Journal: bioRxiv

Article Title: Androgen receptor condensates as drug targets

doi: 10.1101/2022.08.18.504385

Figure Lengend Snippet: A) Structure of AR predicted with AlphaFold. The model is coloured by structure prediction confidence from high confidence (dark-blue) to low confidence (orange-yellow). The known AR domains are highlighted. B) Live-cell STED imaging of HEK293T cells transfected with the indicated AR constructs tagged with mEGFP. Cells were imaged after treatment with 10 nM DHT for four hours. Scale bar: 5 μm. Dashed line indicates the nuclear periphery. C) Intensity of the NMR resonances of the AR AD as a function of amino acid position, measured for the displayed AR AD concentrations. The position of Transactivation Unit 1 and 5 (Tau-1, Tau-5), and of the 23 FQNLF 27 motif are highlighted. Green circles indicate the positions of residues not assigned or not visible (NA/NV) in the NMR spectrum recorded at 25 μM, including residues in regions of low sequence complexity such as poly-glutamine (pQ), poly-proline (pP) and poly-glycine (pG) tracts. Yellow and orange circles represent the positions of tyrosine (Tyr) residues mutated to serine (Ser) in 8YtoS and 14YtoS, respectively; all residues Tyr were mutated to Ser in 22YtoS. D) Fluorescence microscopy images of 40 µM AR-AD in vitro droplets (WT* and Tyr to Ser mutants) at 1 M NaCl and room temperature. Scale bar: 10 μm. E) Schematic representation of the LCST phase diagram of the AR AD (WT) obtained by determining the cloud points of solutions of increasing NaCl concentration (left) and of how cloud point measurements under two different solution conditions (right), labeled as 1 and 2, allow ranking Tyr to Ser mutants in terms of their phase separate capacity. F) Determination of the cloud points of AR AD (WT* and Tyr to Ser mutants) under two different solution conditions, labeled as 1 and 2. G) Representative merged confocal images of 15 µM MED1-IDR (left column) and 5 µM RNAPII-CTD (right column) droplets obtained at 20 mM NaCl or 50 mM NaCl, respectively, and 10 % ficoll before and after addition of 1 µM AR AD (WT* or 22YtoS). Scale bar: 5 μm. H) Quantification of AR AD partitioning into MED1-IDR (top graph) and RNAPII-CTD droplets (bottom graph), by measuring AR AD fluorescence intensity in droplets. Boxes correspond to the mean and the quartiles of all droplets represented as coloured dots from three image replicates. **** p < 0.0001. I) Representative merged confocal images of MED1-IDR and RNAPII-CTD multiphasic droplets obtained in 125 mM NaCl and 10% ficoll with and without the addition of 1 µM AR AD (WT* or 22YtoS). Scale bar: 5 μm. J) Normalized intensity plot profile of droplet cross-sections from the images shown in panel I. See also .

Article Snippet: KpnI-KpnI fragment with that of the wild-type AR sequence from peGFP-C1-AR. mEGFP constructs: Monomeric EGFP was subcloned into vectors containing human AR (Addgene #29235) and AR-V7 (Addgene #86856) using Gibson assembly to create mEGFP-AR-FL and mEGFP-AR-V7 (referred to as ‘AD+DBD+NLS’ in , ) mammalian expression vectors.

Techniques: Imaging, Transfection, Construct, Sequencing, Fluorescence, Microscopy, In Vitro, Concentration Assay, Labeling

Journal: bioRxiv

Article Title: Androgen receptor condensates as drug targets

doi: 10.1101/2022.08.18.504385

Figure Lengend Snippet: A) Live-cell confocal imaging of the indicated mEGFP construct transfected into HEK293T after treatment with vehicle or 10 nM DHT for four hours. Scale bar: 3 µm. Dashed lines indicate nuclear periphery. B) Quantification of confocal data in . Y-axis indicates the standard deviation, and x-axis indicates the mean intensity of pixels in the corresponding nucleus. Each dot represents measurements from an individual cell, and lines represent standard regression fits to the corresponding data spread (N = 2). C) Distribution of aromatic (Histidine, Phenylalanine, Tryptophan, Tyrosine) and Tyrosine residues along the AR AD sequence, clustered using a 9 amino acid window, where the shaded areas correspond to those represented in . D) Average intensity ratio of the NMR resonances of the AR AD at the tested protein concentrations (57.5, 100.8, 122.5 and 155.0 μM) relative to their intensity at 25 μM grouped by amino acid type. E) Fluorescence microscopy images of in vitro AR AD (WT*) concentration-dependent condensation obtained in AR AD buffer (20 mM NaP, 1 mM TCEP pH 7.4) with 150 mM NaCl and 10% ficoll, where ca 1 % of AR-AD molecules were labeled with the dye Dylight 405. Scale bar: 10 µm. F) AR AD WT* liquid character in vitro by FRAP. Top panel: confocal microscopy images of WT* AR AD droplets labeled with Alexa-647, in 150 mM NaCl and 10 % ficoll before and after photobleaching in FRAP experiment. Scale bar 5 µm. Lower panel: average relative fluorescence intensity curve of WT* AR AD droplets as a function of time following photobleaching. Error bars represent s.d. of n=10 droplets. G) (Left) microcopy images of in vitro droplets formed by the indicated proteins. The signal of the AR AD channel and merged channel are shown. AR AD proteins were used in five-fold higher concentrations than . Scale bar: 1 µm. (Right) the representative droplet’s cross section intensity profile.

