Journal: RSC Chemical Biology

Article Title: High-throughput assay for measuring target occupancy of covalent compounds: a case study with MK2

doi: 10.1039/d5cb00224a

Figure Lengend Snippet: Cascade assay for target engagement confirms the mechanisms of action for reference compounds. (A) Schematic of MK2/3 signaling. (B) Isolated reactions tested in the biochemical cascade assays. (C) Concentration-response profiles for CC-99677 and ATI-450 against active kinases on their own (open symbols) or for cascade assays using active p38α to activate unactive MK2 or MK3 (solid symbols). Phosphorylation of the respective substrates for the individual enzymes, or in the cascade assays for the unactive enzyme (MK2 or MK3), were measured by HTRF.

Article Snippet: Recombinant murine MK2 (TP506027) was purchased from OriGene (Rockville, MD, U.S.A.) whereas active human MK2 (02-142) and MK3 (02-143) were purchased from Carna Biosciences (Natick, MA, U.S.A.).

Techniques: Drug discovery, Isolation, Concentration Assay, Phospho-proteomics

Journal: RSC Chemical Biology

Article Title: High-throughput assay for measuring target occupancy of covalent compounds: a case study with MK2

doi: 10.1039/d5cb00224a

Figure Lengend Snippet: Dual HTRF assay development. (A) Schematic of the dual HTRF assay targeting free MK2 bound to a covalent biotinylated tracer (biotin|cov; see panel (C)). (B) Determination of specific antibody pairs for detecting total MK2. Competition (left) of unlabeled monoclonal antibodies for the epitope of anti-MK2 D1E11 terbium (Tb)-cryptate, enabling the identification of a minimally competitive antibody for quantification of total MK2. Unlabeled antibodies were titrated into reactions containing 0.5× anti-MK2 D1E11 Tb-cryptate and 10 nM biotin|cov tracer paired with streptavidin Alexa Fluor 488. Mouse anti-MK2 7H4.2 was chosen for further assay development and directly conjugated to Alexa Fluor 633 for enhanced HTRF signal. Specificity of this antibody pair for MK2 was tested against recombinant human MK2 or MK3 (right). The antibody pair anti-MK3 D54E4 Tb-cryptate with anti-MK3 2B5 Alexa Fluor 568 was used as a positive control for detection of MK3. Note the cross-reactivity of anti-D1E11 Tb-cryptate for MK3. (C) Structure of biotin|cov, the tracer used for the dual HTRF assays (left). The specificity of this tracer for recombinant human MK2 over MK3 is shown in the associated plot (right), using 0.5× anti-MK2 D1E11-Tb cryptate as the energy donor. (D) CC-99677 target engagement on 10 nM recombinant human MK2 using the dual HTRF assay reagents (anti-MK2 pair plus biotin|cov with streptavidin Alexa Fluor 488). The cause of the observed incomplete target occupancy by this compound is unknown. (E) Quantification of endogenous MK2 protein abundance in human cell lines (left) and murine RAW264.7 cells or splenocytes (right). Specificity of the chosen anti-MK2 pair was demonstrated by the absence of signal from genetic knockout samples (human U937 and mouse splenocytes). HCC1428 possessed the highest detectable concentration of MK2 among the adherent human cell lines (family of red symbols), so it was used for studies to measure endogenous target engagement.

Article Snippet: Recombinant murine MK2 (TP506027) was purchased from OriGene (Rockville, MD, U.S.A.) whereas active human MK2 (02-142) and MK3 (02-143) were purchased from Carna Biosciences (Natick, MA, U.S.A.).

Techniques: HTRF Assay, Bioprocessing, Assay Development, Recombinant, Positive Control, Drug discovery, Quantitative Proteomics, Knock-Out, Concentration Assay

