EMBO/soda-nel huggingface dataset:
EGR2 phosphatase regulates OST1 kinase activity and freezing tolerance in Arabidopsis (2018)
Figure 3

Figure 3. Freezing phenotypes of egr2-3 mutant and EGR2-overexpressing plants.

A-C Freezing phenotype (A), survival rate (B) and ion leakage (C) of the egr2-3 mutant and egr2/EGR2:EGR2-Myc complementation lines under non-acclimated (NA) and acclimated (CA; 4 d at 4°C) conditions. Two-week-old seedlings grown on MS medium containing 0.8% agar were treated at −5°C for 0.5 h (NA) or −8°C for 0.5 h (CA).

D-F Freezing phenotype (D), survival rate (E), and ion leakage (F) of EGR2-overexpressing plants. Two-week-old Super:EGR2-Myc plants were treated as described in (A-C).

G-J Expression of CBFs (G, I) and their target genes (H, J) in egr2-3 mutant and EGR2-overexpressing plants. Two-week-old seedlings were treated at 4°C for the indicated period and subjected to qRT-PCR analysis.

Data information: In (B, C, E-J), each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).
EGR2 phosphatase regulates OST1 kinase activity and freezing tolerance in Arabidopsis (2018)
Figure 4

Figure 4. N-myristoylated modification of EGR2 is important for its function under cold stress.

A Possible conserved N-myristoylated motif in EGR2.

B N-myristoylation of EGR2 and EGR2G2A in N. benthamiana leaves. Protein extracts prepared from N. benthamiana leaves expressing EGR2-Myc or EGR2G2A-Myc were immunoprecipated by anti-Myc agarose beads. Precipitated proteins were detected by anti-Myc and anti-myristic acid antibodies.

C Diagram of the protocol for detecting EGR2 N-myristoylation in planta.

D Immunoblot analysis showing that the total level of myristoylated proteins in wild type plants. Total proteins were prepared from two-week-old wild type seedlings treated with or without 80 μM myristic acid alkyne at 22°C for 72 h and then subjected to click-reaction with the biotin azide. The reaction was protected from light at room temperature for 3 h. The products were detected by anti-streptavidin-HRP antibody.

E N-myristoylation of EGR2 in plants. Total proteins were prepared from two-week-old EGR2-Myc seedlings treated as described in (D). The products were immunoprecipated by anti-Myc agarose beads and detected by anti-streptavidin-HRP antibody. EGR2-Myc detected by anti-Myc antibody was used as a loading control.

F Immunoblot assay of myristoylated EGR2 under cold stress. Total proteins were prepared form EGR2-Myc plants treated with 4°C for the indicated period, and immunoprecipated by anti-Myc agarose beads. Precipitated proteins were detected by anti-Myc and anti-myristic acid antibodies. The ratio of band intensity detected by anti-myristic acid antibody to anti-Myc antibody without cold treatment was set to 1.00. Representative pictures are shown (left) and quantitative analysis is shown (right). Each bar represents the mean ± SEM of three biological repeats. **P < 0.01 (two-tailed t-test).

G Expression of EGR2 in egr2-3 EGR2:EGR2G2A-Myc complementation transgenic plants. Total RNAs were extracted from 2-week-old seedlings and subjected to RT-PCR analysis. EF1α was used as a loading control.

H, I Freezing tolerance assays of the egr2-3 mutant and EGR2:EGR2G2A-Myc transgenic plants in egr2-3 mutant. Two-week-old seedlings grown on MS medium containing 0.8% agar at 22°C were treated at −5°C for 0-1.5 h (NA) or −8°C for 0-1.5 h (CA). Representative photographs (-5oC, 0.5 h; -8oC, 0.5 h) are shown in (H), survival rate is shown in (I). Data shown in (I) are mean values ± SEM of three biological replicates, each of which has three technical replicates. Asterisks indicate significant differences compared with wild type for the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test); n. s., not significant.
EGR2 phosphatase regulates OST1 kinase activity and freezing tolerance in Arabidopsis (2018)
Figure 5

Figure 5. Cold stress disrupts the interaction of NMT1 and EGR2.

