NGF sensitizes TrkA SH-SY5Y neuroblastoma cells to TRAIL-induced apoptosis

We report a novel pro-apoptotic function for nerve growth factor (NGF) and its tropomyosin-related kinase A (TrkA) receptor in sensitizing TRAIL (TNF-related apoptotis-inducing ligand)-resistant SH-SY5Y neuroblastoma (NB) cells to TRAIL-induced apoptosis, resulting in the abrogation of anchorage-independent tumourigenic growth in vitro. We show that the TRAIL-resistant SH-SY5Y phenotype is cFLIP (cellular FLICE-like inhibitory protein) dependent and not due to low-level functional TRAIL receptor or caspase expression or an inhibitory equilibrium between functional and decoy TRAIL receptors or B-cell lymphoma 2 (Bcl-2) and BH3-only (Bcl-2 homology domain 3-only) family proteins. NGF sensitization of SH-SY5Y cells to TRAIL-induced apoptosis was dependent upon TrkA expression, activation and subsequent sequestration of cFLIP. This reduces cFLIP recruitment to TRAIL-activated death receptors and increases the recruitment of caspase-8, leading to TRAIL-induced, caspase-dependent, type II apoptosis via the intrinsic mitochondrial pathway. This effect was temporary, inhibited within 6 h by nuclear factor-κ binding (NF-κB)-mediated increase in myeloid cell leukaemia-1 (Mcl-1) expression, abrogated by transient cFLIP or B-cell lymphoma-extra large (Bcl-xL) overexpression and optimized by NF-κB and Mcl-1 inhibitors. This novel mechanism adds an important pro-apoptotic immunological dimension to NGF/TrkA interaction that may not only help to explain the association between TrkA expression, better prognosis and spontaneous remission in NB, but also provides a novel potential pro-apoptotic therapeutic use for NGF, TRAIL and inhibitors of NF-κB and/or Mcl-1 in favourable and unfavourable NBs that express TrkA and exhibit cFLIP-mediated TRAIL resistance.

INTRODUCTION During sympathetic nervous system (SNS) development, neuroblasts delaminate from the neural crest and migrate to form paraaortic sympathetic ganglia before differentiating into the various cellular components of the SNS. This finely tuned process is associated with both resistance and sensitivity to apoptosis, ensuring sufficient cell numbers to form the fully functional SNS, while eliminating cellular excess. Within this context, neuroblast resistance and sensitivity to apoptosis is regulated by neurotrophins (nerve growth factor (NGF), BDNF, NT3 and NT4/5) and their corresponding receptors (tropomyosin-related kinase (Trk)-A, TrkB, TrkC, p75 NTR and Sortilin). Active neurotrophins ligate TrkA, TrkB and TrkC to protect neuroblasts from apoptosis; TrkC in the absence of p75 NTR and TrkA in the presence of p75 NTR are proapoptotic in the absence of active neurotrophins, whereas p75 NTR and Sortilin are pro-apoptotic upon pro-neurotrophin ligation in the absence of Trk receptor expression. This variety of potential pro-and anti-apoptotic outcomes is regulated by spatial and temporal changes in neurotrophin and neurotrophin receptor expression. 1 Apoptosis is activated via the extrinsic and/or intrinsic pathways. 2,3 The identification of tumour necrosis factor-α (TNFα) as the pro-apoptotic cytokine responsible for endotoxin-induced tumour necrosis 4 paved the way for characterization of the extrinsic pathway and the subsequent identification of the TNFα pro-apoptotic cytokine family comprising TNFα, Apo1/Fas, Apo2L/TRAIL (TNF-related apoptotis-inducing ligand) and Apo3L that induce two types of apoptotic response through their cognate TNFR1, Apo1/FasL, FasR, Apo3L/DR3, Apo2L/DR4 and DR5 death receptors. 