EMBO/SourceData huggingface dataset:
Figure 5(A) Fork velocity as measured by DNA combing assay in HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). More than 200 tracks were considered except for RAD1 +RNH1 (n=125), ATR +RNH1 (n=182) and RAD18 +RNH1 (n=146). Median values are indicated. *p<0.05, **p<0.01, ***p<0.001 (Mann-Whitney U test). Black stars denote significant increases whereas red stars denote significant decreases.(B) Fork asymmetry as measured by DNA combing assay in HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). From 40 to 200 total measurements were considered for each candidate. Median values are indicated. *p<0.05, **p<0.01, ***p<0.001 (Mann-Whitney U test). Black stars denote significant increases whereas red stars denote significant decreases.
Figure 7(A) DNA-RNA hybrids, DNA damage and fork stalling in 9-1-1/ATR/CHK1, ATM/CHK2 and PRR deficient cells. DNA-RNA hybrids represent the mean ± SEM of all the DRIP data ***p<0.001, **p<0.01, *p<0.05 (Paired t-test). DNA damage represents the mean of medians ± SEM of all the comet tail moment data ***p<0.001, **p<0.01, *p<0.05 (paired t-test). Fork stalling represents the median ± SEM of all the fork asymmetry data ***p<0.001, **p<0.01, *p<0.05 (Mann-Whitney U test). Black stars denote significant increases whereas red stars denote significant decreases.
Figure 1IF by confocal microscopy performed on KRAB-HA overexpressing cells. Staining was performed with anti-HA (Alexa-488, green), anti-HSPA5 (Alexa-647, red) and DNA was stained with Dapi (blue).
Figure 2HA immunoprecipitation of stably expressed ZNF282-HA in cells previously transfected with ΔDUF3669-ZNF398-GFP and WT-ZNF398-GFP followed by detection of ZNF398 constructs in the IPs through Western-Blot using an anti-GFP antibody. Bottom: Western-Blot using an anti-HA antibody on the IPs. Input = Cellular lysate, IP = ImmunoprecipitateLeft: DUF3669-only and KRAB domain constructs used. Right: HA immunoprecipitation in cells previously co-transfected with ZNF398-DUF3669-HA and either ZNF282-KRAB-Flag or ZNF282-DUF3669-Flag followed by detection of either of these protein constructs in the IPs through Western-Blot using an anti-Flag antibody. Bottom: Western-Blot using an anti-HA antibody on the IPs.
Figure 3Histograms representing the percentage of KZFPs (identified in Fig. 3B) binding sites falling in TEs (red) or TSSs (blue).KZFPs binding sites on L1PA5 and L1PA6. Top: boxes containing the interactors of KZFPs found enriched on L1PA5,6. Middle: plot showing the average ChIP-exo signals (scaled between 0 and 1) for each selected KZFP plotted on top of L1PA5 and L1PA6 multiple sequence alignment (MSA). Bottom: schematic representation of L1PA5,6 different domains.
Figure 4Human KZFPs KRAB domain A-boxes protein phylogenetic tree associated with their corresponding amino acid sequences colored according to the Clustal ZAPPO color scheme (residues sharing common physicochemical properties display the same color: http://www.jal- view.org/ help/html/colourSchemes/zappo.html). This figure also displays a zoom-in on the variant KRAB domain cluster (right). On this zoom-in, the tested vKZFPs, whose interactomes were defined in our study, were marked by an asterisk. D. Box plot representing KAP1FC values in function of KZFPs age. On a superimposed swarm plot, the individual vKZFPs corresponding KAP1FC values were represented by red dots and for their sKZFPs counterparts, by blue dots. Mann-Whitney two-sided rank test.
Figure 6Immunoprecipitations of HA-tagged KRAB domains in order to check  interaction with cKAP1. Co-transfection of Flag-tagged cKAP1 (upper panel) or Flag-tagged hKAP1 (lower panel) and HA-tagged ZNF398 DUF3669 domain negative control, H2ZYM4 and M3XKG1 cKRAB domains and ZNF93 hKRAB domain in KAP1 KO HAP1 cells followed by HA-immunoprecipitation. cKAP1 and hKAP1 presence was revealed by Western-Blot using an anti-Flag antibody. Input = Cellular lysate, IP = Immunoprecipitate. Western-Blot using an HA antibody on the IPs at the bottom.