A In vitro pull-down assay showing the interaction of NMT1 and EGR2. Recombinant GST-NMT1 was incubated with immunoprecipated MBP-His-EGR2 or MBP-His at 4°C for 2 h. Anti-GST and anti-MBP were used to detect NMT1, EGR2 and MBP-His, respectively.

B Co-localization of NMT1 and EGR2 in N. benthamiana leaves. Scale bar: 50 μm

C, D Effect of cold stress on the interaction of EGR2 and NMT1 determined by co-IP assay. Arabidopsis protoplasts expressing HF/NMT1-Myc and HF-EGR2/NMT1-Myc were treated with or without 4°C for 2 h. Total proteins were immunoprecipated by anti-HA agarose beads. Anti-Myc and anti-HA were used to detect NMT1-Myc and HF-EGR2, respectively. In (C), relative band intensity is shown below the co-IP blot. In (D), each bar represents the mean ± SEM of three biological experiments (**P < 0.01, two-tailed t-test).

E In vitro pull-down assay showing decreased interaction intensity of EGR2 (4°C) and NMT1 compared with that of EGR2 (22°C) and NMT1. Total proteins prepared from two-week-old EGR2-Myc plants grown at 4°C for 0 h and 6 h were immunoprecipated by anti-Myc agarose beads and incubated with GST-NMT1 in pull down buffer for 2 h at 4°C. Anti-Myc and anti-GST antibodies were used to detect EGR2 and NMT1 proteins. Relative band intensity is shown below the output blot.

F Expression of NMT1 in NMT1-Myc overexpression plants. EF1α was used as a loading control.

G, H Freezing phenotype (G) and survival rate (H) of NMT1-Myc overexpression transgenic plants. Two-week-old Super:NMT1-Myc plants were treated at −5°C (NA) or −8°C for 0.5 h (CA). In (H), each bar represents the mean ± SEM of three biological experiments, and asterisks indicate significant differences compared with the wild type for the same treatment (*P < 0.05, two tailed t-test).

I, J OST1 kinase activity in NMT1-Myc overexpression plants under cold stress. Top, autoradiograph; bottom, CBB staining. Representative pictures are shown in (I), and the relative kinase activity is shown in (J). In (I): the ratio of band intensity of OST1 to RuBisCO in wild type with cold treatment for 2 h was set to 1.00. In (J): each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two tailed t-test).
EGR2 phosphatase regulates OST1 kinase activity and freezing tolerance in Arabidopsis (2018)
Figure 6

Figure 6. Cold stress attenuates the interaction between EGR2 and OST1.

A, B Pull-down assay. Total proteins prepared from EGR2-Myc or EGR2G2A-Myc transgenic plants was immunoprecipated with anti-Myc agarose beads, and then incubated with GST-OST1. Anti-Myc and anti-GST antibody were used to detect GST-OST1 and EGE2-Myc. Representative pictures are shown in (A). Relative interaction is shown in (B).

C LCI of EGR2 and EGR2G2A with OST1 in N. benthamiana leaves. EGR2-cLuc/OST1-nLuc and EGR2G2A-cLuc/OST1-nLuc were expressed in N. benthamiana leaves for 48 h. EGR2-cLuc/nLuc, EGR2G2A-cLuc/nLuc, and cLuc/OST1-nLuc were used as negative controls. Scale bar: 2 cm.

D, E Co-IP assay. EGR2-Myc/HF-OST1, EGR2G2A-Myc/HF-OST1, HF/EGR2-Myc and HF/EGR2G2A-Myc were expressed in N. benthamiana leaves. Total proteins were immunoprecipated by anti-HA agarose beads. Anti-HA and anti-Myc antibodies were used to detect HF-OST1, EGR2-Myc and EGR2G2A-Myc, respectively. Representative pictures are shown in (D), and relative interaction is shown in (E).

