.

Bars in (A), (H) and (I) represented mean ± SEM of triplicate samples. *p < 0.05, **p < 0.01 and ***p < 0.001; NS, not significant.
The Ulp2 SUMO Protease Promotes Transcription Elongation through Regulation of Histone Sumoylation (2019)
Figure 2

Figure 2. Ulp2 promotes transcription elongation and RNAPII localization.

A Sensitivity of the indicated mutants to 6-AU. All strains carried a URA3 plasmid, pRS316, and were spotted on SD-Ura with or without 6-AU (100 μg/ml); plates were incubated for 2-4 days at 30°C. ULP2 and ulp2(C624A) represent ulp2Δ::HIS3 cells containing either pRS314-ULP2-FLAG or pRS314-ulp2(C624A)-FLAG.

B, C ChIP analyses using anti-Rpb3 (B) and anti-SUMO (C) antibodies in ulp2Δ cells as in Fig 1F. Error bars indicate the SD from three independent experiments.

D qRT-PCR analysis of CUP1 mRNA levels in ulp2Δ cells. Data were normalized to ACT1 mRNA levels. CUP1 gene induction was performed as in Fig 1F. Error bars represent the SD from three RNA samples.

E, F ChIP assays using anti-Rpb3 (E) and anti-SUMO (F) antibodies in ulp2Δ cells as done in Fig 1E. "Pro" denotes the #1 PCR product of PMA1, ADH1 and PYK1 genes described in Fig 1D, while "ORF" indicates the #3 products of PMA1 and #2 of ADH1 and PYK1; these are used in all ensuing figures except where specified. Error bars indicate the SD from four (E) and three (F) independent assays.

Data information: Asterisks indicate statistically significant differences determined by pairwise comparisons between WT and ulp2Δ using a two-tailed Student's t test (*, P <0.05; **, P <0.01). See Dataset EV2 for qPCR raw data.
The Ulp2 SUMO Protease Promotes Transcription Elongation through Regulation of Histone Sumoylation (2019)
Figure 3

Figure 3. Ulp2 C-terminal domain is required for efficient association with nucleosomes.

A Sensitivity to 6-AU of the indicated cells expressing chromosomally integrated ULP2-Myc derivatives. Assays were performed as in Fig 2A. The schematic diagram below shows full-length Ulp2 with a 9Myc epitope tag and the segments defining the N- and C-terminal domains and the catalytic ULP domain (UD).

B Immunoblot assay of the Ulp2-Myc derivatives expressed in the strains used in (A).

C ChIP analysis of Flag-Ulp2 derivatives performed with the same PCR probes used in Fig 2E. W303a cells carrying pRS424-GAL1 plasmids that expressed N-terminally Flag-tagged Ulp2 derivatives-specifically, full-length, NTD (1-403) and CTD (667-1034)-were grown to mid-log phase in SD-Trp medium containing 2% galactose. Error bars indicate the SD from three independent experiments.

D Co-IP assay for interaction between Ulp2-Myc derivatives and Flag-H2B in strains from (A) transformed with a pRS314 plasmid expressing Flag-tagged H2B. Immunoprecipitated (IP) proteins from anti-Myc agarose beads were analyzed by immunoblotting (IB) using anti-Myc or anti-Flag antibodies. The upper and middle panels show anti-Myc blotting and anti-Flag blotting, respectively. The levels of bound H2B proteins were measured relative to INPUT H2B levels shown in the lower panel.

E Chromatin association assays using strains from (A). After fractionation into chromatin (pellet) and soluble (supernatant) components, fractions were analyzed by immunoblotting using the indicated antibodies.

F Quantitation of chromatin association of Ulp2-Myc derivatives in (E). The levels of Ulp2 protein in each fraction were measured by ImageJ and then each signal was divided by the total values for each Ulp2 derivative.

Data information: Asterisks indicate statistically significant differences compared with WT, full length of Ulp2 (*, P <0.05). See Dataset EV2 for qPCR raw data.
The Ulp2 SUMO Protease Promotes Transcription Elongation through Regulation of Histone Sumoylation (2019)
Figure 4

Figure 4. Histone ubiquitylation and sumoylation and Ulp2 function.