Journal: RSC Chemical Biology

Article Title: High-throughput assay for measuring target occupancy of covalent compounds: a case study with MK2

doi: 10.1039/d5cb00224a

Figure Lengend Snippet: NanoBRET assay development. (A) Emission spectrum of NanoLuciferase measured live in dimethyl sulfoxide-treated HeLa cells over-expressing either amino- or carboxy-terminal MK2 fusions, with or without 0.17 mM (0.004%) Triton X-100 permeabilization (dashed or solid lines, respectively). The filter band passes used to measure NanoBRET signal are highlighted (centered at 452 nm for NanoLuciferase and 600 nm for BODIPY585). Magnified portion of the spectrum (right) shows the fraction of light from the NanoLuciferase donor that overlaps with –and should be subtracted from– the BODIPY acceptor emission signal. (B) Characterization of BODIPY|rev and BODIPY585|cov tracers, which compete for the same binding pocket as CC-99677. Structures of tracers (left) are shown alongside magnified emission spectra when added to HeLa cells transfected with plasmids encoding either amino- or carboxy-terminal MK2 fusions (middle) as well as the ratiometric NanoBRET signal following a titration of each tracer (right). Signal was measured 1 hour after tracer addition to the live intact (solid) or permeabilized (open/dashed line) cells using a CLARIOstar Plus (average of 5 replicates with standard deviation is shown). Line weight and style follow the legend in panel (A). (C) and (D) NanoBRET signal kinetics in the presence of BODIPY585|cov tracer, using well-by-well acquisitions (C) on the CLARIOstar Plus (CLARIOstar) or full-plate imaging (D) with a GNF Systems luminescence plate reader (LPR). NanoLuciferase signal (blue profiles in shaded background) decayed over time whereas the NanoBRET ratio (red profiles in white background) increased with exposure time to tracer. Continuous measurements for longer than 1 hour were achieved by supplementing with Endurazine substrate. (E) and (F) Measurement of k -on (E) and k -off (F) for CC-99677 using NanoBRET with BODIPY585|cov on a luminescence plate reader. The time points denote when CC-99677-containing medium was exchanged for medium containing excess BODIPY585|cov, which irreversibly quenched the system by saturating free binding sites. The profile for a reversible compound that occupies the same pocket as the covalent CC-99677 (gray, open circles) is included in the k -off plots (F).

Article Snippet: Recombinant murine MK2 (TP506027) was purchased from OriGene (Rockville, MD, U.S.A.) whereas active human MK2 (02-142) and MK3 (02-143) were purchased from Carna Biosciences (Natick, MA, U.S.A.).

Techniques: Assay Development, Expressing, Binding Assay, Transfection, Titration, Standard Deviation, Imaging, Microplate Reader Luminescence Measurement

Journal: RSC Chemical Biology

Article Title: High-throughput assay for measuring target occupancy of covalent compounds: a case study with MK2

doi: 10.1039/d5cb00224a

Figure Lengend Snippet: Comparison of compound target occupancy assays. (A) to (C) Measurement of MK2 target occupancy using dual HTRF, for endogenous protein targeting, in human HCC1428 cells (A) or murine RAW264.7 cells (B) versus NanoBRET in HeLa cells transfected with NanoLuciferase amino-terminally fused to human MK2 (C). Cells were treated with compound for 3 hours before washout. Curve fits are shown for the “free” signal (the ratio of “tracer”/“total”) for CC-99677 treatment, with regression ( R 2 ) values of 0.96 (RAW264.7 in HTRF); 0.92 (HCC1428 in HTRF), and 0.99 (HeLa in NanoBRET). (D) and (E) NanoBRET target occupancy data for 3-hour CC-99677 exposure in the presence of increasing concentrations of fetal bovine or human serum (D) to model in vivo compound availability using the NanoBRET assay. The right-shifted phenotype of CC-99677 is independent of human serum lot (E), suggesting that the free-fraction of CC-99677 compound will be reduced in circulation.

Article Snippet: Recombinant murine MK2 (TP506027) was purchased from OriGene (Rockville, MD, U.S.A.) whereas active human MK2 (02-142) and MK3 (02-143) were purchased from Carna Biosciences (Natick, MA, U.S.A.).