F, G Effect of EGR2G2A on the interaction of OST1 and EGR2 in Arabidopsis protoplasts expressing HF-OST1/EGR2-GFP/EGR2-Myc, HF-OST1/EGR2-GFP/EGR2G2A-Myc, or HF-OST1/EGR2-GFP. Total proteins were immunoprecipated by anti-HA agarose beads. Anti-Myc, anti-GFP, and anti-HA antibodies were used to detect EGR2-Myc, EGR2-GFP, and HF-OST1, respectively. Representative pictures are shown in (F), and relative interaction is shown in (G).

H, I Effect of cold stress on the interaction of OST1 and EGR2. Arabidopsis protoplasts expressing EGR2-Myc/HF-OST1 and EGR2-Myc/HF were treated at 4°C for 0 and 2 h. Total proteins were immunoprecipated by anti-HA agarose beads. The co-immunoprecipitated products were detected by anti-HA and anti-Myc antibodies, respectively. Representative pictures are shown in (H), and relative interaction is shown in (I).

Data information: In (A, D, F and H): Relative band intensities are shown. In (B, E, G and I): each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two-tailed t-test).
EGR2 phosphatase regulates OST1 kinase activity and freezing tolerance in Arabidopsis (2018)
Figure 7

Figure 7. Genetic Interaction of OST1 and EGR2.

A, B Freezing phenotype (A), survival rate and ion leakage (B) of wild type, ost1-3, egr2-3, and ost1 egr2 mutants. Two-week-old seedlings grown on MS medium were treated at −5°C (NA) or −8°C for 0.5 h (CA).

C Expression of CBFs in wild type, ost1-3, egr2-3, and ost1 egr2-3 plants under cold stress. Two-week-old seedlings grown at 22°C were treated at 4°C for 0 h and 3 h.

D Model of EGR2-OST1 modulation of cold stress responses in Arabidopsis.

At warm temperature (22°C), EGR2 can be modified by myristoylation, which allows it to recognize OST1 and inhibit its activity to ensure plant growth. Under cold stress (4°C), the interaction of NMT1 and EGR2 is decreased, thereby causing the accumulation of unmyristoylated EGR2, which shows reduced interaction with OST1 and disrupts the interaction of EGR2-OST1 as well. This ultimately leads to activating OST1 protein kinases and enhancing plant freezing tolerance. Clade A PP2Cs, such as ABI1, also partially contributes to regulating OST1 activity in response to cold stress. EGR2-M and u-EGR2 represent myristoylated and unmyristoylated EGR2, respectively.

Data information: In (B, C), each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).

Expanded View Figures
A virus-derived microRNA targets immune response genes during SARS-CoV-2 infection (2021)
Figure 2

Figure 2: RNA secondary structure of SARS-CoV-2 miR-O7a precursor and sequence conservation among different SARS coronaviruses and within SARS-CoV-2 variants.

(A) Predicted RNA secondary structure for the CoV2-miR-O7a and flanking sequence using the first 70 nt of the open reading frame of the ORF7a. The arrows indicate the sites of the miRNAs possibly cleaved by Dicer. The stem-loop structure is not conserved in SARS-CoV. The colors indicate the base-pair probabilities.

(B) Conservation of the first 70 nt of the ORF7a sequence among different SARS coronaviruses. The underlined sequences are related to the position of the SARS-CoV-2 miR-O7a. The conserved ribonucleotides of the CoV2-miR-O7a sequence are marked in red and in blue all the non-conserved ribonucleotides across the 70nt sequence. The bat and pangolin coronaviruses closely related to SARS-CoV-2 are marked in purple.

