EMBO/soda-nel huggingface dataset:
IGF1R regulates retrograde axonal transport of signalling endosomes in motor neurons (2020)
Figure 2

Figure 2- The IGF1R pathway influences retrograde axonal transport of signalling endosomes

PMN were treated with 1 µM PPP (blue) or 50 ng/ml IGF1 (green) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT.

Example images of DIV6 PMN treated with 1 µM PPP or 50 ng/ml IGF1. PMN were stained with 30 nM AlexaFluor555-HcT. The scale bar is 50 µm.

Example of HcT-containing organelle tracking in the axon of PMN. Each colour denotes a single endosome tracked over time. The scale bar is 10 µm.

Graph shows the average velocity of HcT-containing organelles, after PPP treatment compared to controls (DMSO: 138 endosomes, 24 axons, 6,433 movements; PPP: 130 endosomes, 23 axons, 5,222 movements) (** P= 0.0061, Student's t-test, N=4 independent experiments, boxplot shows median, first and third quartiles. Upper/lower whiskers extend to 1.5 * the interquartile range).

Percent of time HcT-containing organelles pausing per axon. There was no difference in pausing between PPP-treated neurons and controls (P=0.45, Student's T-test, N=4 independent experiments data shown are mean ± SEM).

Graph shows the average velocity of HcT-containing organelles after IGF1 treatment compared to control (water: 138 endosomes, 22 axons, 5,119 movements; IGF1: 106 endosomes, 20 axons, 5,202 movements; *** P=2.83x10-5, Student's T-test, N=3 independent experiments, boxplot shows median, first and third quartiles. Upper/lower whiskers extend to 1.5 * the interquartile range).

Percent of time HcT-containing organelles spent pausing per axon. IGF1 significantly increased the pausing time of HcT-containing organelles (* P=0.011, Student's T-test, N=3 independent experiments, data shown are mean ± SEM).

Speed distribution profile of PPP-treated neurons and controls. PPP caused an increase in instantaneous velocities of HcT-containing organelles (N=3 independent experiments, data shown are mean ± SEM).

Speed distribution profile of IGF1-treated neurons and controls. IGF1 caused a decrease in instantaneous velocities of HcT-containing organelles compared to controls (N=3 independent experiments , data shown are mean ± SEM).
IGF1R regulates retrograde axonal transport of signalling endosomes in motor neurons (2020)
Figure 3

Figure 3- PPP downregulates Akt signalling

A) Western blot showing the levels of pAKT (Ser473), total Akt, pErk1/2 (T202/Y204) and total Erk1/2 after treatment with 1 µM PPP or 50 ng/ml IGF1 for 60 min. Protein was normalised to GAPDH.

B) Western blot quantification. PMN treated with PPP showed a significant decrease in Akt activation (* P=0.049, N=5 independent experiments, Student's T-test), but no significant difference was seen on Erk1/2 activation (P=0.07, Student's T-test, N=5 independent experiments). 50 ng/ml IGF1 treated neurons showed a significant increase in Akt activation (** P=0.008, Student's T-test, N=3 independent experiments). In contrast, no change in Erk1/2 activation (P=0.88, Student's T-test, N=3 independent experiments) was observed (data shown are mean ± SEM).

C) Graph shows the average velocity of HcT-containing organelles treated with Capivasertib or Ipatasertib (10 nM) for 30 min compared to controls (DMSO: 483 endosomes, 34 axons, 41,897 movements; Capivasertib: 658 endosomes, 43 axons, 49,841 movements; Ipatasertib: 605 endosomes, 32 axons, 41,485 movements) (** P=2 x10-16, One-way ANOVA, Tukey's post-hoc test: DMSO-Capivasertib and DMSO-Ipatasertib *** P <0.0001; N=6 independent experiments, boxplot shows median, first and third quartiles. Upper/lower whiskers extend to 1.5 * the interquartile range).