Techniques: Comparison, Transfection, In Vivo

Journal: Cell Death and Differentiation

Article Title: Primidone blocks RIPK1-driven cell death and inflammation

doi: 10.1038/s41418-020-00690-y

Figure Lengend Snippet: A Human U937 cells were stimulated at 37 °C for up to 8 h with 100 ng/ml TNFα + 1 μM SMAC mimetic SM-164 + 25 μM zVAD (TSZ) in the absence or presence of 1 mM primidone, wherein the drug was added 30 min before the induction of necroptosis. B U937 cells were treated for 8 h with 100 ng/ml TNFα + 1 μM SMAC mimetic SM-164 + 25 μM zVAD (TSZ) in the absence or presence of primidone (concentrations indicated), wherein the drug was added 30 min before the induction of necroptosis. Necroptotic cell death was quantified by FACS analysis using 7-AAD and phosphatidylserine accessibility (Annexin V staining) as markers. Each graph shows the mean ± SD; n = 3 independent repeats. Each sample analyzed in the time course of this experiment was split for the corresponding western blotting [illustrated in ( C )]. C The expression levels and activation status (phosphorylation) of the indicated necrosome member RIPK1, RIPK3, and MLKL after the induction of necroptosis in the absence or presence of primidone were analyzed by western blotting, using the indicated specific antibodies. The blots were stripped and re-probed with an antibody against HSP90 as the loading control. For each cell line illustrated herein, one representative cropped blot of three independent experiments is shown. Human HT-29 cells ( D ) and primary MEFs ( G ) were treated as described under ( A ) and the activation status of the indicated necrosome members was analyzed as described under ( C ). Murine NIH3T3 ( E ) and L929 cells ( F ) were stimulated at 37 °C for up to 8 h with 100 ng/ml TNFα + 25 μM zVAD (TZ) in the absence or presence of 1 mM primidone, wherein primidone was added 30 min before the induction of necroptosis. Cell death was quantified and detection of the activation status (phosphorylation) of the indicated necrosome members was detected as described before.

Article Snippet: The following primary antibodies were used for immunoblotting at 1:1000 dilution unless specified otherwise: anti-RIPK1 (3493), anti-phospho-RIPK1 (human) (65746), anti-phospho-RIPK1 (murine) (31122), anti-RIPK3 (human) (13526), anti-phospho-RIPK3 (human) (93654), anti-phospho-RIPK3 (murine) (57220), anti-IκBα (9242), anti-phospho-IκBα (2859), anti-NF-κB (6956), anti-phospho-NF-κB (3033), anti-MK2 (3042), anti-phospho-MK2 (3007), anti-p38 (9212), anti-phospho-p38 (9215), anti-JNK (9252), anti-phospho-JNK (4668), anti-caspase-8 (9746), anti-CYLD (8462) all from Cell Signaling Technology (Frankfurt, Germany), anti-MLKL (MABC604) from Merck Millipore GmbH, anti-SHARPIN (14626-1-AP) from Proteintech (Manchester, UK), anti-TRADD (610572) from BD Biosciences (Wokingham, UK), anti-human phospho-MLKL (ab187091), anti-murine phospho-MLKL (ab196436), anti-TNFR1 (ab205718) all from Abcam (Berlin, Germany), anti-HSP90 (ADI-SPA-835, dilution 1:3000) from Enzo Life Sciences, anti-RIPK3 (murine) (2283, dilution 1:3000) from ProSci (via Hölzel Diagnostika), anti-FADD (sc-6036), anti-caspase-8 (sc-6136) both from Santa Cruz Biotechnology (Heidelberg, Germany), anti-β-Actin (A5441), anti-FLAG (F3165) both from Sigma-Aldrich and anti-HA (11867423001) from Roche Pharma AG (Reinach, Switzerland).

