Journal: Nature Communications

Article Title: An organelle-specific protein landscape identifies novel diseases and molecular mechanisms

doi: 10.1038/ncomms11491

Figure Lengend Snippet: ( a ) Detailed network of IFT-B1/2 and MAK/ICK/KIAA0556; IFT-B is linked to IFT-A by NUDC, and to complex KIF3 by SSNA1. The IFT-B protein TTC30B interacts with multiple membrane proteins. One of those, TMEM41B, was further analysed and shows a ciliary length phenotype upon modulation of expression by siRNA knock-down and overexpression. For both, knockdown and overexpression, biological triplicates were analysed and a t -test was performed. P values below 0.01 are represented by ** and below 0.001 by ***. Error bars represent the s.e.m. ( b ) Detailed interaction network of the KIF3 complex and Complex:15, with SLC9A2R2 bridging ciliary processes. ( c ) Detailed interactions between Dynein and Dynactin intermediated by the HSF1/HSPA1L/MAPRE2 linker complex. ( d ) Muskelin/RanBP9/CTLH complex (GID complex in Yeast) network showing core, attachments and links to several other complexes, mediated by RAB8A. Immunofluorescence demonstrates the localization of two GID components, GID8 and MKLN1 (red arrows in the network) to the ciliary base: MKLN1 in kidney tubule epithelial cells (anti-MKLN1, left panel, red); GID8 (right panel, green) in multi-ciliated brain ependymal cells. DAPI staining (blue) marks the nucleus, GT335 co-staining (green or red) marks the cilium. Scale bars represent 10 μm. ( e ) Complex:21 and 31 containing several ubiquitin conjugating or modifying enzymes in interaction with the GID and exocyst complexes. ( f ) Elaborated view of the sub-network involving the NPHP1-NPHP4-RPGRIPL/PDE6D/RPGR complex, and its association with the complexes IQ/CALM1, KIF3, COP9 signalosome, and Complex:15. ( g ) Ciliogenesis transcription factor FOXJ1 stably interacts with PLK1 (Polo-like kinase 1) and CFAP20, and is linked to the FOXJ/RFX complex and Complex:15.

Article Snippet: After the blocking step, the cryosections were incubated overnight with the primary rabbit polyclonal antibody targeting GID8 (c20orf11 (N1C3), Genetex, cat. no. GTX106672; 1:100) or MKLN1 (Sigma-Aldrich, cat. no. HPA022817; 1:100) in combination with the monoclonal antibody GT335 (Adipogen, cat. no. AG-20B-0020-C100; 1:1,000), diluted in blocking buffer.

Techniques: Membrane, Expressing, Knockdown, Over Expression, Immunofluorescence, Staining, Ubiquitin Proteomics, Stable Transfection

Journal: Cell Research

Article Title: Twa1/Gid8 is a β-catenin nuclear retention factor in Wnt signaling and colorectal tumorigenesis

doi: 10.1038/cr.2017.107

Figure Lengend Snippet: Twa1 promotes β-catenin nuclear accumulation and target gene expression in response to Wnt signaling. HEK-293 cells treated with lentivirus-based shRNAs targeting different regions of Twa1 mRNA (sh-Twa1 and sh-Twa1-2), β-catenin mRNA (sh-β-catenin) or control shRNA (sh-ctr) were transfected with an RNAi-resistant human Twa1 (Twa1 * ) construct or not. The cells were then treated with Wnt3a-conditioned medium (Wnt3a-CM) or control medium (Ctr-CM), and subjected to the following analyses. (A-D) Effect of Twa1 depletion on Wnt-induced luciferase reporter activity (A , C) or Wnt target gene expression (B , D) . Quantitative data are expressed as the mean ± SEM (at least three independent experiments). * P < 0.05 and ** P < 0.01, Student's t -test. (E) Western analysis of endogenous β-catenin and Twa1 from cytosolic and nuclear fractions of HEK-293 cells. Lamin B and α-tubulin were used as loading controls for nuclear and cytoplasmic fractions, respectively. (F , G) Confocal microscopy of β-catenin nuclear localization. Green signals represent cells infected with lentiviruses. DNA was visualized with DAPI (blue). The intensity of red signals in the nuclei of GFP-positive cells was plotted. The nuclear intensity in the GFP-positive cells treated with both sh-ctr and Wnt3a-CM is set as 100%. More than 50 nuclei are measured in each group. Scale bars, 10 μm. ** P < 0.01, Student's t -test. (H) Co-IP analysis with the indicated antibodies showing the interaction between endogenous β-catenin and TCF4. (I) Western analysis with the indicated antibodies showing the effect of β-catenin depletion on Twa1 nuclear accumulation upon Wnt3a stimulation. IB, immunoblotting; IP, immunoprecipitation.

Article Snippet: Anti-β-catenin (BD Bioscience), Twa1, lamin B (Proteintech), GSK3β, TCF4, Axin1 (Cell Signal Technology), Myc, GAPDH, GST, His, HA, Flag (Santa Cruz), actin and α-tubulin (Sigma) antibodies were acquired commercially.