Figure 1Immunoblot analysis of mouse intestinal crypts treated with Wnt3a (60 ng/mL) for indicated time. The relative protein level was quantified and shown under each band.Polysome profiles of mouse intestinal crypt treated with or without Wnt3a for 30 min. The right panels show the distributions of Ythdf1 and Actb in polysome fractions. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (3 biological replicates, t-test).Immunoblot analysis of SW620 cells with or without APC overexpression. The relative protein level was quantified and shown under each band.Polysome profiles of SW620 cells with or without APC overexpression. The right panels show the distributions of YTHDF1 and ACTB in polysome fractions. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (3 biological replicates, t-test).Dual-Luciferase assay wih a construct bearing the 5'UTR of YTHDF1 in SW620 cells with or without APC overexpression. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (3 biological replicates, t-test).YTHDF1 protein expression in intestinal tissue from 4 pairs of Apc+/+ and Apcmin/+ mice. N, normal tissue. T, tumor tissue. A, tumor adjacent tissue.YTHDF1 staining in matched samples of human CRC tissue and adjacent non-tumor tissue. Scale bar, 450 μm. The right panel shows the statistics of IHC scores. Chi-square test; n = 75 for each group.
Figure 2The representative jejunum from wildtype (Ythdf1CTL) and Ythdf1cKO mice. Left: hematoxylin and eosin staining (H&E); middle: BrdU staining; right: quantification of crypt height and BrdU+ cells. Scale bar, 50 μm. Data are represented as mean ± SEM. (9 biological replicates, t-test).Immunoblot analysis of small intestines from Ythdf1CTL and Ythdf1cKO mice after 12 Gy IR.YTHDF1 (left) and Ki67 (right) staining of small intestine from wildtype mice 72 hr after 12 Gy IR. Scale bar, 50 μm. Red arrows indicate the proliferating cells.Small intestines from Ythdf1CTL and Ythdf1cKO mice 72 hr after 12 Gy IR. Left: H&E staining; middle: Ki67 staining; right: quantification of crypt height and Ki67+ cells. Scale bar, 50 μm. Vertical bars indicate the length of the crypts. Data are represented as mean ± SEM. ****p<0. 0001 (9 biological replicates, t-test).RT-qPCR analysis of small intestines from Ythdf1CTL and Ythdf1cKO mice 72 hr after 12 Gy IR. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (5 biological replicates, t-test).
Figure 3Small intestines from 24-week-old Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice.H&E staining of small intestines from 24-week-old Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice. Scale bar, 1 mm. The black arrows indicate the tumors.Tumor number (C) and size distribution (D) from the small intestines of 24-week-old Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice. Data are shown as mean ± SEM (6 mice for each group). **p<0. 01, ***p<0. 001 (t-test).Colons from Ythdf1CTL and Ythdf1cKO mice on day 84 of AOM/DSS induction.H&E staining of colons from Apcmin/+; Ythdf1CTL and Apcmin/+; Ythdf1cKO mice after AOM/DSS induction. Scale bar, 200 μm.Colon tumor number (H) and size distribution (I) from Ythdf1CTL and Ythdf1cKO mice on day 84 of induction. Data are shown as mean ± SEM (6 mice for each group). **p<0. 01 (t-test).
Figure 4Fluorescent in situ hybridization for Ythdf1 mRNA in Ythdf1CTL and Ythdf1cKO small intestines. Scale bar, 50 μm.Immunoblot analysis of fractionated murine intestine enriched for villus cells and crypt cells.Flowcytometry analysis of YTHDF1 protein expression in GFPhigh/low/neg populations from Lgr5-GFP-IRES-creERT2 mice.Morphology of organoids from Lgr5-creERT2:Ythdf1fl/fl mice in Wnt3a-conditioned medium without or with 4-OHT induction and infected with lentivirus expressing YTHDF1 or YTHDF1 mutant. Scale bar, 250 μm.Quantification of differentiated versus undifferentiated organoids from (D). Data are represented as mean ± SEM. **p<0. 01 (4 biological replicates, t-test).Relative mRNA levels of intestinal stem cell and differentiation marker genes in organoids described in (D). Data are represented as mean ± SEM. 4 biological replicates. *p<0. 05, **p<0. 01, ***p<0. 001 (t-test).