Techniques: Staining, Western Blot, Expressing, Activation Assay

Journal: Cell Death and Differentiation

Article Title: Primidone blocks RIPK1-driven cell death and inflammation

doi: 10.1038/s41418-020-00690-y

Figure Lengend Snippet: A Human U937 cells were stimulated with 100 ng/ml TNFα in the absence or presence of 1 mM primidone for the indicated durations. Primidone was added 30 min before the addition of TNFα. Western blotting analysis of the cell lysates was performed using the indicated antibodies. B TNFα-induced complex I immunoprecipitation (IP) of human HT-1080 cells treated in the absence or presence of 1 mM primidone with 1 μg/ml FLAG-tagged TNFα (FLAG-TNFα) for the indicated durations, followed by anti-FLAG IP and western blotting analysis using the indicated antibodies. The results presented in A , B indicate that the biological effect of primidone is manifested in death-signaling complex II. C In murine cells such as NIH3T3 cells, primidone prevented the assembly of complex II. The macromolecular association of FADD, RIPK1, and RIPK3, which served as a platform for subsequent cell death was directly affected by primidone, blocking the activation (phosphorylation) of RIPK1. NIH3T3 cells were treated with 100 ng/ml TNFα + 25 μM zVAD (TZ) in the absence or presence of the RIPK1 inhibitor Nec-1 s and 1 mM primidone, respectively, for the indicated durations. Nec-1 s and primidone were added each 30 min before the addition of TNFα. TZ-induced complex II was immunoprecipitated with α-FADD antibody from cell lysates. Lysates pre-IP (Input) were also analyzed by western blotting using the indicated antibodies. D In human cells such as U937 cells, primidone did not prevent the assembly of the cytosolic death-inducing signaling complex II, but affected RIPK1 and RIPK3 activation, preventing TNFα-induced cell death. U937 cells were treated with 100 ng/ml TNFα + 1 μM SMAC mimetic SM-164 + 25 μM zVAD (TSZ) in the absence or presence of the RIPK1 inhibitor Nec-1 s and 1 mM primidone, respectively. Nec-1 s and primidone were each added 30 min before the addition of TNFα. The TSZ-induced complex II was immunoprecipitated with anti-caspase-8 antibody from the cell lysates. Lysates pre-IP (Input) were also analyzed by western blotting using the indicated antibodies. A – D Each blot was stripped and re-probed with an antibody against HSP90 or β-Actin (β-ACTIN) as the loading control. For each, one representative cropped blot of three independent experiments is shown.

Article Snippet: The following primary antibodies were used for immunoblotting at 1:1000 dilution unless specified otherwise: anti-RIPK1 (3493), anti-phospho-RIPK1 (human) (65746), anti-phospho-RIPK1 (murine) (31122), anti-RIPK3 (human) (13526), anti-phospho-RIPK3 (human) (93654), anti-phospho-RIPK3 (murine) (57220), anti-IκBα (9242), anti-phospho-IκBα (2859), anti-NF-κB (6956), anti-phospho-NF-κB (3033), anti-MK2 (3042), anti-phospho-MK2 (3007), anti-p38 (9212), anti-phospho-p38 (9215), anti-JNK (9252), anti-phospho-JNK (4668), anti-caspase-8 (9746), anti-CYLD (8462) all from Cell Signaling Technology (Frankfurt, Germany), anti-MLKL (MABC604) from Merck Millipore GmbH, anti-SHARPIN (14626-1-AP) from Proteintech (Manchester, UK), anti-TRADD (610572) from BD Biosciences (Wokingham, UK), anti-human phospho-MLKL (ab187091), anti-murine phospho-MLKL (ab196436), anti-TNFR1 (ab205718) all from Abcam (Berlin, Germany), anti-HSP90 (ADI-SPA-835, dilution 1:3000) from Enzo Life Sciences, anti-RIPK3 (murine) (2283, dilution 1:3000) from ProSci (via Hölzel Diagnostika), anti-FADD (sc-6036), anti-caspase-8 (sc-6136) both from Santa Cruz Biotechnology (Heidelberg, Germany), anti-β-Actin (A5441), anti-FLAG (F3165) both from Sigma-Aldrich and anti-HA (11867423001) from Roche Pharma AG (Reinach, Switzerland).

Techniques: Western Blot, Immunoprecipitation, Blocking Assay, Activation Assay

Journal: Cell Death and Differentiation

Article Title: Primidone blocks RIPK1-driven cell death and inflammation

doi: 10.1038/s41418-020-00690-y

Figure Lengend Snippet: A The possibility that the protective effect of primidone in RIPK1-mediated cell death is due to avoiding RHIM- and DD-mediated dimerization and/or oligomerization of RIPK1 was ruled out via expression experiments with tagged recombinant RIPK1 proteins. B – D The Drug Affinity Responsive Target Stability (DARTS) assay identified RIPK1 as a target of primidone. U937 cells were lysed, cleared, and incubated for 20 min at room temperature in the presence of the indicated molecules. Subsequently, protein lysates were digested for 5 min using thermolysin. B Increased resistance to proteolysis of RIPK1 was detected upon binding of primidone as well as Nec-1 s by western blotting using anti-RIPK1 antibody. Nonselective interference of small-molecules with this assay was excluded by the addition of GSK'872, a selective inhibitor of the structurally related RIPK3 that does not affect digestion of RIPK1. Simultaneous proteolysis of RIPK3 and MLKL in this setting by primidone or Nec-1 s was excluded by western blots using anti-RIPK3 ( C ) and anti-MLKL ( D ) antibody, respectively, in which the suitability of the DARTS assay was confirmed by the addition of GSK’872 ( C ) and NSA ( D ) as selective inhibitors of RIPK3 and human MLKL, respectively [ , ]. All blots were stripped and re-probed with an antibody against HSP90 as the loading control. One representative cropped blot of three independent experiments each is shown. E The ADP-Glo ™ Kinase Assay used to measure the kinase activity of recombinant human RIPK1 (aa 1-327) revealed that primidone is an effective kinase inhibitor of RIPK1 (RLU = relative light units). In parallel, Nec-1 s (a confirmed kinase inhibitor of RIPK1) and GSK’872 (a specific kinase inhibitor of RIPK3) were integrated in the assay as internal controls. The graph shows the mean ± SD of three independent experiments.