5,6 In type I cells, activated death receptors recruit FADD (Fas-associated protein with death domain) and caspase-8 (or caspase-10) to induce apoptosis via the activation of caspase-7 and caspase-3. In type II cells, low-level caspase-8 activation does not result in the direct activation of caspase-3 but cleaves the pro-apoptotic BH3-only (Bcl-2 homology domain 3-only) protein BID (BH3 interacting domain death agonist). Cleaved tBID translocates to the mitochondria and induces permeabilization of the outer mitochondrial membrane (OMM). This results in the release of the pro-apoptotic mitochondrial proteins cytochrome C, Omi/Htr2a and SMAC/DIABLO (second mitochondria-derived activator of caspases) into the cytosol that results in caspase-3-dependent apoptosis 3,7 and/or caspaseindependent death through the release of mitochondrial apoptosis-inducing factors (AIFs) and endonuclease G. 8 Apo2L/TRAIL is a promising chemotherapeutic agent because of its selective pro-apoptotic action on tumour but not nontransformed cells 9-12 that exhibit a multiplicity of redundant TRAIL resistance pathways. 13 In tumour cells, TRAIL induces apoptosis by ligating functional DR4 and/or DR5 TRAIL receptors that recruit FADD and initiator caspase-8 (or -10) to the death-inducing complex DISC. 5,6,14 In both type I and type 2 tumour cells, TRAILinduced apoptosis is regulated by the equilibrium between functional (DR4 and DR5) and decoy (DcR1, DcR2 and OPG) TRAIL receptors, the level of caspase-3, caspase-8, caspase-9 and caspase-10 expression and the equilibrium between caspase-8 and its inhibitory analogues cFLIP-S and cFLIP-L. In type 2 but not type I cells, TRAIL-induced apoptosis is also regulated by the equilibrium between pro-apoptotic BH3-only and anti-apoptotic B-cell lymphoma 2 (Bcl-2) family proteins that regulate OMM permeability. 2,3,[5][6][7][8][9]15 Neuroblastoma (NB) originates from neural crest-derived neuroblasts transformed at different stages along the sympathoadrenal lineage, blocked in differentiation and selected for resistance to apoptosis by a combination of oncogene activation, oncosuppressor inactivation and physiological apoptosis protection mechanism(s) conserved at the time of transformation. 1 The characterization of these mechanisms is critical for developing novel pro-apoptotic therapeutic strategies.

RESULTS
NGF sensitizes TRAIL-resistant TrkA SH-SY5Y cells to TRAIL-induced apoptosis In two-dimensional culture, TRAIL at concentrations from 10 ng to 1 μg/ml did not induce significant death of either nontransfected (NT), duplicate empty pcDNA3.1 vector (pcDNA)-transfected or duplicate TrkA-transfected SH-SY5Y cell lines, confirming their TRAIL-resistant phenotypes (Figures 1a and b). NGF (100 ng/ml) also failed to induce significant death of NT SH-SY5Y, pcDNA SH-SY5Y or TrkA SH-SY5Y cells (Figures 1a and b). In contrast, the combination of NGF (100 ng/ml) and TRAIL (200 ng/ml) induced a mean (± S.D.) of 68.7 ±13.8 and 66 ± 14.3% death in the two TrkA SH-SY5Y cell lines but did not induce the death of either NT SH-SY5Y or pcDNA SH-SY5Y cells (Figures 1a and b). TrkA SH-SY5Y cells that survived the effects of NGF plus TRAIL exhibited neuritelike projections, suggesting the initiation of neural differentiation (Figure 1c). In concentration studies, the death of TrkA SH-SY5Y cells in the presence of NGF (100 ng/ml) was detected at TRAIL concentrations ≥ 50 ng/ml and plateaued with a mean (± S.D.) of 68.3 ±15.3% death at a concentration of 200 ng/ml (Figures 1a and  d). The TRAIL-induced death of TrkA SH-SY5Y cells was confirmed to be apoptotic by Tunel assay (Figure 1a), detection of DNA laddering ( Figure 1e) and was abrogated by the caspase inhibitors z-DEVD-fmk (caspase-3), z-IETD-fmk (caspase-8) and z-LEHD-fmk (caspase-9) (see Figures 4d and e).

NGF/TRAIL inhibits TrkA SH-SY5Y tumourigenic growth in vitro
The effect of TRAIL and NGF upon the tumourigenic activity of NT, pcDNA and TrkA SH-SY5Y cell lines was assessed in substrateindependent tumourigenesis assays in vitro.