Techniques: Targeted Gene Expression, Control, shRNA, Transfection, Construct, Luciferase, Activity Assay, Western Blot, Confocal Microscopy, Infection, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: Cell Research

Article Title: Twa1/Gid8 is a β-catenin nuclear retention factor in Wnt signaling and colorectal tumorigenesis

doi: 10.1038/cr.2017.107

Figure Lengend Snippet: TCF4 competes with Twa1 for β-catenin binding. (A , B) HEK-293 cells were transfected with the Flag-Twa1 construct or not, treated with Wnt3a-CM or Ctr-CM, and then subjected to ChIP analysis with the indicated antibodies. The ChIP-enriched DNAs were measured by PCR with the primers specific for the Axin2 or Cyclin D1 promoter (WREs). The region located in the corresponding ORF (open reading frames) was used as a negative control. (C-E) HEK-293 cells transfected with the Flag-Twa1 construct were treated with Wnt3a-CM or Ctr-CM, and then used for co-IP and subsequent western analysis with the indicated antibodies. (F) HEK-293 cells transfected with the Flag-Twa1, Myc-β-catenin and increasing amounts of HA-TCF4 plasmids were incubated in Wnt3a-CM for 4 h. The nuclear fractions were extracted and subjected to co-IP analysis using anti-Myc antibody. (G) His pull-down analysis of purified His-β-catenin, GST-Twa1 and increasing amounts of Flag-TCF4 protein immunoprecipitated from HEK-293 cells transfected with the Flag-TCF4 construct.

Article Snippet: Anti-β-catenin (BD Bioscience), Twa1, lamin B (Proteintech), GSK3β, TCF4, Axin1 (Cell Signal Technology), Myc, GAPDH, GST, His, HA, Flag (Santa Cruz), actin and α-tubulin (Sigma) antibodies were acquired commercially.

Techniques: Binding Assay, Transfection, Construct, Negative Control, Co-Immunoprecipitation Assay, Western Blot, Incubation, Purification, Immunoprecipitation

Journal: eLife

Article Title: Modular UBE2H-CTLH E2-E3 complexes regulate erythroid maturation

doi: 10.7554/elife.77937

Figure Lengend Snippet: Figure 2. Erythroid maturation stage-dependent modulation of RANBP9- and RANBP10-assembled CTLH complexes. (A) Cartoon of the supramolecular RANBP9-CTLH assembly indicating the catalytic (blue), core (grey/yellow), and supramolecular assembly (orange) modules. (B) Model of remodelling RANBP9- and RANBP10-CTLH complexes. (C) HUDEP2 cell lysates from differentiation days 0 and 6 were separated on sucrose gradients, and fractions analysed by immunoblotting with indicated antibodies. Fractions containing supramolecular CTLH assemblies are boxed in

Article Snippet: For immunoblot analysis, lysates were denatured with SDS sample buffer, boiled at 95°C for 5 min, separated on SDS- PAGE, and proteins were visualized by immunoblotting using indicated primary antibodies: RMND5A (Santa Cruz), MAEA (R&D Systems), RANBP9 (Novus Biologicals), RANBP10 (Invitrogen, #PA5- 110267), TWA1 (Thermo Fisher), ARMC8 (Santa Cruz), WDR26 (Bethyl Laboratories), MKLN1 (Santa Cruz), YPEL5 (Thermo Fisher), GID4 (described in Sherpa et al., 2021), CD235a/GYPA (Abcam), HBD (Cell Signaling), HBG1/2 (Cell Signaling), and Flag (Sigma).

Techniques: Western Blot

Journal: eLife

Article Title: Modular UBE2H-CTLH E2-E3 complexes regulate erythroid maturation

doi: 10.7554/elife.77937

Figure Lengend Snippet: Figure 3. RANBP9 and RANBP10 form similar CTLH complex structures that cooperate with UBE2H to promote ubiquitin transfer. (A) Chromatograms (top) and Coomassie-stained SDS-PAGE gels (bottom) from size-exclusion chromatography of recombinant RANBP10-CTLHSR4 and RANBP9- CTLHSR4 complexes. (B) Cryo-EM map of RANBP10-CTLHSR4 (EMDB: EMD-16242, left) and RANBP9-CTLHSR4 (EMDB: EMD-12537) (right) with Cat- module and SRS-module indicated. (C) Focused refined map of the RANBP10-CTLH SRS-module with coloured subunits: ARMC8, purple; TWA1, salmon;GID4(Δ1–99), red; RANBP10 SPRY-domain, green. (D) Immunoprecipitation (IP) from HUDEP2 cell lysates with IgG control and UBE2H-specific antibody and immunoblot analysis. (E) IP from K562 cell lysates with IgG control and UBE2H-specific antibody and immunoblot analysis. IgG light chain (IgG-LC), IgG heavy chain (IgG-HC). (F) Fluorescence scan of SDS-PAGE gels presenting time course of in vitro ubiquitylation assay with fluorescently labelled model substrate peptide PGLW(X)n-23K with lysine at position 23 (pep*) in the presence of UBE2H, RANBP10-CTLH or RANBP9-CTLH, and GID4.