D) Percent of time HcT-containing organelles pausing per axon. Treatment with Ipatasertib and Capivasertib reduced the amount of time organelles spent pausing (* P=1.14 x10-7, One-way ANOVA, Tukey's post-hoc test: DMSO-Capivasertib P= 9.37x10-5, DMSO-Ipatasertib P= 2 x10-7; data shown are mean ± SEM, N=6 independent experiments).
IGF1R regulates retrograde axonal transport of signalling endosomes in motor neurons (2020)
Figure 4

Figure 4- Knockdown of IGF1R increases retrograde axonal transport of signalling endosomes

A) PMN were treated with either an shRNA targeting IGF1R (1 µl) or a scrambled control 6 h after plating. At DIV 6-7, retrograde axonal transport was analysed using 30 nM AlexaFluor555-HcT. Knockdown of IGF1R caused a significant increase in the average velocity of signalling endosomes compared to controls (IGF1R shRNA: 108 endosomes, 21 axons, 801 movements; scrambled shRNA: 94 endosomes, 20 axons, 777 movements, * P=0.011, Student's T-test, N=3 independent experiments, boxplot shows median, first and third quartiles. Upper/lower whiskers extend to 1.5 * the interquartile range).

B) Speed distribution profile including pausing events of PMN treated with a shRNA targeting IGF1R or a scrambled control. IGF1R knockdown caused an increase in instantaneous velocities of HcT-containing organelles and decreased the amount of pausing (N=3 independent experiments, data shown are mean ± SEM).
IGF1R regulates retrograde axonal transport of signalling endosomes in motor neurons (2020)
Figure 5

Figure 5- IGF1R inhibition alters the expression level of BICD1

A) Western blot of PMN treated with 1 µM PPP or 50 ng/ml IGF1 for 60 min. Cells were lysed and the extracts assessed for expression levels of dynein adaptor proteins, all proteins were assessed from the same blot.

B) Hook3 expression didn't change after treatment with PPP or IGF1 compared to controls (DMSO, 100%; PPP, 102.16 ± 4.3%, P=0.67, Student's T-test, N=3 independent experiments; water, 100%; IGF1, 93.48 ± 4.2%, P=0.26, N=3 independent experiments, data shown are mean ± SEM).

C) However, BICD1 expression was significantly increased in PMN treated with PPP compared to control (DMSO, 100%; PPP, 137.1 ± 7.3%, **P=0.002, Student's T-test, N=7 independent experiments). In contrast, IGF1R stimulation by IGF1 had no effect on BICD1 expression (water, 100%; IGF1, 88.1 ± 14.0%, P=0.49, Student's T-test, N=3 independent experiments, data shown are mean ± SEM).

D) BICD2 expression didn't change after treatment with PPP or IGF1 compared to controls (DMSO, 100%; PPP, 104.44 ± 6.5%, P=0.51, N=3 independent experiments: water, 100%; IGF1, 106 ± 7.5%, P=0.51, Student's T-test, N=3 independent experiments, data shown are mean ± SEM).
IGF1R regulates retrograde axonal transport of signalling endosomes in motor neurons (2020)
Figure 6

Figure 6- BICD1 expression in axons is upregulated by de-novo synthesis

A) Western blot of PMN pre-treatment with cycloheximide (50 µg/ml) or MG-132 (10 µM) for 30 min before incubation with 1 µM PPP for 60 min. Cycloheximide causes a significant decrease in BICD1 levels compared to PPP treated cells. (DMSO, grey: 100%, N=7 independent experiments; PPP, blue: 137.1 ± 7.3%, N=7 independent experiments; CHX, pink: 89.5 ± 12.7%, N=5 independent experiments; MG-132, teal: 124 ± 28.7, N=3 independent experiments; P=0.009, one-way ANOVA; Tukey's post-hoc test: DMSO-PPP * P=0.04, PPP-PPP+CHX * P=0.01; data shown are mean ± SEM).

B) Schematic of puromycin-PLA assay. Cells were treated with DMSO, PPP or PPP + anisomycin for 30 min in the axonal compartment. After this puromycin was added to the medium for an additional 30 min. Cells were then fixed and the PLA protocol was performed (see Materials and Methods). Puromycin- Purple triangle, BICD1-Grey circles, Anti-puromycin antibody- green, Anti-BICD1 antibody- blue, Secondary antibodies- black.

C) Puromycin-PLA signal in PMN grown in microfluidics devices. Images are magnifications from the white boxes in Appendix Fig S6 taken from the axonal compartment. The scale bars are 10 µm. White arrows point to puromycin-PLA puncta within the β3-tubulin mask; the empty arrow shows a PLA puncta not associated with neurons.