Article Snippet: KpnI-KpnI fragment with that of the wild-type AR sequence from peGFP-C1-AR. mEGFP constructs: Monomeric EGFP was subcloned into vectors containing human AR (Addgene #29235) and AR-V7 (Addgene #86856) using Gibson assembly to create mEGFP-AR-FL and mEGFP-AR-V7 (referred to as ‘AD+DBD+NLS’ in , ) mammalian expression vectors.

Techniques: Imaging, Construct, Transfection, Standard Deviation, Sequencing, Fluorescence, Microscopy, In Vitro, Concentration Assay, Labeling, Confocal Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Chemical Proteomics Identifies Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1 as the Molecular Target of Quercetin in Its Anti-cancer Effects in PC-3 Cells *

doi: 10.1074/jbc.M114.553248

Figure Lengend Snippet: Schematic depiction of the workflow used to identify and characterize quercetin-binding proteins. Quercetin-specific binding proteins were captured by quercetin-Sepharose beads, and eluted fractions were resolved by SDS-PAGE. Distinct proteins in gel-eluted bands were identified using MS and validated by immunoblotting analyses and surface plasmon resonance binding assays. Specific targets were further characterized using a series of approaches, including confocal microscopy, IP, RIP, RT-qPCR, and immunoblotting analysis. Q, quercetin; T, total cell lysates; W, proteins that did not bind quercetin; E, bound proteins eluted.

Article Snippet: Quercetin was from Sigma-Aldrich, and CNBr-Sepharose 4B was obtained from GE Healthcare.

Techniques: Binding Assay, SDS Page, Western Blot, SPR Assay, Confocal Microscopy, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A and C) HEK293T cells stably expressing the indicated TRIM21 mutants (EV = empty vector) were transfected with WT His-ubiquitin and infected with AdV5 ± 9C12 or 9C12(H433A). Ubiquitinated proteins were isolated by denaturing His-pulldown using Ni-NTA beads in 6M Guanidine buffer before immunoblot analysis with anti-TRIM21 antibody. (B) A co-crystal structure of TRIM21 RING:Ube2N∼ubiquitin complex (PDB: 6S53) showing the tri-anionic anchor motif (E12, E13 and D21) and second site residues (R67 and N71). (D) Immunoblot of TRIM21 following denaturing His-ubiquitin pulldown from HEK293T cells overexpressing the indicated ubiquitin mutant at 30 min post infection with AdV5 ± 9C12 or 9C12(H433A). See also

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Transfection, Ubiquitin Proteomics, Infection, Isolation, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: Denaturing TRIM21-His pulldown from HEK293T cells stably expressing TRIM21-His and infected with AdV5 ± 9C12 or 9C12(H433A) which does not bind TRIM21. Cells were lysed in buffer containing 4M urea and where indicated, the Nickel beads were treated with the deubiquitinase USP2 before boiling and immunoblotting with anti-TRIM21 antibody.

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Stable Transfection, Expressing, Infection, Western Blot

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A) Sequence of TRIM21 with RING domain in gray, B Box in red, coiled-coil in cyan, L2 linker helices in orange & green and PRYSPRY in magenta. (B) SEC-MALS chromatograms of TRIM21 CC (129-235) loaded at concentrations of 15 (black), 5 (green), 0.55 (blue) and 0.25 mg/ml (red) are shown, in which the refractive index is indicated by the solid lines while the molar mass evaluated from the light scattering analysis is indicated with the corresponding coloured dotted lines. (C) Using a MicroCal iTC200 calorimeter, 140 µM TRIM21 coiled-coil was added in 2 µl injections into buffer at 25 °C. Integrated heats were then fit to a dimer dissociation model reveal an enthalpy of 65 kcal/mol and Kd of 7 µM. (D-F) SAXS data on TRIM21 constructs. (D) SAXS data were collected at the beam line P12 of the EMBL at the Petra-III storage ring (DESY, Hamburg). Protein expression, purification, data analysis and collection are in Supplementary Information. Data statistics are shown in Table 1. (D) Concentration-normalised scattering plots and DAM fits for CC235 (cyan, χ = 1.00), MBP-CC235 (green, χ = 1.31) and RBCC (purple, χ = 1.05). (E) The linear Guinier regions from (D). (F) Derived P( r ) curves. The CC235 P( r ) curve is consistent with an elongated rod, while the two peaks observed for MBP-CC235 and RBCC are consistent with ‘dumbbell’-shaped molecules. (G-I). SAXS data on TRIM21:Fc complex. (G) Scattering plot with DAM fit (red line, 1.00), TRIM21:Fc atomic model fit (blue, χ = 1.04), and apo-TRIM21 atomic model fit (brown dashed, χ = 2.00). (H) The linear Guinier regions from (G). (I) Derived P( r ) curves.