Figure 5Scatterplot of mRNA expression and ribosome profiling data from control and YTHDF1 knockdown cells. The upregulated (red) and downregulated (blue) genes in translational efficiency (TE) are highlighted.The relative m6A peak coverage in control cells for transcripts grouped according to TE changes after YTHDF1 knockdown. POI: peak over input. Down, genes significantly downregulated in YTHDF1 knockdown cells (<0.5 fold). Unchanged, genes not significantly changed. Up, genes significantly upregulated (>2 fold). The upper and lower quartiles and the median are shown for each group. Mann-Whitney test. **p<0. 01. ***p<0. 001.Violin plots showing TE change between control and YTHDF1 knockdown cells for not-methylated (non-m6A) and methylated (m6A) transcripts. The upper and lower quartiles and the median are indicated for each group. Mann-Whitney test.Cumulative distributions of TE change for non-m6A and m6A transcripts as in (C). Kolmogorov-Smirnov test.Heatmap showing the TE of Wnt signaling components in control and YTHDF1 knockdown cells. The values show the fold change of the indicated color of the heatmap.
Figure 6Immunoblot analysis of HCT116 cells with YTHDF1 knockdown. Two major bands were detected for human TCF7L2 due to alternative splicing.Polysome profiles of HCT116 cells with or without YTHDF1 knockdown. The right panels show the distributions of TCF7L2, and ACTB in polysome fractions. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01 (3 biological replicates, t-test).Immunoblot analysis of crypts from Ythdf1CTL and Ythdf1cKO mice. Multiple bands were detected for mouse TCF7L2 representing different isoforms.Immunoblot analysis of mouse intestinal crypts treated with Wnt3a for 60 min.MeRIP-qPCR analysis of m6A levels of TCF7L2 in crypts treated with or without Wnt3a. Data are represented as mean ± SEM. *p<0. 05 (3 biological replicates, t-test).RIP analysis of the interaction of YTHDF1 with TCF7L2 mRNA. The enrichment was measured by qPCR and normalized to input. Data are represented as mean ± SEM. *p<0. 05 (3 biological replicates, t-test).β-catenin/TCF4 reporter activity in YTHDF1 knockdown cells infected with lentivirus expressing TCF7L2. Data are represented as mean ± SEM. *p<0. 05, **p<0. 01, ***p<0.001 (3 biological replicates, t-test).Morphology of organoids from Lgr5-creERT2:Ythdf1fl/fl mice in Wnt3a-conditioned medium without or with 4-OHT induction and infected with lentivirus expressing TCF7L2. Scale bar, 250 μm. Quantification of differentiated versus undifferentiated organoids from (H). Data are represented as mean ± SEM. **p<0. 01, ***p<0. 001 (3 biological replicates, t-test).
Figure 7Colons from mice in (A) treated with or without TAM on day 80 of AOM/DSS induction.Colon tumor number from mice treated with or without TAM on day 80 of AOM/DSS induction. Data are shown as mean ± SEM (7 mice for each group). ****p<0. 0001 (t-test).Immunoblot analysis of 2 pairs of colon tumors from mice treated with or without TAM.Kaplan-Meier survival plots for Apcmin/+; Lgr5-creERT2:Ythdf1fl/fl mice (8 mice for each group) treated with or without TAM. Log-rank (Mantel Cox) test. **p<0. 01 (t-test).