(C) Percentage of conservation along the nucleotide positions in the ORF7a among 4,055,609 sequenced SARS-CoV-2 genomes. The first 70 nt are shown in red and shows a higher percentage of conservation compared to the rest of the sequence of ORF7a. Boxplot shows the distribution of conservation percentage for each nucleotide either in the first 70 nt or 71-366 nt of ORF7a among 4,055,609 sequenced SARS-CoV-2 genomes. Box plots display median (line), first and third quartiles (box), and 5th /95th percentile value (whiskers). Each dot represents the outliers. Two-tailed P values were calculated using the student's t-test.
A virus-derived microRNA targets immune response genes during SARS-CoV-2 infection (2021)
Figure 3

Figure 3: The SARS-CoV-2 miR-O7a produces two isoforms processed by DICER and loaded onto AGOs.

(A) Log2 fold changes of the levels of hsa-miR-let-7a, CoV2-miR-O7a.1 and COV2-miR-O7a.2 in DICER1 knockdown SARS-CoV-2-infected A549-ACE2 cells compared to control cells analyzed by stem-loop RT-qPCR. Results from three independent replicates are shown.

(B) In vitro kinetics of DICER1 mediated cleavage of pre-CoV2-miR-O7a stem-loop precursor, pre-CoV2-miR-O7a with mutations that prevent the formation of stem-loop structure and pre-hsa-miR21. Folded conformations for the three precursor molecules are shown. Fold change in the production of mature CoV2-miR-O7a.2 is shown in presence of DICER1 compared to control reaction (no DICER1) as measure by stem-loop RT-qPCR. Bars and error bars represent the average and standard deviation from three independent experiments.

(C) Expression levels of the virus-derived miRNAs as a percentage of the viral genome. Absolute quantification of virus-derived miRNAs and viral genome from infected A549-ACE2 cells was performed using two spike-in (see methods). Line represents the average and individual dots represent data from two independent experiments.

(D) copy number per cell of hsa-miR-let-7a, CoV2-miR-O7a.1 and CoV2-miR-O7a.2 in infected A549-ACE2 cells quantified using a synthetic small RNA spike-in standard curve by stem-loop RT-qPCR. Levels of hsa-miR-let-7a were normalized for the percentage of infected cells. Bars and error bars represent the average and standard deviation from at least three independent experiments.

(E) Loading of hsa-miR-let-7a, CoV2-miR-O7a.1 and COV2-miR-O7a.2 into AGOs as measured by stem-loop RT-qPCR and analyzed as a percentage of input from the immunoprecipitates (IPs) of either pan-AGO IP or control IgG IP from infected A549-ACE2 cells. Levels of miRNA were normalized for the percentage of infected cells.
A virus-derived microRNA targets immune response genes during SARS-CoV-2 infection (2021)
Figure 4

Figure 4: SARS-CoV-2 miR-O7a.2 represses the activation of transcription factor BATF2.

(A) Base-pairing of CoV2-miR-O7a.2 to complementary 3´UTR site or site with mutations in seed region cloned in 3'UTR of firefly luciferase mRNA in pmirGLO Dual-Luciferase miRNA Target Expression Vector. The seed region required for binding of miRNAs with its target is underlined.

(B) Relative firefly luciferase normalized to Renilla luciferase of pmirGLO Dual-Luciferase miRNA Target Expression Vector with either CoV2-miR-O7a.2 target site or CoV2-miR-O7a.2 site with mutations in seed region at 24 h on co-transfection with CoV2-miR-O7a.2 or control miRNA mimics. Activity on transfecting control miRNA mimic was considered 100 %. Bars and error bars represent the average and standard deviation from three independent experiments.

(C) Base-pairing of CoV2-miR-O7a.2 to complementary 3´UTR site of BATF2 mRNA. The seed region required for binding of miRNAs with its target is underlined. One mismatch in the seed region of CoV2-miR-O7a.1 is observed.

(D) Kinetics of BATF2 and LAMP3 mRNA levels in non-induced and interferon IFN-α-induced (for 8, 16, and 24 h) A549-ACE2 cells transfected with either control or CoV2-miR-O7a.2 mimics. Normalized read abundances in transcript per million (TPM) are shown for two individual experiments (n=2).