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Sequencing, Refractive Index, Construct, Expressing, Purification, Concentration Assay, Derivative Assay

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A) Dummy atom model (DAM) structures of TRIM21 calculated from SAXS data shown in . Overlay of reconstructions of constructs comprising TRIM21 residues 1-235 (RBCC, purple spheres) and TRIM21 residues 129-235 (CC235, blue spheres). A structural model of the TRIM21 RBCC region based on atomic models of the TRIM21 RING (yellow) and B Box (orange) domains and TRIM25 coiled-coil domain (cyan) using PDBs 5OLM and 4CFG respectively. (B) SAXS-derived model of TRIM21:Fc complex. Averaged and filtered DAM’s (white and green spheres, respectively) overlaid with an atomic model of the TRIM21:Fc complex. The RING, B Box and coiled-coil regions are as above. The PRYSPRY (magenta) and IgG Fc (grey) domains are taken directly from PDB 2IWG. (C) Crystal structure of TRIM21 RING dimer showing the important residues M10 and M72 at the dimer interface. (D) Immunoblot of TRIM21 following denaturing His-ubiquitin pulldown from TRIM21 lentivector reconstituted HEK293T cells overexpressing WT His-ubiquitin at 30 min post infection with AdV5 ± 9C12 or 9C12(H433A). (E) Trim-away of IKKα in TRIM21 lentivector reconstituted HEK293T cell lines by electroporation of anti-IKKα IgG (anti-IKKα). Cell lysates were immunoblotted for the indicated proteins. (F) in vitro ubiquitination assay using the indicated TRIM21 constructs and Ube2N. Samples were taken at the indicated timepoints and analysed by immunoblotting with anti-ubiquitin antibody. (G) Immunoblot analysis of ubiquitin discharge from Ube2N by the indicated TRIM21 constructs over a course of 20 minutes. (H) Quantification of the Ube2N∼Ub band in G relative to time point 0 in each reaction. See also

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Construct, Derivative Assay, Western Blot, Ubiquitin Proteomics, Infection, Electroporation, In Vitro

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A) Schematic illustration of multimeric TRIM21 assembly on the surface of a virus-antibody complex. (B) Remaining infectivity of AdV5-GFP particles on HEK293T cells following incubation with saturating concentrations of 9C12. For 1/e neutralization, approximately 12% of antibodies must bear an intact TRIM21 binding site which is present on 9C12(WT) but lacking in 9C12(H433A). This equates to 24 out of the maximum ∼200 antibodies bound to AdV5 at saturation. (C, E, J and L) Neutralisation of AdV5 by 9C12 in lentivector reconstituted HEK293T cells expressing the indicated TRIM21 mutants. Data normalised to the virus only condition and presented as the mean ± SEM. (D and F) AdV5-9C12 immune complex-induced NF-kB activation in HEK293T cells stably expressing the indicated TRIM21 mutants. Data normalised to the virus only condition and presented as the mean ± SEM. (G) In vitro ubiquitination assay using the indicated TRIM21 RING-linker-RING (RLR) constructs and Ube2N. Samples were taken at the indicated timepoints and analysed by immunoblotting with anti-ubiquitin antibody. (H) Trim-away of IKKα in TRIM21 lentivector reconstituted HEK293T cell lines by electroporation of anti-IKKα IgG (anti-IKKα). Cell lysates were immunoblotted for the indicated proteins. (I) Crystal structure of TRIM21-RING-B-box (PDB: 5OLM) showing residue S80 at situated at the RING:B Box interface. (K) Structure of the hydrophobic core at the TRIM21 RING:Ube2N interface (PDB: 6S53). See also Figures S3 and S4

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Virus, Infection, Incubation, Neutralization, Binding Assay, Expressing, Activation Assay, Stable Transfection, In Vitro, Ubiquitin Proteomics, Construct, Western Blot, Electroporation, Residue

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A) Upper panel: TRIM5α (rhesus) residues involved in mediating the three layers of box-box interactions (PDB:5EIA). Lower panel: model of TRIM21 B box domain structure (PDB:5OLM) onto the trimeric box structure of TRIM5α (PDB:5EIA). Residues that could mediate B box: B box interactions are highlighted. (B and C) Neutralisation of AdV5 by 9C12 in lentivector-reconstituted HEK293T cells expressing the indicated TRIM21 mutants. Data normalised to the virus only condition and presented as the mean ± SEM. (D) AdV5-9C12 immune complex-induced NF-kB activation in HEK293T cells stably expressing the indicated TRIM21 mutants. Data normalised to the virus only condition and presented as the mean ± SD. (E) Neutralisation of AdV5 by 9C12 in HEK293T cells stably expressing TRIM21-ΔBox construct with or without MG132 (20 µM). Data normalised to the virus only condition and presented as the mean ± SD.