Article Snippet: The following primary antibodies were used for immunoblotting at 1:1000 dilution unless specified otherwise: anti-RIPK1 (3493), anti-phospho-RIPK1 (human) (65746), anti-phospho-RIPK1 (murine) (31122), anti-RIPK3 (human) (13526), anti-phospho-RIPK3 (human) (93654), anti-phospho-RIPK3 (murine) (57220), anti-IκBα (9242), anti-phospho-IκBα (2859), anti-NF-κB (6956), anti-phospho-NF-κB (3033), anti-MK2 (3042), anti-phospho-MK2 (3007), anti-p38 (9212), anti-phospho-p38 (9215), anti-JNK (9252), anti-phospho-JNK (4668), anti-caspase-8 (9746), anti-CYLD (8462) all from Cell Signaling Technology (Frankfurt, Germany), anti-MLKL (MABC604) from Merck Millipore GmbH, anti-SHARPIN (14626-1-AP) from Proteintech (Manchester, UK), anti-TRADD (610572) from BD Biosciences (Wokingham, UK), anti-human phospho-MLKL (ab187091), anti-murine phospho-MLKL (ab196436), anti-TNFR1 (ab205718) all from Abcam (Berlin, Germany), anti-HSP90 (ADI-SPA-835, dilution 1:3000) from Enzo Life Sciences, anti-RIPK3 (murine) (2283, dilution 1:3000) from ProSci (via Hölzel Diagnostika), anti-FADD (sc-6036), anti-caspase-8 (sc-6136) both from Santa Cruz Biotechnology (Heidelberg, Germany), anti-β-Actin (A5441), anti-FLAG (F3165) both from Sigma-Aldrich and anti-HA (11867423001) from Roche Pharma AG (Reinach, Switzerland).

Techniques: Expressing, Recombinant, Incubation, Binding Assay, Western Blot, Kinase Assay, Activity Assay

Journal: bioRxiv

Article Title: MK2/p38/p53 negatively regulate basal IL-1β and non-canonical NF-κB signaling

doi: 10.1101/2025.09.30.679163

Figure Lengend Snippet: The non-canonical NF-κB pathway is activated in i MK2 -KO cells (mRNA) (A) Inhibition of the canonical NF-κB pathway using the IKKβ inhibitors Takinib (10 µM, 2h) and sc-514 (50 µM, 2h) reduced Il1b mRNA levels in IL-1α- and LPS-treated i MK2 -KO cells, but did not affect basal Il1b levels. Inhibiting IKKα and IKKβ with HPN-01 (10 µM, 2h) reduced Il1b mRNA levels in untreated (UT) and IL-1α (5 ng/ml, 1h)- or LPS-stimulated cells (100 ng/ml, 1h). Inhibition of the non-canonical NF-κB pathway by IKKα inhibitor B022 (5 µM, 2h) reduced mainly basal Il1b mRNA. (B) The level of Map3k14 mRNA is increased in i MK2 -KO+ GFP . (C) Relb mRNA level is increased in UT i MK2 -KO+ GFP cells. (D) Relb and (E) Nfkb2 mRNA levels are increased in IL-1α (5ng/ml, 1h)-stimulated i MK2 -KO+ GFP cells. (F) Basal Traf2 mRNA is reduced in i MK2 -KO+ GFP cells. (G) The Traf3 mRNA level is not changed significantly. mean ± SEM, students t-test, * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: BMDM (5x10 5 cells/well), i MK2 -KO or RAW 264.7 cells (both 2x10 5 cells/well) were seeded, and treated one day later with the indicated concentrations and durations of recombinant murine IL-1α (211-11A, Peprotech), LPS (Escherichia coli 0127:B8; Sigma-Aldrich), Actinomycin D (Cayman Chemicals), B022 (MedChemExpress), BMS 345541 (Axon Medchem), CAY10657 (Cayman Chemicals), SML1160 (Sigma-Aldrich), IKK inhibitor XII (HPN-01, Sigma Aldrich), sc-514 (Cayman Chemicals), T-5224 (Cayman Chemicals), Takinib (MedChemExpress) or Nutlin-3 (Santa Cruz Biotechnology).