D) Quantification of puromycin-PLA signal. PPP causes an increase in PLA puncta in the axon of PMN, which is abolished by treatment with 40 µM anisomycin (DMSO, grey: 100%; PPP, blue: 139.3 ± 7.7%; PPP + anisomycin, purple: 86.3 ± 10.0; P=0.005, one-way ANOVA, Tukey's post-hoc test; DMSO vs PPP, * P=0.02; PPP vs PPP + anisomycin, ** 0.005, N=3 independent experiments, 27-57 images, data shown are mean ± SEM).
IGF1R regulates retrograde axonal transport of signalling endosomes in motor neurons (2020)
Figure 7

Figure 7- IGF1R inhibition increases the rate of retrograde axonal transport in an ALS mouse model

A) The graph shows the average velocity of HcT-containing organelles in vivo in D72/73 SOD1G93A mice and wild type littermate controls. PPP (blue) treatment increased the transport rate of signalling endosomes in wild type and SOD1G93A mice compared to animals treated with vehicle control (grey) (WT control: cargo 371, movements 15,272, N=4 independent experiments; WT + PPP: cargo 388, movements 14,512, N=4 independent experiments; SOD1G93A control: cargo 322, movements 15,425, N=4 independent experiments; SOD1G93A + PPP: cargo 404, movements 17,643, N=4 independent experiments; P=2.24x10-12 (treatment), P=2.22 x10-16 (genotype), two-way ANOVA, Tukey's post-hoc test: all conditions *** P<0.005, boxplot shows median, first and third quartiles. Upper/lower whiskers extend to 1.5 * the interquartile range).

B) PPP treatment does not alter the pausing of signalling endosomes (WT: 16.1 ± 1.5%, WT + PPP: 13.1 ± 1.5%; SOD1G93A: 16.6 ± 1.9%, SOD1G93A + PPP: 16.2 ± 2.1%; P=0.36 (treatment), P=0.32 (genotype), N=4 independent experiments for each condition, data shown are mean ± SEM.

C) Treatment with PPP had no effect on Akt levels in the sciatic nerve (WT: Control-100 ± 8.6, PPP-91.5 ± 14.6%; SOD1: Control-101.4 ± 13.5%, PPP-117.6 ± 32.9%, P=0.81 (treatment), P=0.53 (genotype), two-way ANOVA, N=5 independent experiments, data shown are mean ± SEM, Appendix Fig S7D).

D) Erk1/2 activation was down-regulated after treatment with PPP in the sciatic nerve of wild type and SOD1G93A mice (WT: control-100 ± 15.4%, PPP-64.4 ± 3.2%; SOD1: Control-78.0 ± 11.9 %, PPP-51.2 ± 8.2%, * P=0.013 (treatment), P=0.13 (genotype), two-way ANOVA, N=5 independent experiments, data shown are mean ± SEM, Appendix Fig S7D). Protein was normalised to GAPDH protein levels.

Expanded view figure legends
Crystal structure of the 2019-nCoV spike receptor-binding domain bound with the ACE2 receptor (2020)
Fig. 1

Overall structure of 2019-nCoV RBD bound with ACE2.(a) Overall topology of 2019-nCoV spike monomer. NTD, N-terminal domain. RBD, receptor-binding domain. RBM, receptor-binding motif. SD1, subdomain 1. SD2, subdomain 2. FP, fusion peptide. HR1, heptad repeat 1. HR2, heptad repeat 2. TM, transmembrane region. IC, intracellular domain. (b) Sequence and secondary structures of 2019-nCoV RBD. The RBM is colored red. (c) Overall structure of 2019-nCoV RBD bound with ACE2. ACE2 is colored green. 2019-nCoV RBD core is colored cyan and RBM is colored red. Disulfide bonds in the 2019-nCoV RBD are shown as stick and indicated by yellow arrows. The N-terminal helix of ACE2 responsible for binding is labeled.
Crystal structure of the 2019-nCoV spike receptor-binding domain bound with the ACE2 receptor (2020)
Fig. 2

Structural comparisons of 2019-nCoV and SARS-CoV RBDs and their binding modes to ACE2 receptor.(a) Alignment of the 2019-nCoV RBD (core in cyan and RBM in red) and SARS-CoV RBD (core in orange and RBM in blue) structures. (b) Structural alignment of 2019-nCoV RBD/ACE2 and SARS-CoV RBD/ACE2 complexes. 2019-nCoV RBD is colored cyan and red, its interacting ACE2 is colored green. SARS-CoV RBD is colored orange and blue, its interacting ACE2 is colored salmon. The PDB code for SARS-CoV RBD/ACE2 complex: 2AJF.
Crystal structure of the 2019-nCoV spike receptor-binding domain bound with the ACE2 receptor (2020)
Fig. 3