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Expressing, Virus, Activation Assay, Stable Transfection, Construct

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A) Schematic of myc-mEGFP constructs. (B-D) HEK293T-mCherry-TRIM21 cells were electroporated with mRNA encoding the indicated myc-mEGFP constructs together with either control IgG (9C12), ant-Myc (9E10) or anti-GFP (polyclonal) antibodies. 8 hours post-electroporation cellular GFP fluorescence was imaged (B) and quantified (C) using the IncuCyte system, or total GFP protein levels analysed by immunoblotting (D) cell extracts with the indicated antibodies. Scale bar 100 µm. (E) HEK293T-mCherry-TRIM21 cells were electroporated with mRNA encoding the indicated myc-mEGFP constructs together with control IgG or increasing concentrations of anti-Myc antibody and GFP fluorescence quantified 8 hours later. (F) HEK293T-mCherry-TRIM21 cells were electroporated with mRNA encoding 2myc-mEGFP together with the indicated antibodies and GFP fluorescence quantified 8 hours later. (G-I) The indicated antibodies (50 nM) either alone, or mixed with GFP protein (100 nM), were analysed by mass photometry. (J) RPE-1 cells expressing mEGFP were electroporated with the indicated antibodies and cell extracts blotted 3 hours later for the indicated proteins. See also

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Construct, Control, Electroporation, Fluorescence, Western Blot, Expressing

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: RPE-1 TRIM21 KO cells expressing membrane-localised GFP (mem-mEGFP) were electroporated with PBS, control IgG or the indicated anti-GFP antibodies. Cells were fixed 3 hours post-electroporation and stained with alexa 647-conjugated anti-IgG secondary antibodies and imaged by confocal microscopy. Scale bar 10 µm.

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Expressing, Membrane, Control, Electroporation, Staining, Confocal Microscopy

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A) Schematic of light-induced clustering of TRIM21. (B-C) Drosophila S2 cells expressing the indicated constructs were incubated with or without MG132 and RFP fluorescence quantified by live imaging. Time shows minutes (min) from onset of blue light exposure. Scale bar 5 µm. Pseudo-coloured kymographs show fluorescence intensity in regions defined by red dotted lines. Graph shows mean fluorescence intensity (± SD) of RFP-CRY2-TRIM21 (n = 23) and RFP-CRY2-TRIM21+MG132 (n = 20) normalised for the respective controls (RFP-CRY2 (n=16) and RFP-CRY2 + MG132 (n=15)). (D-G) RPE-1 cells expressing the indicated constructs together with mem-mEGFP were incubated with or without MG132 and blue light for 3 hours prior to immunoblotting for the indicated proteins. See also Movie S2

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Expressing, Construct, Incubation, Fluorescence, Imaging, Western Blot

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A) Catalysis of unanchored ubiquitin chains by TRIM21 RING (R), RING-Box (RB), RING-Box-Coiled-Coil (RBCC) and full length MBP-tagged TRIM21 (FL-hT21). (B-C) Catalysis of ubiquitin discharge from ubiquitin-conjugated Ube2N by RING (R), RING Box (RB), RING Box coiled-coiled (RBCC) and full length MBP-tagged TRIM21 (FL-hT21).

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A) Schematic of TRIM21, nanobody-Fc and TRIM21-nanobody chimeric constructs. (B) NIH3T3-Caveolin-1-GFP cells were electroporated with mRNA encoding the indicated constructs. 16 hours later cell extracts were immunoblotted for the indicated proteins. (C-D) NIH3T3-Caveolin-1-GFP and (F-G) RPE-1H2B-mEGFP-FKBP cells were electroporated with water (control) or mRNAs encoding the indicated TRIM21-nanobody chimeric constructs and GFP fluorescence was imaged (C and F) and quantified (D and G) using the IncuCyte system. Time shows hours:minutes (h:min) post-electroporation. Scale bar 50 µm (C) and 30 µm (F). (E) NIH3T3-Caveolin-1-GFP cells and (H) RPE-1-H2B-mEGFP-FKBP cells were electroporated with mRNA encoding mCherry-tagged versions of the indicated constructs and GFP and mCherry fluorescence quantified using the IncuCyte system. TRIM21-nanobody chimera expression levels (mCherry fluorescence) are plotted against GFP fluorescence. See also and Movie S2.

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Construct, Control, Fluorescence, Electroporation, Expressing

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A-D) NIH3T3-Caveolin-1-GFP (A and B) and RPE-1-H2B-mEGFP-FKBP (C and D) cells were electroporated with mRNA encoding mCherry-T21R-vhhGFP4, incubated with either DMSO (control) or MG132 and imaged (A and C) and GFP fluorescence quantified (B and D) with the IncuCyte system. Scale bar 20 µm. (E and F) NIH3T3 cells (E) and RPE-1 cells (F) were electroporated with mRNA encoding mCherry-tagged versions of the indicated TRIM21-nanobody constructs and mCherry fluorescence quantified using the IncuCyte system. (G and H) NIH3T3-Caveolin-1-GFP cells were electroporated with the indicated concentrations of T21R-vhhGFP4 (G) or T21RB-vhhGFP4 (H) proteins and GFP fluorescence quantified with the IncuCyte system. (I) Data from G and H plotted as protein concentration against GFP fluorescence at 4 hours post-electroporation.