Techniques: Inhibition

Journal: bioRxiv

Article Title: MK2/p38/p53 negatively regulate basal IL-1β and non-canonical NF-κB signaling

doi: 10.1101/2025.09.30.679163

Figure Lengend Snippet: p53 inactivates the non-canonical NF-κB pathway via RelB degradation (A-B) Nutlin-3 (20 µM, 4h) treated i MK2 -KO cells accumulate p53 and harbor reduced RelB protein, but not NF-κB2, TRAF2 or p38α. (C) RelB cleavage products appear in Western blots after Nutlin-3 (20 µM) treatment in i MK2 -KO cells. (D) Il1b mRNA and (E) TP53 mRNA are reduced in Nutlin-3 (20 µM)-treated i MK2 -KO cells. N=3-4, mean ± SEM, B) students t-test, D-E) 2W-RM-ANOVA with Bonferroni posttests, * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: BMDM (5x10 5 cells/well), i MK2 -KO or RAW 264.7 cells (both 2x10 5 cells/well) were seeded, and treated one day later with the indicated concentrations and durations of recombinant murine IL-1α (211-11A, Peprotech), LPS (Escherichia coli 0127:B8; Sigma-Aldrich), Actinomycin D (Cayman Chemicals), B022 (MedChemExpress), BMS 345541 (Axon Medchem), CAY10657 (Cayman Chemicals), SML1160 (Sigma-Aldrich), IKK inhibitor XII (HPN-01, Sigma Aldrich), sc-514 (Cayman Chemicals), T-5224 (Cayman Chemicals), Takinib (MedChemExpress) or Nutlin-3 (Santa Cruz Biotechnology).

Techniques: Western Blot

Journal: bioRxiv

Article Title: MK2/p38/p53 negatively regulate basal IL-1β and non-canonical NF-κB signaling

doi: 10.1101/2025.09.30.679163

Figure Lengend Snippet: p53 activates mitochondrial caspase-3, which cleaves RelB (A-B) Nutlin-3 (20 µM, 4h) treated i MK2 -KO cells have reduced levels of full-length Pro- caspase-3 protein. (C-D) The appearance of RelB cleavage products after Nutlin-3 (20 µM, 4h) treatment in i MK2 -KO cells can be blocked by the caspase-3 inhibitor z-DEVD-fmk (100 µM, 1h), the pan-caspase inhibitor z-VAD-fmk (25 µM, 1h), and (E-F) the inhibitor of p53 mitochondrial translocation Pifithrin-µ (10 µM, 1h), but not by the p53 transcriptional inhibitor Pifithrin-α (10 µM, 1h) in i MK2 -KO cells. A-F) N=3-5, mean ± SEM, students t-test, * p<0.05, ** p<0.01, *** p<0.001.

Article Snippet: BMDM (5x10 5 cells/well), i MK2 -KO or RAW 264.7 cells (both 2x10 5 cells/well) were seeded, and treated one day later with the indicated concentrations and durations of recombinant murine IL-1α (211-11A, Peprotech), LPS (Escherichia coli 0127:B8; Sigma-Aldrich), Actinomycin D (Cayman Chemicals), B022 (MedChemExpress), BMS 345541 (Axon Medchem), CAY10657 (Cayman Chemicals), SML1160 (Sigma-Aldrich), IKK inhibitor XII (HPN-01, Sigma Aldrich), sc-514 (Cayman Chemicals), T-5224 (Cayman Chemicals), Takinib (MedChemExpress) or Nutlin-3 (Santa Cruz Biotechnology).

Techniques: Translocation Assay