The 2019-nCoV RBD/ACE2 binding interface compared with that of SARS-CoV RBD/ACE2.(a) Contacting residues shown as stick at the 2019-nCoV RBD/ACE2 and SARS-CoV RBD/ACE2 interfaces. Positions in both RBDs involved in ACE2 binding are indicated by red labels. (b) Sequence alignment of 2019-nCoV RBD and SARS-CoV RBD. Contacting residues in the 2019-nCoV RBD are indicated by black dots; contacting residues in the SARS-CoV RBD are indicated by red dots. (c) Electrostatic potential map of 2019-nCoV RBD (left panel), and SARS-CoV RBD (right panel). The position of K417 in the 2019-nCoV RBD is indicated by black arrow. The N-terminal helix of ACE2 is shown as green ribbon. The PDB code for SARS-CoV RBD/ACE2 complex: 2AJF.
Differential functions of FANCI and FANCD2 ubiquitination stabilize ID2 complex on DNA (2020)
Figure 1

Figure 1. FANCD2 ubiquitination enhances ID2-dsDNA binding.

IRDye700-labelled 32 base pair dsDNA was used to assess ID2-DNA binding, when neither protein is ubiquitinated (I + D2), when only FANCD2 is ubiquitinated (I + D2Ub) and when both FANCD2 and FANCI are ubiquitinated (IUb +D2Ub).

A. Left: Fluorescence changes of IRDye700 labelled dsDNA (at 125 nM) when incubated at increasing ID2 (I+D2, IUb+D2Ub, I+D2Ub) or ubiquitinated FANCD2 (D2Ub) concentrations (ranging from 1.3 nM to 5.9 µM). Measurement of fluorescence enhancement for each ID2 complex was conducted for two separately prepared complexes (two technical repeats) and all data points for each protein-combination were used in fitting of a one-site binding model. Right: Bar graph showing mean apparent Kd values calculated from model fitting. Error bars: Asymmetric 95% confidence intervals from non-linear regression (23-24 data points each).

B. Assessment of protein-DNA interactions using Electro-mobility shift assays (EMSA). IRDye700 labelled dsDNA (at 2 nM) was incubated with indicated amounts of non/single/double-ubiquitinated ID2 (His6-TEV-V5-FANCI and FLAG-FANCD2) protein complexes (I+D2, IUb+D2Ub, I+D2Ub­) or ubiquitinated FLAG-FANCD2 (D2Ub). Mixes were ran on non-denaturing gels, and the resolved free- and protein-bound DNA bands were visualized using an infrared scanner. EMSA gels of ID2 complexes are representative of 3 replicate experiments.
Differential functions of FANCI and FANCD2 ubiquitination stabilize ID2 complex on DNA (2020)
Figure 2

Figure 2. FANCD2 ubiquitination creates a secondary ID2 "Arm" interface, resulting in a closed ID2 conformation.

A. Left: CryoEM 2D classes of ID2Ub and IUbD2Ub-dsDNA. Right: 3D reconstructions of ID2Ub (EMD-10843) and IUbD2Ub-DNA (EMD-10844). Both structures exhibit a torus-like shape.

B. Left: Rigid body fit of the mouse ID2 atomic structure (3S4W) into the human IUbD2Ub-dsDNA cryoEM map. Right: Flexible fit of the mouse ID2 atomic model into the human IUbD2Ub-dsDNA cryoEM map (iMODFIT). The C-termini "arms" of FANCD2 and FANCI close in the flexible fitting. A tube-like volume in the IUbD2Ub-DNA cryoEM map, which is not occupied by the ID2 iMODFIT structure, is present just beneath the Arm ID2 interface and likely represents bound DNA (solid density).
Differential functions of FANCI and FANCD2 ubiquitination stabilize ID2 complex on DNA (2020)
Figure 3

Figure 3. The pathological FANCI R1285Q mutation disrupts ID2 ubiquitination, likely via disturbing the closed ID2 conformation.

A. Arginine 1285 of FANCI, which is mutated in some FA patients, is located in the Arm ID2 interface in our cryoEM IUbD2Ub-dsDNA structure.

B. Both wild-type (IWT) and R1285Q mutant (IR1285Q) FANCI can be efficiently ubiquitinated in the presence of DNA. Reactions were performed at 2 μM FANCI substrate for 60 min in the absence or presence of 4 µM ssDNA.