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Incubation, Control, Fluorescence, Construct, Protein Concentration, Electroporation

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A) Bacterially-expressed 6His-T21R-vhhGFP4 was purified using a two-step protocol of NiNTA-followed by size exclusion-chromatography and analysed by Coomassie blue staining of SDS-PAGE. (B) NIH3T3-Caveolin-1-GFP cells were electroporated with PBS (control) or the indicated concentrations of T21R-vhhGFP4 protein and GFP fluorescence was quantified using the IncuCyte system. (C) H2B-GFP primary MEFs were electroporated with PBS (control) or T21R-vhhGFP4 in the form or mRNA or protein and GFP fluorescence quantified using the IncuCyte system. Time shows hours (h) post-electroporation. (D-G) Drosophila S2 cells expressing GFP-aPKC and (D, E) vhhGFP4-RFP-CRY2-TRIM21 or (F, G) a LARIAT module that includes RFP-CRY2 fused with vhhGFP4 and which enables clustering in the absence of TRIM21. (E, G) Graphs show mean ± SD fluorescence intensity quantified by live imaging (n = 30 in E and n=24 in G). Time shows minutes (min) from onset of blue light exposure. Scale bar 10 µm.

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Purification, Size-exclusion Chromatography, Staining, SDS Page, Control, Fluorescence, Electroporation, Expressing, Imaging

Journal: eLife

Article Title: Filopodia powered by class x myosin promote fusion of mammalian myoblasts

doi: 10.7554/eLife.72419

Figure Lengend Snippet: ( A ) Myo10 protein content increases during myoblast differentiation, as measured by immunoblotting (n = 3 individual experiments). ( B ) Fractionation of differentiation day 5 myoblast cultures into soluble and insoluble cellular fractions reveals that the slight majority of Myo10 content exists in the soluble fraction. ( C ) Immunofluorescence (IF) of differentiated myoblast insoluble fractions shows that Myo10 of the insoluble cellular fraction is associated with the actin cytoskeleton (as shown by phalloidin staining) and can be found at the tips of thin cellular projections. ( D ) Schematic of the consensus E-box-binding motifs (CANNTG) identified in the Myo10 promoter. ( E–F ) Activation of the Myo10 promoter reporter plasmid in differentiating myoblasts co-transfected with constitutively expressed GFP-CAAX and mApple (RFP) driven by the Myo10 promoter depicted in ( D ) (n = 4 individual experiments). ( G ) Expression of RFP in a differentiating myoblast following 1 day of differentiation. Efficient shRNA-mediated knockdown (KD) of myoblast Myo10 gene expression in ( H ) undifferentiated and ( I ) differentiated myoblasts, whereas muscle differentiation is not affected by Myo10 KD, as indicated by Myh2 expression, a gene encoding a mature myosin heavy chain (MHC) expressed by skeletal muscle (n = 3 individual experiments). ( J ) Representative images of MHC IF of control shRNA cells, Myo10 KD cells expressing a control RFP plasmid after 7 days of differentiation, and Myo10 KD cells expressing an RFP-Myo10 rescue plasmid. Data are presented as box-and-whisker plots depicting second and third quartiles with minimum and maximum values. Data of ( A ) were analyzed using one-way ANOVA followed by Tukey post hoc tests (α = 0.05; *p < 0.05 vs. day 1 values; # p < 0.05 vs. day 3 values; effect size is presented as eta-squared (η 2 )). Data of ( E ) and ( H–I ) were analyzed using two-tailed Welch’s t-tests with effect size presented as Cohen’s d ( d ). Scale bars represent ( G ) 10 or ( C, J ) 25 µm. Figure 2—figure supplement 1—source data 1. Source data file for . Figure 2—figure supplement 1—source data 2. Source data file for . Figure 2—figure supplement 1—source data 3. Source data file for . Figure 2—figure supplement 1—source data 4. Source data file for . Figure 2—figure supplement 1—source data 5. Source data file for . Figure 2—figure supplement 1—source data 6. Source data file for .

Article Snippet: The RFP-Myo10 construct was prepared by cloning mApple (from Addgene No. 54631) to the N-terminus of human Myo10 (NCBI Accession No. NP_036466) using a G-G-R linker, similar to as previously described , in pCDNA3.1(+) vector (Thermofisher No. V79020).

Techniques: Western Blot, Fractionation, Immunofluorescence, Staining, Binding Assay, Activation Assay, Plasmid Preparation, Transfection, Expressing, shRNA, Knockdown, Gene Expression, Control, Whisker Assay, Two Tailed Test