Figure 2(A) Motility assay of C.c. ΔpseI cells expressing predicted Leg or Pse synthases (LegI or PseI, in yellow or red, respectively) from Plac on pSRK-Gm. Overnight cultures were spotted on PYE soft (0.3%) agar plates with gentamycin and IPTG (0.5 mM) and incubated for 3 days at 30°C. Only predicted PseI-like synthase coding sequences (CDSs) restore motility of the C.c. ΔpseI cells. Note that the predicted synthases labeled in white do not share similarities with LegI or PseI of C. jejuni. Motility assays are representative of three independent experiments.(B) Immunoblot of cell extracts from cells in (A) probed with antibodies to C. crescentus FljK (FljKCc). The migration of flagellins in ΔpseI cells is shifted towards lower molecular mass suggesting that post-translational modification of flagellin is defective in ΔpseI mutant. Molecular sizes are indicated by the blues lines (in kDa). Bs: Brevundimonas subvibrioides, Bl: Brevundimonas lutea, Bv: Brevundimonas viscosa, Dv: Dermabacter vaginalis, Li: Leptospira interrogans, Tp: Treponema pallidum, Td: Treponema denticola, AbACICU: Acinetobacter baumannii ACICU, AbLUH: Acinetobacter baumannii LUH, AbLAC: Acinetobacter baumannii LAC-4, Lp: Legionella pneumophila, PaY82: Pseudomonas aeruginosa Y82, PaPAPS475: Pseudomonas aeruginosa PAPS475, Sj: Shewanella japonicum, Basu: Bacillus subtilis, Ks: Kurthia sibirica, Cb: Clostridium botulinum, Gk: Geobacillus kaustophilus, Mh: Moorella humiferrea, Myco: Mycobacterium sp. KS0706, HrrPV6: Halorubrum sp. PV6, Ms: Methanobrevibacter smithii. All the genes come from synthetic fragments codon optimized for E. coli (except the synthase CDS from Gk that is codon optimized for C.c.). Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Yellow boxes indicate PseI functional orthologs. Immunoblots are representative of at least two independent experiments.
Figure 3(A) and (B) Motility assays of WT, ΔlegI (A) and ΔflmG (B) B.s. cells. Overnight cultures of WT and mutants harboring the empty pSRK-Gm vector (+Plac) or the corresponding complementing plasmid were spotted on PYE soft agar plates with gentamycin and IPTG (0.5 mM) and incubated for 7 days at 30°C. Motility assays are representative of three independent experiments. The same original picture was used to generate Figs 3B and EV2C.(C) and (D) Immunoblots of cell extracts (cells) and supernatants (SN) of cultures from WT and mutant cells probed with antibodies to FljKCc, that detect the B.s. flagellins. The estimated molecular mass (in kDa) are indicated by the blue lines on the left. Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Immunoblots are representative of at least two independent experiments.(F) Flagellum length measurements determined by TEM of WT and mutants B.s. cells: WT, ΔlegX (ΔBresu_3267), ΔlegB (ΔBresu_3266) expressing legX in trans from Plac on a plasmid, ΔlegH (ΔBresu_0506), ΔlegI (ΔBresu_0507), ΔflmG (ΔBresu_2406) and ΔlegI ΔflmG. Box plots represent the distribution of the flagellum lengths. The cross indicates the average length, the central band indicates the median, the upper and lower sides of the box indicate the upper and lower quartiles, respectively, and the whiskers indicate the minimum and the maximum length measured. Twenty-five independent flagella were measured different cells (biological replicates) for each strain from TEM images.
Figure 4(A) Motility assays of B.s. ΔlegI cells complemented with LegI-type (yellow) or PseI-type (red) synthases expressed from Plac on pSRK-Gm. Motility assays are representative of three independent experiments.(B) Immunoblots probed with antibodies to FljKCc, revealing the cell extracts levels of flagellin in B.s. ΔlegI derivatives shown in (A). The blue lines represent the migration of the molecular size standards (in kDa). Bl: Brevundimonas lutea, Bv: Brevundimonas viscosa, Dv: Dermabacter vaginalis, Li: Leptospira interrogans, Tp: Treponema pallidum, Td: Treponema denticola, AbACICU: Acinetobacter baumannii ACICU, AbLUH: Acinetobacter baumannii LUH, AbLAC: Acinetobacter baumannii LAC-4, Lp: Legionella pneumophila, PaY82: Pseudomonas aeruginosa Y82, PaPAPS475: Pseudomonas aeruginosa PAPS475, Sj: Shewanella japonicum, Basu: Bacillus subtilis, Ks: Kurthia sibirica, Cb: Clostridium botulinum, Gk: Geobacillus kaustophilus, Mh: Moorella humiferrea, Myco: Mycobacterium sp. KS0706, HrrPV6: Halorubrum sp. PV6, Ms: Methanobrevibacter smithii. All the genes come from synthetic fragments codon optimized for E. coli (except the synthase CDS from Gk that is codon optimized for C.c.). Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Red boxes indicate LegI functional orthologs. Immunoblots are representative of at least two independent experiments.