NT, pcDNA and TrkA SH-SY5Y cells formed similar numbers of similar-sized tumour spheroids over 14 days in soft agar tumourigenesis assays, under basal conditions (Figures 2a and b,  identical results were obtained for duplicate cell lines and results  are displayed for single NT, pcDNA and TrkA SH-SY5Y cell lines). The addition of TRAIL (200 ng/ml) to soft agar did not significantly reduce the tumourigenic activity of NT SH-SY5Y, pcDNA SH-SY5Y or TrkA SH-SY5Y cells (Figures 2a and b). NGF (100 ng/ml) added to soft agar significantly reduced the number of tumour spheroids formed by TrkA SH-SY5Y cells by a mean (± S.D.) of 58.5 ±9.1% (P o 0.0001, d.f. = 10) but did not inhibit the number of tumour spheroids formed by either NT SH-SY5Y or pcDNA SH-SY5Y cells over 14 days (Figures 2a and b). Furthermore, tumour spheroids formed by TrkA SH-SY5Y cells in the presence of NGF (100 ng/ml) appeared to be less well formed (Figure 2c). The addition of NGF (100 ng/ml) plus TRAIL (200 ng/ml) to soft agar abrogated tumour spheroid growth by TrkA SH-SY5Y cells, resulting in a mean (± S.D.) of 96.8% (P o 0.0001, d.f. = 10) inhibition when compared with untreated TrkA SH-SY5Y cells and 92.5% (P o0.0001, d.f. = 10) inhibition when compared with NGF-treated TrkA SH-SY5Y cells (Figures 2a and b). In contrast, the combination of NGF plus TRAIL did not reduce the number of tumour spheroids formed by NT SH-SY5Y or pcDNA SH-SY5Y cells (Figures 2a and b). NGF plus TRAIL induce type II apoptosis in TrkA SH-SY5Y cells TRAIL-induced apoptosis of NGF-sensitized TrkA SH-SY5Y cells was associated with the cleavage of BID, caspase-3, caspase-8 and caspase-9, increased cytosolic levels of the mitochondrial proteins cytochrome C and Htr2a/OMI and reduced cytosolic levels of the caspase inhibitor XIAP (X-linked inhibitor of apoptosis) (Figure 4c Figure 5f). These data identify cFLIP as a major mediator of the TRAIL-resistant SH-SY5Y phenotype and regulator of TRAIL-induced apoptosis in NGF-sensitized TrkA SH-SY5Y cells.
NGF induces TrkA-cFLIP binding NGF (100 ng/ml for 1-6 h) did not reduce cFLIP mRNA or protein expression in TrkA SH-SY5Y cells nor induce cFLIP degradation (Figure 4a), suggesting an alternative mechanism for sensitization to TRAIL-induced apoptosis. NGF (100 ng for 1 and 6 h), however, induced a binding interaction between TrkA and endogenous cFLIP ( Figure 6a) and also between TrkA and exogenous FLAG-tagged cFLIP transiently overexpressed in TrkA SH-SY5Y cells (Figure 6b). The TrkA tyrosine kinase inhibitor CEP-701 (100 nM) reduced the binding interaction between TrkA and FLAG-tagged cFLIP detected following 6 h of incubation with NGF, consistent with a requirement for TrkA tyrosine kinase activity (Figure 6b).
NGF reduces cFLIP recruitment to activated TRAIL death receptors in TrkA SH-SY5Y cells The possibility that NGF sensitization of TrkA SH-SY5Y cells to TRAIL-induced death results from cFLIP sequestration was confirmed by comparing cFLIP and caspase-8 levels associated with TRAIL-ligated DR4 death receptor complexes purified from TrkA SH-SY5Y cells preincubated for 6 h in the presence or absence of NGF (100 ng/ml) before 1 h of treatment with biotinylated-TRAIL (500 ng/ml). Ligand-affinity purified, TRAILligated, DR4-positive death receptor complexes from TrkA SH-SY5Y cells that had been pretreated for 6 h with NGF (100 ng/ml) before being treated for 1 h with biotinylated-TRAIL (500 ng/ml) contained lower levels of cFLIP, higher levels of caspase-8 and similar levels of DR4 when compared with complexes purified from TrkA SH-SY5Y cells treated with biotinylated-TRAIL (500 ng/ml), confirming that NGF reduces cFLIP recruitment and increases caspase-8 recruitment to TRAILactivated death receptors in TrkA SH-SY5Y cells. Similar complexes  were not purified from untreated TrkA SH-SY5Y cells or TrkA SH-SY5Y cells treated with NGF (100 ng/ml) alone (Figure 6c).