A Immunoblot analysis of immunoprecipitated Flag-tagged histone H2B with anti-Flag (bottom panel) or anti-SUMO (upper panel) antibodies in WT, ulp2Δ, SUMO-all KR or ulp2Δ SUMO-all KR strains carrying empty vector or pRS426-FLAG-HTB1. One, two or three asterisks represent mono, di and polysumoylated histones, respectively. Note that the apparent diSUMO-H2B species (**) in ulp2Δ SUMO-all KR cells migrates more slowly than H2B linked to a SUMO dimer, suggesting that this species likely represents a low level of H2B with SUMO attached to two different histone lysines (lane 8; also see weaker band in lane 6).

B Yeast strains of the indicated genotypes and carrying a YCplac33-ULP2 cover plasmid were streaked on SD+5-FOA to evict YCplac33-ULP2; cells were grown at 30°C for 3 days.

C Mutant htb1-K123R ulp2Δ and htb1-K123R ULP2 strains with a pRS316-ULP2 cover plasmid were grown on the indicated plates at 30°C for 2-3 days. Three double mutant isolates were tested in parallel.

D Mutant ulp2Δ bre1Δ strains carrying a pRS426-ULP2 (URA3) cover plasmid and the indicated plasmids were grown on SD-Ura and SD+5-FOA at 30°C for 2-3 days.

E Yeast strains of the indicated genotypes and carrying a YCplac33-ULP2 cover plasmid were streaked on SD+5-FOA to evict YCplac33-ULP2; cells were grown at 30°C for 3 days.
The Ulp2 SUMO Protease Promotes Transcription Elongation through Regulation of Histone Sumoylation (2019)
Figure 5

Figure 5. Histone H2B ubiquitylation promotes histone sumoylation.

A Immunoblot analysis of immunoprecipitated Flag-tagged histone H2B using anti-Flag or anti-SUMO antibodies in the indicated mutants, as described in Fig 4A. The upper and lower panels show Flag-H2B-SUMO and Flag-H2B-ub, respectively. One, two or three asterisks indicate mono-, di-, and poly-sumoylated histones (upper panel), respectively, and an arrowhead and bullet point represent mono-ubiquitylated H2B, and a non-specific band, respectively.

B Immunoblot assay of sumoylated proteins in extracts prepared from the strains in (A). Anti-Flag blotting for Flag-H2B was used to verify the similar loading.

C Chromatin double immunoprecipitation (ChDIP) of Flag-H2B-ub in the indicated strains expressing Flag-H2B. The first ChIP was performed with anti-Flag agarose, and the eluted samples were immunoprecipitated with an antibody against ubiquitin. PCR signals from the indicated genes were normalized to an internal control and the input DNA. The error bars indicate the SD from three independent chromatin samples.

D ChDIP of Flag-H2B-SUMO in the indicated strains expressing both Flag-H2B and HA-SUMO. Chromatin samples were sequentially immunoprecipitated with anti-HA agarose followed by anti-Flag agarose. PCR signals were normalized to the input DNA. The error bars indicate the SD from three independent experiments.

E ChDIP of Flag-H2B-SUMO in the indicated mutant strains during CUP1 gene induction, analyzed as in (D). The CUP1 induction was performed as in Fig 1F. CUP1 OFF and ON indicate uninduced and induced (5 min) conditions, respectively. The error bars represent the SD from three independent ChDIP assays.

F Immunoblot assay of Flag-tagged Ulp2 in the indicated strains. Anti-PGK blotting shows similar loading.

G-I ChIP assays in the indicated strains expressing Flag-tagged Ulp2 as in Fig 1E. The error bars indicate the SD from three experiments.

Data information: Asterisks indicate statistically significant differences compared with WT in (C, D, G, H, I) and significant differences between uninduced and induced cells in (E) (*, P <0.05; **, P <0.01). See Dataset EV2 for qPCR raw data.
Early 5-HT6 receptor blockade prevents symptom onset in a model of adolescent cannabis abuse (2020)
Figure 2