Figure 5(A) Motility assays of WT B.s. and ΔBresu_3266 (ΔlegB) cells expressing Bresu_3266 (LegB), Bresu_3267 (LegX) or Bresu_3266-Bresu_3267 (LegB-LegX, Bresu_3266-67) from Plac on plasmid pSRK-Gm. Motility assays are representative of three independent experiments. (D) Motility assay of WT B.s. and ΔBresu_0506 (ΔlegH) cells complemented with a plasmid expressing Bresu_0506 (LegH) from Plac on pSRK-Gm. Motility assays are representative of three independent experiments.(B) Immunoblots probed with anti-FljKCc antibodies to reveal flagellins in cell extracts (cells) and supernatants (SN) of the strains described in (A). (E) Immunoblots probed with anti-FljKCc antibodies to reveal flagellins in cell extracts (cells) and supernatants (SN) of the strains described in (D). Immunoblots are representative of two independent experiments.
Figure 6(A) Motility assay of ΔBresu_3267 (ΔlegX) B.s. cells compared to WT B.s. cells harbouring the empty pSRK-Gm vector (+Plac) or a complementing derivative with PtmE/LegX orthologs from C. jejuni (Cj), Tanerella forsythia (Tf) or P. aeruginosa NMI1897 (PaNMI). Motility assays are representative of three independent experiments.(B) Immunoblots probed with anti-FljKCc antibodies from cell lysates (cells) and supernatants (SN) of WT B.s. and ΔBresu_3267 (ΔlegX) cultures harbouring either the empty pSRK-Gm vector (+Plac) or a complementing plasmid expressing different PtmE/LegX orthologs from C. jejuni (Cj), Tanerella forsythia (Tf) or P. aeruginosa NMI1897 (PaNMI). Molecular size standards are indicated by the blue lines with the corresponding value in kDa. Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Immunoblots are representative of two independent experiments.
Figure 7(B) Anti-FljKCc immunoblot analyses of whole cell lysates (cells) from C. crescentus (C.c.) mutant cultures expressing fljKBssyn (codon optimized for E. coli) and flmGBs from the replicative pMT463 plasmid, in the presence or absence of a compatible integrative plasmid carrying a legBs synthetic operon (pXGFP4-legBs) and in the presence or absence of an additional compatible replicative plasmid carrying Bresu_3267 (pMT375). In the presence of pXGFP4-legBs (integrated at the xylX locus) and pMT375-Bresu_3267, FljKBssyn migration is shifted toward higher molecular mass, indicative of glycosylation. Note that in the C.c. Δfljx6 background, the six flagellin-encoding genes have been deleted and the protein detected by the antibodies only corresponds to FljKBssyn. The legBs synthetic operon (in red) is composed of Bresu_3266, Bresu_0765, Bresu_0506, Bresu_3264, Bresu_0507 (legI) and Bresu_3265. Molecular masses are indicated in kDa by the blue lines. Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Asterisk indicates a modification of flagellin by FlmGBs that does not require Pse or Leg. Immunoblots are representative of at least two independent experiments.(C) Anti-FljKCc immunoblot analyses of whole cell lysates from C. crescentus mutant cultures expressing FlmGCc, FlmGBs and FlmGCc-Bschim chimera from the replicative pSRK-Gm plasmid (Plac) in the presence of a compatible integrative plasmid carrying a legBs synthetic operon (pXGFP4-legBs) and in the presence or absence of an additional compatible replicative plasmid carrying Bresu_3267 (pMT375). Molecular masses are indicated in kDa by the blue lines. Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Immunoblots are representative of at least two independent experiments.