Journal: eLife

Article Title: Filopodia powered by class x myosin promote fusion of mammalian myoblasts

doi: 10.7554/eLife.72419

Figure Lengend Snippet: Clonal lines of C2C12 cells expressing control or Myo10 -targeted short-hairpin RNA (shRNA) were validated for efficacy of Myo10 knockdown (KD) and myogenic differentiation potential. ( A ) Immunoblotting for Myo10 protein and the myogenic differentiation marker, myosin heavy chain (MHC; loading control visualized by Ponceau Red staining). KD of Myo10 myoblasts results in loss of filopodia during differentiation compared to control shRNA cells, as demonstrated by ( B ) immunofluorescence (day 3), ( C ) scanning electron microscopy (day 5), and ( D ) live-cell confocal microscopy (day 5), as well as loss of ( E ) cellular extension lengths (n = 31–152 cellular extensions). Myoblast differentiation assays (n = 3 individual experiments) reveal loss of multinucleated myotubes formation in Myo10 KD cells after 7 days of differentiation compared to control cells, quantified as ( F ) population distribution of myotube nuclear content. ( G–H ) Loss of fusion ability by Myo10 KD cells can be partially rescued by transfection of a full-length Myo10 construct with an N-terminal mApple fluorescent tag (RFP-Myo10; n = 3–6 individual experiments). Data analysis performed using ( E–F ) Welch’s two-tailed t-test (α = 0.05) with effect size displayed as Cohen’s d ( d ) or ( H ) one-way ANOVA followed by Tukey post hoc tests (α = 0.05; *p < 0.05 vs. control values; # p < 0.05 vs. RFP values; effect size is presented as eta-squared (η 2 )). Unless otherwise noted, scale bars represent 25 µm. Figure 3—source data 1. Source data file for . Figure 3—source data 2. Source data file for . Figure 3—source data 3. Source data file for . Figure 3—source data 4. Source data file for .

Article Snippet: The RFP-Myo10 construct was prepared by cloning mApple (from Addgene No. 54631) to the N-terminus of human Myo10 (NCBI Accession No. NP_036466) using a G-G-R linker, similar to as previously described , in pCDNA3.1(+) vector (Thermofisher No. V79020).

Techniques: Expressing, Control, shRNA, Knockdown, Western Blot, Marker, Staining, Immunofluorescence, Electron Microscopy, Confocal Microscopy, Transfection, Construct, Two Tailed Test

Journal: eLife

Article Title: Filopodia powered by class x myosin promote fusion of mammalian myoblasts

doi: 10.7554/eLife.72419

Figure Lengend Snippet:

Article Snippet: The RFP-Myo10 construct was prepared by cloning mApple (from Addgene No. 54631) to the N-terminus of human Myo10 (NCBI Accession No. NP_036466) using a G-G-R linker, similar to as previously described , in pCDNA3.1(+) vector (Thermofisher No. V79020).

Techniques: Transfection, Construct, Membrane, Activation Assay, Control, shRNA, Sequencing

Journal: Scientific Reports

Article Title: The acetylation of cyclin-dependent kinase 5 at lysine 33 regulates kinase activity and neurite length in hippocampal neurons

doi: 10.1038/s41598-018-31785-9

Figure Lengend Snippet: Acetylation of CDK5 at K33 causes a loss of kinase activity due to impaired ATP binding. ( a , b ) HEK293 cells were transfected with either FLAG-tagged wild type mouse CDK5 (WT), an acetyl-null mutant (K33R; KR) or a mimetic mutant (K33Q; KQ) of CDK5 in the presence of ( a ) p35-HA or ( b ) p25-HA. Lysates were immunoprecipitated (IPed) with an anti-FLAG antibody and then subjected to an in vitro phosphorylation assay using histone H1 as a substrate. The resulting phosphorylated H1 (P-H1) was visualized via immunoblot analysis (IB) with an anti-phospho-H1 antibody. Coomassie brilliant blue (CBB) staining for H1 was used as a loading control. Immunoprecipitates or whole cell lysates (WCLs) were resolved by SDS-PAGE and subjected to IB with the indicated antibodies. Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control for WCL. ( c ) Bacterially purified, recombinant His-tagged CDK5 WT or K33-acetylated CDK5 (Ac-CDK5; Ac) was subjected to an in vitro phosphorylation assay in the presence of H1, [γ- 32 P]ATP and the indicated doses of recombinant p25. The resulting phosphorylated H1 was visualized by autoradiography. Inputs were resolved by SDS-PAGE and subjected to IB with the indicated antibodies. ( d ) Recombinant His-CDK5 WT or His-Ac-CDK5 was incubated with or without resin conjugated to ATP. After washing, the resulting ATP-bound CDK5 was resolved by SDS-PAGE and visualized by IB with an anti-His antibody. Input signals were measured by IB with the indicated antibodies. ( e ) Recombinant His-CDK5 WT (blue-filled circles) or His-Ac-CDK5 (magenta-filled rectangles) was titrated with increasing concentration of mant-ATP. Nonlinear regression was performed to obtain a best-fit curve for a specific binding [Y = Bmax*X/(Kd + X)] and the summary of binding parameters were shown in Supplementary Table . X-axis represents the varying concentration of mant-ATP as indicated. Y-axis represents the relative fluorescence intensity of specific binding, where Bmax is maximum specific binding and Kd is equilibrium binding constant. Wilcoxon matched-pairs rank test was employed to test the binding difference between CDK5 WT and Ac-CDK5 ( ** P = 0.004; Spearman correlation coefficient, rs = 0.976; n = 3). ( f ) Lysates from HEK293 cells expressing p35-FLAG or p25-FLAG were incubated with recombinant His-CDK5 WT or His-Ac-CDK5 bound to Ni-NTA beads. Reaction mixtures were subjected to pull-down and subsequent IB with the indicated antibodies. An anti-FLAG antibody was employed to visualize the extent of CDK5-bound p35 or p25. WCLs were subjected to IB with the indicated antibodies. ( g ) Recombinant His-CDK5 WT plus increasing amounts of recombinant His-Ac-CDK5 was subjected to an in vitro phosphorylation assay in the presence of H1 and [γ- 32 P]ATP. Phosphorylated H1 levels were visualized by autoradiography.