NGF sensitization of TrkA SH-SY5Y cells to TRAIL-induced apoptosis is temporary and eventually inhibited by NF-κB and Mcl-1 In preincubation studies, NGF sensitization of TrkA SH-SY5Y cells to TRAIL-induced apoptosis was time dependent and was detected following preincubation with NGF for 1 h but not 6 h ( Figure 7a). This loss of sensitivity to TRAIL-induced apoptosis following 6 h of preincubation with NGF did not associate with loss of TrkA Y674/675 phosphorylation (Figure 4a), considered to be an index of TrkA tyrosine kinase activity, 44 nor with a reduction in the NGF-induced binding interaction between TrkA and FLAGtagged cFLIP (Figure 6b). It did associate, however, with a marked increase in the expression of the mitochondrial apoptosis inhibitor Mcl-1, 45 and to a lesser extent Bcl-xL but not Bcl-2 ( Figure 4a). Furthermore, NGF also stimulated the expression of functional DR4 and DR5 but not decoy DcR1 or DcR2 TRAIL receptors, caspase-3, caspase-8, caspase-9, BID or BAX (Bcl-2-associated X protein) (Figures 4a and b, BAX not shown).   (Figure 7a). Mcl-1 knockdown also prevented loss of sensitivity to TRAIL-induced apoptosis, following 6 h of preincubation with NGF (Figures 7b and c). Mcl-1 knockdown, however, did not sensitize NT SH-SY5Y, pcDNA SH-SY5Y or TrkA SH-SY5Y cells to TRAIL-induced apoptosis in the absence of NGF (data not displayed), confirming that Mcl-1 was not directly responsible for the TRAIL-resistant SH-SY5Y phenotype despite being largely responsible for the loss of sensitivity to TRAIL, observed following 6 h of preincubation with NGF.

DISCUSSION
In the present study, we show that NGF sensitizes TrkA-expressing SH-SY5Y cells to TRAIL-induced apoptosis, unveiling a novel proapoptotic immunological dimension to NGF interaction with the TrkA receptor in transformed human neuroblasts. This mechanism depends upon TrkA expression, TrkA activation, caspase and BID cleavage and results in type II apoptosis through the intrinsic mitochondrial pathway. We identify cFLIP as the major mediator of the TRAIL-resistant SH-SY5Y phenotype and provide evidence that NGF sensitization to TRAIL-induced apoptosis results from cFLIP sequestration by NGF-activated TrkA. This reduces cFLIP and increases caspase-8 recruitment to TRAIL-activated death receptors, resulting in the cleavage of caspase-8 and BID, in turn leading to OMM permeabilization and type II cell apoptosis through the intrinsic mitochondrial pathway. We identify NF-κB and Mcl-1 as endogenous inhibitors and NF-κB and Mcl-1 inhibitors as exogenous optimizers of this novel NGF/TrkA-mediated TRAIL sensitization response.
The TRAIL-resistant SH-SY5Y phenotype could not be explained by the absence of any single component of the TRAIL-induced apoptotic pathway and, in contrast to some reports, 19,20,27 our SH-SY5Y cell lines exhibited constitutive functional TRAIL receptors and caspase-8 expression. Furthermore, the relatively low level of decoy (DcR1 and DcR2) relative to functional (DR4 and DR5) TRAIL receptors expression observed suggests that the TRAIL-resistant SH-SY5Y phenotype was not the result of an antiapoptotic equilibrium between functional and decoy TRAIL receptors. Indirect IF and ligand affinity TRAIL receptor purification confirmed that functional TRAIL receptors were expressed at the cell surface, indicating that differential localization of functional and decoy TRAIL receptors was unlikely to be responsible for the TRAIL-resistant phenotype, as reported previously. 51 SH-SY5Y cell lines also exhibited low-level expression of the Bcl-2 family proteins, Bcl-2, Bcl-xL and Mcl-1, suggesting that TRAIL resistance was unlikely to depend upon an anti-apoptotic equilibrium between Bcl-2 and BH3-only family proteins.