Figure 2. Localization of 5-HT6 and CB1 receptors in the prefrontal cortex

A-E. The PFC was stained with CB1 receptor (A1-D1, green) or 5HT6 receptor (E1-H1, green), Bassoon (A2 and E2, red), GAD65 (B2 and F2, red), PSD-95 (C2 and G2, red), or SERT (D2 and H2, red) antibodies. Merge images are also depicted (A3-H3). CB1 receptors were mainly co-localized with the presynaptic marker Bassoon (white arrows, A3), mostly within GABAergic boutons (white arrows, B3) and to a lesser extend with PSD-95 (white arrows, C3) and SERT (D3), while 5-HT6 receptors were mainly co-localized with PSD-95 (white arrows, G3). No co-localization of CB1 and 5-HT6 receptors was detected (white arrows indicate the 5-HT6 staining, H3). Scale bar: 10 μm. The scatter plot represents the co-localization analysis for CB1 and 5-HT6 receptor immunostainings. Graph on the top, Mander's split coefficient was used to identify the fraction of CB1 receptors that co-localizes with Bassoon (Mander's coefficient: 0.66 ± 0.02, n=4), GAD65 (Mander's coefficient: 0.56 ± 0.01, n=4), PSD95 (Mander's coefficient: 0.15 ± 0.01, n=4) or SERT (Mander's coefficient: 0.082 ± 0.004, n=4). Graph on the bottom. Mander's split coefficient was used to identify the fraction of 5-HT6 receptors that co-localizes with Bassoon (Mander's coefficient: 0.09 ± 0.01, n=4), GAD65 (Mander's coefficient: 0.059 ± 0.004, n=4), PSD95 (Mander's coefficient: 0.58 ± 0.01, n=4) or CB1 receptors (Mander's coefficient: 0.0020 ± 0.0004, n=4). Error bars correspond to the mean ± SEM.
Early 5-HT6 receptor blockade prevents symptom onset in a model of adolescent cannabis abuse (2020)
Figure 3

Figure 3. THC-induced long-lasting mTOR activation and cognitive deficits are not inhibited by the administration of SB258585 or rapamycin in adulthood

A. Schema of the experimental paradigm used for drug administration. Mice were injected daily with THC (5 mg/kg) or vehicle (Veh) during adolescence, from PND 30 to 45. Vehicle and THC-injected mice were treated daily with either vehicle or SB258585 (SB, 2.5 mg/kg) or rapamycin (Rapa, 1.5 mg/kg) from PND 60 to 75. Biochemical and behavioral experiments were performed from PND 90.

B. Top: representative Western blots assessing mTOR activity in PFC are illustrated. Bottom: data represent the ratios of immunoreactive signals of the anti phospho-mTOR (S2448) or anti phospho-p70S6K (T389) antibodies to the immunoreactive signal of the anti-β-actin antibody and are expressed in % of values in vehicle-injected mice. They are the means ± S.E.M. of results obtained in six mice per group. * p < 0.05; ** p < 0.01, one-way ANOVA followed by Newman-Keuls test.

C. The plots represent the discrimination index measured in each condition. *** p < 0.001, one-way ANOVA followed by Bonferroni test. The discrimination index for the novel object recognition task is 0.34 ± 0.06 (N=14) and -0.08 ± 0.07 (N= 20), for mice injected with Vehicle and THC, respectively, p < 0.001 and -0.09 ± 0.08 (N=21) and 0.00 ± 0.06 (N=18), for mice treated with THC+SB258285 and THC+Rapa, respectively, p > 0.05 vs. THC-injected mice (error bars correspond to the mean ± SEM, the dotted line to a discrimination index equal to zero).
A revised model of TRAIL-R2 DISC assembly explains how FLIP(L) can inhibit or promote apoptosis (2020)
Figure 1

Figure 1: Assessment of TRAIL-R2 DISC stoichiometry. (A) Western blot analysis of Caspase 8, FLIP and FADD recruitment to the TRAIL-R2 DISC in A549, HCT116 and DU145 cell lines following incubation with increasing concentrations (1x, 2x, or 4x) of anti-TRAIL-R2 (AMG655)-conjugated magnetic beads for 90 minutes. (B) Western blot analysis of the unbound soluble fraction from (A). An untreated input of each cell line is included for comparison. (C) Quantification of Western blot analysis from panel (A) for FLIP, FADD, Procaspase 8 and their respective cleavage fragments at the TRAIL-R2 DISC. Proteins were quantified by densitometry and normalised to known protein standards (see Fig EV1). (D) Caspase-8-like (IETDase) activity assay in the unbound soluble fraction from the DISC IP in panel (A). An untreated control (0) was used for each cell line to show basal caspase-8 activity. (E) Ratios of DED proteins quantified at the TRAIL-R2 DISC from panel (C) in A549, HCT116 and DU145 cells; increasing concentrations of AMG655 are indicated by colour intensity. (F) Correlation between caspase-8 activity and ratio of FADD:FLIP(L).