Article Snippet: To assess ATP binding affinity, purified recombinant His-CDK5 WT and His-Ac-CDK5 (1 μg) were reacted for 2 hrs with 20 μl of ATP-conjugated agarose (Jena Bioscience, #AC-101) as previously described .

Techniques: Activity Assay, Binding Assay, Transfection, Mutagenesis, Immunoprecipitation, In Vitro, Phosphorylation Assay, Western Blot, Staining, SDS Page, Purification, Recombinant, Autoradiography, Incubation, Concentration Assay, Fluorescence, Expressing

Journal: Scientific Reports

Article Title: The acetylation of cyclin-dependent kinase 5 at lysine 33 regulates kinase activity and neurite length in hippocampal neurons

doi: 10.1038/s41598-018-31785-9

Figure Lengend Snippet: GCN5 acetylates CDK5 at K33 in the nucleus. ( a ) Lysates obtained from HEK293 cells expressing FLAG-CDK5 plus one of the indicated KAT vectors were subjected to IP with an anti-FLAG antibody followed by IB with an anti-Ac-CDK5 antibody or anti-FLAG antibody. The intensity of the Ac-CDK5 band was measured using Image-J software and normalized to FLAG-CDK5. The fold change over the control (value = 1) is indicated at the bottom of the blot. WCLs were subjected to IB analysis with the indicated antibodies. Each KAT band is marked by the indicated letters. The asterisk indicates non-specific bands. After normalization to FLAG-CDK5, the fold intensity of Ac-CDK5 versus the control (value = 1) was indicated. The bar represents the mean ± S.D from three independent experiments. *** p < 0.001; * p < 0.05; n.s, not significant. ( b ) Lysates harvested from HEK293 cells expressing FLAG-CDK5 alone or in combination with p35-MYC and/or FLAG-GCN5 were subjected to IP with an anti-FLAG antibody. The bound CDK5 was incubated in the presence of H1 and [γ- 32 P]ATP and visualized by autoradiography. The relative kinase activity of CDK5/p35 was expressed as the fold change over the control (value = 1). The bar represents the mean ± S.D from 3 independent experiments. * p < 0.05. ( c ) HEK293 cells were immunostained with an anti-Ac-CDK5 antibody. Staining specificity was confirmed by pre-incubating with the blocking peptide (EIVAL(acK)RVRLD) that was used to raise the antibody. The nuclei were counterstained with Hoechst dye. Confocal microscopy images are shown. The scale bar represents 10 μm. ( d ) HEK293 cells transfected with the indicated combinations of constructs were subjected to cellular fractionation. The resulting nuclear fractions were IPed with an anti-FLAG antibody and subsequently subjected to either IB with an anti-Ac-CDK5 or anti-FLAG antibody or an in vitro phosphorylation assay in the presence of H1 and [γ- 32 P]ATP. Signals from the phosphorylated H1 were visualized by autoradiography. The fold change over the control (value = 1) is indicated. Nuclear fractions were subjected to IB with the indicated antibodies. Anti-SOD-1 and anti-lamin A/C antibodies were employed to verify the purity of the nuclear fractions.

Article Snippet: To assess ATP binding affinity, purified recombinant His-CDK5 WT and His-Ac-CDK5 (1 μg) were reacted for 2 hrs with 20 μl of ATP-conjugated agarose (Jena Bioscience, #AC-101) as previously described .

Techniques: Expressing, Software, Incubation, Autoradiography, Activity Assay, Staining, Blocking Assay, Confocal Microscopy, Transfection, Construct, Cell Fractionation, In Vitro, Phosphorylation Assay

Journal: Scientific Reports

Article Title: The acetylation of cyclin-dependent kinase 5 at lysine 33 regulates kinase activity and neurite length in hippocampal neurons