We identify cFLIP as the major mediator of the TRAIL-resistant SH-SY5Y phenotype, based upon the observations that: TRAIL-resistant cell lines exhibited high-level expression of  cFLIP (S and L); TRAIL alone did not induce the cleavage of either caspase-8 or BID consistent with a block at the death receptor level; siRNA cFLIP knockdown sensitized TrkA SH-SY5Y cells to TRAIL-induced apoptosis in the absence of NGF; and transient cFLIP overexpression inhibited TRAIL-induced apoptosis in NGF-sensitized TrkA SH-SY5Y cells. However, NGF did not reduce cFLIP expression or induce cFLIP degradation in TrkA SH-SY5Y cells, implicating an alternative sensitization mechanism. A sensitization mechanism based upon cFLIP sequestration was suggested by NGF stimulation of a CEP-701-sensitive binding interaction between NGF-activated TrkA and cFLIP and was confirmed by a reduction in cFLIP and increase in caspase-8 recruitment to TRAIL-activated death receptor complexes. This adds to a recent report that TrkA binds cFLIP during NGF-induced neuritogenesis, 52 characterizes TrkA as a TRAILactivated TRAIL receptor competitor and supports reports that cFLIP competes with caspase-8 to bind TRAIL-activated TRAIL receptors. 53 This adds a novel dimension to TrkA function as a regulator of cFLIP involvement in death receptor-mediated apoptosis. The sequestration of cFLIP may also extend to MAPKs  that also bind cFLIP, 54 but it remains to be determined whether this may extend to other NT receptors. NGF sensitization of TrkA SH-SY5Y cells to TRAIL-induced apoptosis was dependent upon TrkA tyrosine kinase activity and was prevented by the TrkA inhibitors CEP-701, 41 Gö6976, 42 and GW441756. 43 Apoptosis was also caspase dependent and prevented by the caspase-3 inhibitor z-VAD-fmk, 55 the caspase-8 inhibitor z-IETD-fmk 56 and the caspase-9 inhibitor z-LEHD-fmk. 57 Bcl-xL overexpression inhibited apoptosis, confirming intrinsic mitochondrial apoptosis pathway involvement that was also supported by the induction of BID cleavage, increased cytosolic levels of the pro-apoptotic mitochondrial proteins cytochrome C and OMI/Htr2a and reduced cytosolic levels of the anti-apoptotic protein XIAP, consistent with OMM permeabilization and type II cell apoptotic behaviour. 3,7,58,59 NGF sensitization of TrkA SH-SY5Y cells to TRAIL-induced apoptosis was temporary and in preincubation studies was detected following preincubation with NGF for 1 h but not 6 h. This time-dependent loss of sensitivity to TRAIL did not associate with the loss of TrkA tyrosine phosphorylation or a reduction in the binding interaction between TrkA and cFLIP. Furthermore, it was also associated with an increase in the expression of functional (DR4 and DR5) over decoy (DcR1 and DcR2) TRAIL receptors, excluding a potential role for an inhibitory equilibrium between functional and decoy TRAIL receptors. Loss of sensitivity did, however, associate with a marked increase in the expression of the mitochondrial apoptosis inhibitors Mcl-1 and to a lesser extent Bcl-xL. Mcl-1 involvement was confirmed by siRNA knockdown that not only prevented NGF stimulation of Mcl-1 expression but also prevented the loss of TrkA SH-SY5Y cell sensitivity to TRAIL-induced apoptosis following 6 h of preincubation with NGF. In contrast to its effects upon TRAIL receptor, Mcl-1 and Bcl-xL expression, NGF did not alter the expression of caspases, Bcl-2, BID or BAX.
The transcription factor NF-κB regulates Mcl-1 expression 46,47 and is activated by NGF in TrkA SH-SY5Y cells. 48 A role for NF-κB in the regulation of sensitivity to TRAIL was also confirmed using the NF-κB inhibitors dn-NF-κB and PDTC, 49,50 both of which reduced NGF stimulation of Mcl-1 expression and prevented the loss of TrkA SH-SY5Y cell sensitivity to TRAIL-induced apoptosis following 6 h of preincubation with NGF. Interestingly, NGF stimulated Mcl-1 and Bcl-xL but not Bcl-2 expression in TrkA SH-SY5Y cells. Although all three genes have been reported to be NF-κBregulated, 46,47,60,61 Bcl-2 is differentially regulated by the transcription factor CREB (cAMP response element-binding protein) 62 that is inhibited in SH-SY5Y cells. 63 We are further investigating this differential regulation of Mcl-1, Bcl-xL and Bcl-2.
The combination of NGF plus TRAIL inhibited TrkA SH-SY5Y tumourigenicity by 495%, more than expected, considering that NGF plus TRAIL induced no more than 70% cell death in a temporary sensitization effect. This can be explained by the differentiation promoting effects of NGF (see this study, and Poluha et al. 64 and Tacconelli et al. 65 ) that, despite not inducing apoptosis, caused 50% inhibition of TrkA SH-SY5Y tumourigenicity in the absence of TRAIL.