C. Both wild-type (IWT) and R1285Q mutant (IR1285Q) FANCI efficiently associate with FANCD2. Fluorescence changes occurring when RED-tris-NTA-labelled FANCI (IWT or IR1285Q; both at 60 nM) is incubated at increasing concentrations of FANCD2 (ranging from 2.48 nM to 5.08 µM). Titrations were conducted twice (two technical replicates) and all data points for each protein-combination were used in fitting of a one-site binding model. Apparent Kd values (mean ± SEM) derived from fitting of a one-site binding model to the 24 data points of the two technical replicates are shown.

D. FANCD2 within an IR1285QD2 complex is resistant to ubiquitination. Reactions were performed at 4 μM ID2 substrate and 16 μM dsDNA. Progress of FANCD2 and FANCI ubiquitination was monitored by western blotting following SDS-PAGE.

E. FANCD2 ubiquitination cannot robustly enhance ID2-DNA binding when complexed with IR1285Q, as determined by PIFE. Left: Fluorescence changes when IRD700-labelled dsDNA (at 125 nM) is incubated at increasing concentrations of IR1285Q +D2 or IR1285Q +D2Ub (complex concentrations ranging from 1.54 nM to 3.8 µM). Measurement of fluorescence enhancement for each protein combination was conducted for two separately prepared complexes (two technical repeats) and all data points for each protein-combination were used in fitting of a one-site binding model. Right: Bar graph showing mean apparent Kd values calculated from the one-site binding model. Error bars: Asymmetric 95% confidence intervals from non-linear regression (22 data points each). IWT + D2 and IWT + D2Ub previously calculated curves and corresponding Kd values (from data points shown in Figure 1A) are also shown for comparison.

F. FANCD2 ubiquitination cannot robustly enhance ID2-DNA binding when complexed with IR1285Q, as determined by EMSAs. IRDye700-labelled dsDNA (at 2 nM) was incubated with indicated amounts of ID2 (His6-FANCI or His6-FANCIR1285Q mixed with non-ubiquitinated or ubiquitinated FLAG-FANCD2. Mixes were ran on non-denaturing gels, and the resolved free- and protein-bound DNA bands were visualized using an infrared scanner. Gels shown are representative of two replicate experiments.
Differential functions of FANCI and FANCD2 ubiquitination stabilize ID2 complex on DNA (2020)
Figure 4

Figure 4. FANCI ubiquitination of ID2Ub protects D2Ub from USP1-UAF1 mediated deubiquitination.

A. USP1-UAF1 can efficiently deubiquitinate D2Ub in the presence or absence of FANCI (I), but not in the presence of ubiquitinated FANCI (IUb). Ubiquitinated FANCD2 (D2Ub) was mixed with a 50 base pair dsDNA and either His6-TEV-V5-FANCI (I), ubiquitinated His6-TEV-V5-FANCI (IUb) or no protein; protein-DNA mixes were subsequently incubated with USP1-UAF1 (at 25 nM or 100 nM) for indicated time periods. Deubiquitination of D2Ub and IUb was assessed, following SDS-PAGE, by Coomassie staining of the gels, as well as by western blotting of transferred blots with a specific FANCD2 antibody.

B. USP7 and USP2 can deubiquitinate D2Ub, but their activity towards D2Ub is greatly reduced in the presence of FANCI (I) or ubiquitinated FANCI (IUb). Reactions were set-up as in A, but with 100 nM USP7 or USP2. Deubiquitination was assessed, following SDS-PAGE, by Coomassie staining.

C. Location of the four hydrophobic residues mutated to alanine in the ubiquitin structure (PDB: 1ubq). Top: location of F4, I36, I44 and L73 (shown in red) in ubiquitin's surface (shown in yellow). Bottom: same as above but with ubiquitin's surface coloured according to charge (red: negative, blue: positive, white: no charge).

D. Time course of FANCI ubiquitination with various ubiquitin mutants and corresponding sensitivity/resistance to USP1-UAF1 mediated deubiquitination of resulting products. Deubiquitination reactions in the presence or absence of USP1-UAF1 (100 nM) incubated for 30 minutes.