doi: 10.1038/s41598-018-31785-9

Figure Lengend Snippet: SIRT1 is responsible for the deacetylation of Ac-CDK5. ( a ) HEK293 cells transiently expressing FLAG-CDK5 were treated for 24 hrs with increasing doses of nicotinamide (NA, a pan-SIRT inhibitor). Lysates were subjected to IP with an anti-FLAG antibody and probed with the indicated antibodies. After normalization to FLAG-CDK5, the fold intensity of Ac-CDK5 versus control (value = 1) was determined. ( b ) HEK293 cells were transfected with one of the FLAG-tagged SIRTs plus FLAG-CDK5 and GCN5-HA. Lysates were subjected to IP with an anti-FLAG antibody and IB with an anti-Ac-CDK5 antibody. WCLs were subjected to IB with the indicated antibodies. Each band of SIRTs is marked by the indicated letters. After normalization to FLAG-CDK5, the fold intensity of Ac-CDK5 versus the control (value = 1) was indicated. The bar represents the mean ± S.D from three independent experiments. *** p < 0.001; n.s, not significant. ( c ) FLAG-SIRT1 was expressed in HEK293 cells and purified by IP with FLAG beads. SIRT1-bound beads were incubated with recombinant His-Ac-CDK5 supplemented with β-nicotinamide adenine dinucleotide (NAD + ) to activate SIRT1. Reaction mixtures were subjected to IB with an anti-Ac-CDK5 antibody. The fold intensity of Ac-CDK5 versus the control (value = 1) was determined after normalization to the His-Ac-CDK5 inputs. The inputs were probed with the indicated antibodies. ( d , e ) HEK293 cells transfected with FLAG-CDK5 were treated with increasing doses of ( d ) EX527 (a selective SIRT1 inhibitor) or ( e ) SRT1720 (a selective SIRT1 activator) for 24 hrs. Lysates were subjected to IP with an anti-FLAG antibody followed by IB with an anti-Ac-CDK5 antibody. After normalization to FLAG-CDK5, the fold change over the control (value = 1) was determined. ( f ) Lysates were prepared from HEK293 cells transfected with FLAG-CDK5 and p35-HA and exposed to 100 μM EX527 for 24 hrs. Immunoprecipitates purified with an anti-FLAG antibody were subjected either to IB with the indicated antibodies or an in vitro phosphorylation assay in the presence of H1 and cold ATP. Phospho-H1 signals were visualized with an anti-phospho-H1 antibody. After normalization to FLAG-CDK5, the fold intensity of Ac-CDK5 versus the control (value = 1) was indicated. The bar represents the mean ± S.D from three independent experiments. * p < 0.05.

Article Snippet: To assess ATP binding affinity, purified recombinant His-CDK5 WT and His-Ac-CDK5 (1 μg) were reacted for 2 hrs with 20 μl of ATP-conjugated agarose (Jena Bioscience, #AC-101) as previously described .

Techniques: Expressing, Transfection, Purification, Incubation, Recombinant, In Vitro, Phosphorylation Assay

Journal: Scientific Reports

Article Title: The acetylation of cyclin-dependent kinase 5 at lysine 33 regulates kinase activity and neurite length in hippocampal neurons

doi: 10.1038/s41598-018-31785-9

Figure Lengend Snippet: Pharmacological modulation of SIRT1 affects Ac-CDK5 levels and kinase activity in hippocampal neurons. ( a ) Primary cultures of hippocampal neurons were prepared from rat hippocampi isolated in gestational day 18. At the indicated days in vitro (DIV), photomicrographs were captured with an Axiovert 100. The scale bar represents 50 μm. ( b ) Cultured hippocampal neurons at DIV5 were fixed and immunostained with an anti-Ac-CDK5 antibody and an anti-NeuN antibody (a neuronal nuclear marker) followed by incubation with appropriate fluorescence-tagged secondary antibodies. Fluorescent images were obtained with an LSM700 confocal microscope. The scale bar represents 10 μm. ( c ) Hippocampal neurons at DIV3 were treated with SRT1720 or EX527 at the indicated doses for 48 hrs. Immunoprecipitates of cellular lysates purified with an anti-CDK5 antibody or IgG were subjected to IB with an anti-CDK5 antibody or an in vitro phosphorylation assay in the presence of H1 and [γ- 32 P]ATP. Phosphorylated H1 signals were visualized by autoradiography. After normalization to H1 or CDK5, the relative intensities of phospho-H1 and Ac-CDK5 were calculated over the controls (value = 1) and are indicated at the bottom of the blot. WCLs were subjected to IB with the indicated antibodies. ( d , e ) The relative kinase activity of CDK5 was expressed as the fold change over the control (value = 1) in the presence of ( d ) SRT1720 or ( e ) EX527. The bar represents the mean ± S.D from 4 independent experiments. *** p < 0.001. ( f ) Hippocampal neurons at DIV3 were treated with EX527 at 100 μM for 48 hrs, fixed, and then processed for immunofluorescent staining as described in ( b ). The scale bar represents 50 μm. ( g ) The fluorescence intensity of Ac-CDK5 in the nuclei of NeuN-positive neurons was measured using Image-J software. The relative fluorescence intensity was expressed as the fold change over the control (value = 1). The bars represent the mean ± S.D of 35 neurons from at least 5 randomly selected areas. *** p < 0.001.

Article Snippet: To assess ATP binding affinity, purified recombinant His-CDK5 WT and His-Ac-CDK5 (1 μg) were reacted for 2 hrs with 20 μl of ATP-conjugated agarose (Jena Bioscience, #AC-101) as previously described .

Techniques: Activity Assay, Isolation, In Vitro, Cell Culture, Marker, Incubation, Fluorescence, Microscopy, Purification, Phosphorylation Assay, Autoradiography, Staining, Software