Article Snippet: For immunoblot analysis, lysates were denatured with SDS sample buffer, boiled at 95°C for 5 min, separated on SDS- PAGE, and proteins were visualized by immunoblotting using indicated primary antibodies: RMND5A (Santa Cruz), MAEA (R&D Systems), RANBP9 (Novus Biologicals), RANBP10 (Invitrogen, #PA5- 110267), TWA1 (Thermo Fisher), ARMC8 (Santa Cruz), WDR26 (Bethyl Laboratories), MKLN1 (Santa Cruz), YPEL5 (Thermo Fisher), GID4 (described in Sherpa et al., 2021), CD235a/GYPA (Abcam), HBD (Cell Signaling), HBG1/2 (Cell Signaling), and Flag (Sigma).

Techniques: Ubiquitin Proteomics, Staining, SDS Page, Size-exclusion Chromatography, Recombinant, Cryo-EM Sample Prep, Immunoprecipitation, Control, Western Blot, Fluorescence, In Vitro, Ubiquitin Assay

Journal: bioRxiv

Article Title: Proteome-wide C-degron activity profiling connects conditional regulation of the CTLH E3 ligase complex to ribosome biogenesis

doi: 10.64898/2026.01.14.698769

Figure Lengend Snippet: (A-B) Cell-to-cell variability in ZMYND19 stability. (A) ZMYND19 expressed in the context of the GPS system escapes degradation in a variable proportion of the cells. (B) Following isolation of the GFP bright cells by FACS, the population quickly reverted towards the GFP dim state. (C) A chemical screen to identify conditions which impact ZMYND19 degradation. HEK-293T cells expressing ZMYND19 in the context of the GPS vector were subjected to the indicated treatments and the effect on ZMYND19 degradation assessed by flow cytometry. (D-E) Inhibition of mTOR signaling potentiates ZMYND19 degradation. (D) Overnight treatment with the mTOR inhibitor Torin-1 abolishes phosphorylation of p70 S6 kinase. (E) Amino acid withdrawal or Torin-1 treatment enhances ZMYND19 degradation, overriding the effects of TNF-α stimulation. See also . (F) Conditional regulation of the CTLH GID4 substrate HMGCS1. HMGCS1 is more abundant following TNF-α stimulation and less abundant following Torin-1 treatment, as assessed by immunoblot. (G) The association between ZMYND19 and Muskelin is maintained in GFP bright cells where GPS-ZMYND19 escapes degradation. Following overnight TNF-α stimulation, cells expressing GPS-ZMYND19 were partitioned into GFP dim and GFP bright populations by FACS and the interaction between ZMYND19 and Muskelin assessed via co-immunoprecipitation. (H) Effect of TNF-α stimulation and mTOR inhibition on the abundance of CTLH subunits, as assessed by immunoblot. (I-J) Differential requirements for RANBP9 and RANBP10 for CTLH-mediated degradation of ZMYND19 upon mTOR inhibition. Using sgRNAs that result in efficient gene disruption as assessed by immunoblot (I) , the effects of targeting RANBP9 and RANBP10 either individually or simultaneously on ZMYND19 degradation were assessed by flow cytometry (J) . Ablation of RANBP9 prevents ZMYND19 degradation at steady state; upon mTOR inhibition, however, targeting of RANBP9 has little impact on ZMYND19 stability, and dual ablation of RANBP9 and RANBP10 is necessary before abrogated degradation is observed.

Article Snippet: Primary antibodies used were: rabbit α-MAEA (Proteintech, #28363-1-AP), rabbit α-TWA1 (Novus Biologicals, #NBP1-32596), mouse α-Muskelin (Santa Cruz, #sc-398956), mouse α-RanBP9 (Santa Cruz, #sc-271727), rabbit α-RanBP10 (Proteintech, #21107-1-AP), mouse α-ARMC8 (Santa Cruz, #sc-365307), rabbit α-WDR26 (Bethyl, #A302-244A), rabbit α-YPEL5 (Proteintech, #11730-1-AP), rabbit α-AAMP (Abcam, #EPR12369), rabbit α-ISG20L2 (Proteintech, #24639-1-AP), rabbit α-HMGCS1 (Proteintech, #17643-1-AP), mouse α-UBE2H (Santa Cruz, #sc-100620), rabbit α-uL16/RPL10 (Proteintech, #17013-1-AP), rabbit α-uL14/RPL23 (Abcam, #ab264369), mouse α-eS6/RPS6 (Cell Signaling Technology, #2317), mouse α-β-actin (Merck, #A2228), mouse α-vinculin (Merck, #V9131), rabbit α-HA-tag (Cell Signaling Technology, #C29F4), mouse α-FLAG (Merck #F1804), rabbit α-GFP (Abcam, #ab290), mouse α-GFP (Roche, #11814460001) and rabbit α-Phospho-p70 S6 Kinase (Thr389) (Cell Signaling Technology, #9205).

Techniques: Isolation, Expressing, Plasmid Preparation, Flow Cytometry, Inhibition, Phospho-proteomics, Western Blot, Immunoprecipitation, Disruption