In conclusion, we report a novel important pro-apoptotic immunological dimension to the interaction between NGF and the TrkA receptor in sensitizing neuroblastoma cells to TRAILinduced apoptosis. We propose that this mechanism depends upon cFLIP sequestration by activated TrkA receptors and characterizes TrkA as a novel competitive regulator of TRAILinduced apoptosis that competes with death receptor complexes for cFLIP. Our observations may help to explain the association between TrkA expression, better prognosis and spontaneous remission in NB 1 and provide a novel potential pro-apoptotic therapeutic strategy, to be developed in the future, for the use of 'painless' forms of NGF 66 or alternative TrkA agonists, together with TRAIL and inhibitors of NF-κB and/or Mcl-1 in favourable and unfavourable NBs that express TrkA and exhibit cFLIP-mediated TRAIL resistance. This novel approach may work best as a first-line therapy, as TRAIL receptor activation has recently been reported to promote mutated ki-Ras-driven metastatic progression 67 and ki-Ras mutations are detected in relapsed but not primary NBs. [68][69][70]
Cell death was routinely assayed using a modification of previously described methods. 73,74 Briefly, cells were washed once in Ca 2+ -free PBS, detached with ice-cold PBS containing 1 mM EDTA, transferred to sterile 15 ml tubes, centrifuged for 5 min at 1000 × g at 4°C, washed with ice-cold PBS and repelleted by centrifugation at 1000 × g for 5 min at 4°C. Cell pellets were resuspended in 25 μl of PBS containing 2 μl of acridine orange/ethidium bromide solution (100 μg/ml acridine orange and 100 μg/ml ethidium bromide in PBS) plated onto glass slides and examined immediately under a Zeiss (Carl Zeiss, Oberkochen, Germany) 'Axioplan-2' fluorescence microscope. Representative fields were digitally photographed under identical exposure conditions and the number of dead cells (orange/red nuclei) and live cells (green nuclei) counted. In addition to this assay, phase contrast micrographs of parallel cultures were used to confirm changes in the relative percentage of adherent and suspension (predominantly apoptotic) cells following TRAIL treatment.

Tumour growth in soft agar
For substrate-independent tumour growth assays, 1 × 10 4 cells in singlecell suspension (passed through a gauge × 18 syringe needle) were mixed in a 0.3% solution of agar (BiTech Difco-BD, Milan, Italy) in RPMI containing 5% FCS at 37°C, with or without NGF (100 ng/ml) and/or TRAIL (200 ng/ml) and layered onto a solid 0.6% agarose substrate also with and without NGF and/or TRAIL, prepared in the same growth medium. Following top phase agar solidification, complete medium was added and replaced every 2 days. Tumour spheroid growth was monitored over a 14-day period by phase contrast microscopy. Tumour spheroids were counted in 10 random fields at × 10 magnification.
Isolation of TRAIL-activated TRAIL receptor complexes TRAIL-ligated death receptor complexes were purified from TrkA SH-SY5Y cells by ligand affinity precipitation, as previously described. 75 Briefly, biotinylated TRAIL was prepared by incubating TRAIL (1 mg/ml) with Sulfo-NHS-LC-Biotin (1mg/ml; Thermo Fisher Scientific, Waltham, MA, USA) for 1 h on ice. The reaction was stopped by adding 1 : 10 volume of 1 M Tris-HCl (pH 7.5) and unincorporated biotin removed by buffer exchange into 150 mM NaCl, 30 mM HEPES (pH 7.5) using PD-10 columns (Amersham Pharmacia Biotech, Piscataway, NJ, USA). For ligand affinity precipitation, cells (5 × 10 6 cells per sample) were washed twice in RPMI at 37°C and incubated with 1 μg/ml biotinylated TRAIL for 1 h. Death receptor complex formation was stopped by the addition of 15 volumes of ice-cold PBS and cells were then lysed in 4.5 ml of lysis buffer (30 mM TRIS-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, supplemented with complete protease inhibitor cocktail; Roche Diagnostics, Mannheim, Germany). TRAIL receptor protein complexes were precipitated from lysates by co-incubation with 20 μl of Streptavidin beads (Pierce) for 3 h at 4°C with rotation. Ligand affinity precipitates were washed four times in lysis buffer, eluted from beads in reducing SDS-PAGE sample buffer and subjected to SDS-PAGE western blotting.