E. Time course of USP1-UAF1 mediated deubiquitination of IUbD2Ub-DNA complexes consisting of D2Ub a 50 base pair dsDNA and IUb produced with various ubiquitin mutants or wild-type (WT) ubiquitin. Progress of deubiquitination reaction was assessed following SDS-PAGE, by both Coomassie staining of the gels, as well as by western blotting of transferred blots with a specific FANCD2 antibody. FANCI ubiquitination with indicated ubiquitin-mutants (or WT) and subsequent deubiquitination of resulting IUbD2Ub-DNA complexes were conducted twice; the residual FANCD2 ubiquitination, calculated from the FANCD2 blots for each time-point, was plotted for each type of ubiquitin in the protein complex, and corresponding deubiquitination progress curves were fitted using a one-phase decay model.
Thalidomide and its metabolite 5-hydroxythalidomide induce teratogenicity via the cereblon neo-substrate PLZF (2020)
Figure 1

Figure 1. Identification of thalidomide-dependent interactors of CRBN using a cell-free based human TF protein array

(A) Chemical structures of thalidomide, pomalidomide and lenalidomide.

(B) Schematic diagram of the thalidomide-dependent in vitro binding assay between CRBN and substrates using AlphaScreen technology.

(C) Detection of luminescent signals of thalidomide-dependent interactions between bls-CRBN and FLAG-GST-IKZF1. Dose-dependent signals (DMSO, 2.5, 5, 10, 25, 50 or 100 µM thalidomide) were analysed with an in vitro binding assay using the AlphaScreen technology. (D) Results of in vitro high-throughput screening targeting 1,118 human transcription factors. Green and red spots denote known neosubstrates and candidate clones, respectively.

(E) Confirmation of thalidomide dependency on six hit proteins using an in vitro binding assay. Interaction between bls-CRBN and FLAG-GST-protein in the presence of DMSO or 50 µM thalidomide was detected using the AlphaScreen technology.

(F) In vitro binding assay for thalidomide, pomalidomide and lenalidomide. Interaction between bls-CRBN and FLAG-GST-PLZF in the presence of DMSO (3.125, 6.25, 12.5, 25, 50, 100 or 200 µM) thalidomide, pomalidomide or lenalidomide was analysed using the AlphaScreen technology.

Data information: All relative AlphaScreen (AS) signals are expressed as relative luminescent signal with luminescent signal of DMSO as one. Error bars denote ± standard deviation (n=3).
Thalidomide and its metabolite 5-hydroxythalidomide induce teratogenicity via the cereblon neo-substrate PLZF (2020)
Figure 2

Figure 2. PLZF is a substrate of CRL4CRBN with thalidomide and lenalidomide for E3 ubiquitin ligase

(A) Immunoblot analysis of AGIA-PLZF protein levels in AGIA-PLZF and FLAG-CRBN expressing HEK293T cells treated with DMSO or lenalidomide (Len) in the presence of DMSO, MG132 or MLN4924 for 9 h.

(B) Immunoblot analysis of endogenous PLZF protein levels in HEK293T cells or CRBN-/- HEK293T cells treated with DMSO or lenalidomide (Len) for 24 h.

(C) Immunoprecipitation of FLAG-CRBN in FLAG-CRBN and AGIA-PLZF expressing HEK293T cells treated with DMSO or lenalidomide (Len) in the presence of MG132 for 8 h. Components of CRL4FLAG-CRBN and AGIA-PLZF were detected using each specific antibody, as indicated.

(D) Ubiquitination of AGIA-PLZF in AGIA-PLZF and FLAG-CRBN expressing CRBN-/- HEK293T cells treated with DMSO or thalidomide (Tha) in the presence of MG132 for 10 h. AGIA-PLZF was immunoprecipitated using anti-AGIA antibody and the polyubiquitin chain on AGIA-PLZF was analysed by immunoblot.

(E) In vitro binding and ubiquitination assay of AGIA-PLZF. Empty vector, AGIA-PLZF or FLAG-CRBN expressing HEK293T cells were lysed and the lysates were mixed. The first immunoprecipitation with anti-AGIA or anti-FLAG antibodies was performed in the presence of DMSO or 200 µM lenalidomide. The purified AGIA-PLZF or CRL4FLAG-CRBN complex, including AGIA-PLZF and FLAG-CRBN, was incubated with recombinant E1, E2 and HA-ubiquitin in the presence of DMSO or 200 µM lenalidomide. The second immunoprecipitation was performed using anti-AGIA antibody. Ubiquitination of PLZF was analysed by immunoblot.