Figure 1Strep-Tactin pull down of GIDAnt (strep-tagged at Gid8 C-terminus) probing binding of Gid1057-292 and Gid4117-362 to the complex visualized with Coomassie-stained SDS-PAGE. The experiment was performed with WT and C-terminal deletion (∆C) of the substrate receptors (∆289-292 (FEIA) and ∆359-362 (FEFA) for Gid10 and Gid4, respectively) and WT and mutant (mut, Gid5W606A/Y613A/Q649A) GIDAnt.Lysates from yeast strains expressing endogenously tagged 3xFLAG-Gid4 or 3x-FLAG-Gid10 that were grown in SD complete at 30°C (no stress), SE complete for 19 hours (EtOH), SD complete for 1 hour following 19 hour ethanol treatment (EtOH rec.), 42°C for 1 hour (HS), SD complete supplemented with 0.5M NaCl for 1 hour (OS), or SD-N for 1 hour (-N) were run on an SDS-PAGE gel and immunoblotted with αFLAG (two exposures from the same gel are shown) and αPGK.Lysates from yeast strains expressing endogenously tagged 3xFLAG-Gid4 or 3xFLAG-Gid10 that were grown at 42°C for one hour, and then returned to 30°C for the indicated timepoints were run on an SDS-PAGE gel and immunoblotted with αFLAG (two exposures from the same gel are shown) and αPGK.Lysates from wildtype and ∆Gid2 yeast strains expressing endogenously tagged 3xFLAG-Gid10 that were grown at 42°C for one hour, and then returned to 30°C for the indicated timepoints were run on an SDS-PAGE gel and immunoblotted with αFLAG and αPGK.Lysates from wildtype, Gid5W606A,Y613A,Q649A, and Gid10∆C strains expressing endogenously tagged 3xFLAG-Gid10 that were grown at 42°C for one hour, and then returned to 30°C for the indicated timepoints were run on an SDS-PAGE gel and immunoblotted with αFLAG and αPGK.
Figure 2Lysates from wildtype, ∆Gid4, and Gid4∆C strains expressing endogenously tagged 3xFLAG-Gid10 that were grown at 42°C for one hour, and then returned to 30°C for the indicated timepoints were run on an SDS-PAGE gel and immunoblotted with αFLAG and αPGK.Wildtype and Gid10 overexpressing yeast strains expressing endogenously tagged Gid4 were grown for 19 hours in YPE, and transitioned to YPD for the indicated timepoints. Lysates were run on an SDS-PAGE gel and immunoblotted with αFLAG and αPGK. Points represent mean, error bars represent standard deviation (n>3 biological replicates).In vitro competition assay probing the ability of N-terminally 2xStrep-tagged Gid4 (2xS-Gid4) to exchange with Gid10 in the GIDSR10 complex. Proteins after Strep-Tactin pull-down were visualized by Coomassie-stained SDS-PAGE.
Figure 5Art2-3xFLAG was immunocaptured from yeast cells grown in YPD and incubated with GidSR10, GidSR4, GidAnt, or Gid10 for the indicated timepoints. Progress of the reaction was followed by αFLAG immunoblot.Mdh2-3xFLAG was immunocaptured from yeast cells following growth in YPE for 19h and incubated with GidSR10, GidSR4, GidAnt, or Gid4 for the indicated timepoints. Progress of the reaction was followed by αFLAG immunoblot.Art2-3xFLAG or Art2P2S-3xFLAG was immunocaptured from yeast cells grown in YPD and incubated with GidSR10, or GidSR10∆C for the indicated timepoints. Progress of the reaction was followed by αFLAG immunoblot.In vitro ubiquitination assay probing the ability of Gid10 and Gid4 to promote ubiquitination of Art22-29 N-terminus and its P2S mutant. Progress of the reaction was monitored by fluorescent scan of the gel visualizing the Art2 peptide with FAM appended to its C-terminus (pep*).