(E) Genomic view of the human BATF2 gene showing normalized RNA-seq reads from non-induced and IFN-α-induced (for 8 h) A549-ACE2 cells transfected with either control, CoV2-miR-O7a.1 or CoV2-miR-O7a.2 mimics. The complementary site of CoV2-miR-O7a to BATF2 3´UTR is shown.
A virus-derived microRNA targets immune response genes during SARS-CoV-2 infection (2021)
Figure 5

Figure 5: SARS-CoV-2 miR-O7a.2 represses the activation of interferon-stimulated genes with target sites in their 3'UTR.

(A, B) Volcano plots showing the log2 fold change and corresponding significance levels of ISGs upon 8 hours of IFN-α treatment in A549-ACE2 cells transfected with CoV2-miR-O7a.1 (A) or CoV2-miR-O7a.2 (B) compared to control mimic. Significantly downregulated genes are marked in red, and upregulated genes in blue. The orange horizontal line indicates two-tailed P = 0.05. n=2, dashed lines represent +1 and -1 log2 fold change.

(C) Log2 fold change of ISGs at 8, 16, 24 hours of IFN-α treatment and categorized based on CoV2-miR-O7a.2 target sites 8mer, 7mer-m8, 7mer-A1, and no seed as shown in the schematic. The mean and standard error of the mean is shown. Number of ISGs with 8mer site n=8, 7mer-m8 n= 32, 7mer-A1 n=26 and no seed n= 92. The two-tailed p values were calculated using the Mann-Whitney-Wilcoxon test. ISGs were calculated as all the upregulated genes (≥ 3-fold; padj < 0.05) in two replicates of IFN-induced versus non-induced conditions in all time points.
A virus-derived microRNA targets immune response genes during SARS-CoV-2 infection (2021)
Figure 6

Figure 6: Dynamic expression of ISGs upon type I IFN-α treatment at different time points in A549-ACE2 cells and in the presence of CoV2-miR-O7a.2 mimic.

(A) Heat map showing log2 fold change of expression of ISGs in A549-ACE2 cells across 8, 16 and 24 h time points upon IFN-α treatment compared to non-treated controls. The common upregulated genes (≥ 3-fold; padj < 0.05) in IFN-induced versus non-induced conditions at all time points were categorized as ISGs.

(B) Heat map showing log2 fold change of expression of ISGs across 8, 16 and 24 h time points upon IFN-α treatment in A549-ACE2 cells transfected with CoV2-miR-O7a.2 mimic compared to control mimic.

Data information: Genes marked in red and * indicate the ISGs detected by RT-qPCR in Fig. EV5C, D.
A virus-derived microRNA targets immune response genes during SARS-CoV-2 infection (2021)
Figure 7

Figure 7: The two CoV2-miR-O7a isoforms are produced in COVID-19 patients.

(A) Expression levels of CoV2-miR-O7a.1, CoV2-miR-O7a.2 and a 22 nt region from the ORF6 of the viral genome that does not produce detectable levels of small RNAs (Control ORF6) analyzed by stem-loop RT-qPCR from nasopharyngeal swabs of patients tested positive for COVID-19 or another seasonal human coronavirus (HCoV). Relative expression to hsa-miR-let-7a is shown. Ct values in parenthesis refer to the Ct value for the detection of viral genome in patient swab samples.

(B) SARS-CoV-2 genomic view showing the distribution of normalized 22nt small RNA reads from nasopharyngeal swabs of three patients tested positive for COVID-19. The most abundant small RNAs are marked by the red boxes and correspond to the CoV2-miR-O7a.

(C) Model for the production and function of Cov2-miR-O7a in COVID-19 patients.

Expanded View Figure legends
A Heterotypic Assembly Mechanism Regulates CHIP E3 Ligase Activity (2022)
Figure 1

Figure 1: UFD-2 activates CHN-1.

A) Auto-Ub of recombinant CHN-1 and CHN-1-UFD-2 was carried out using the E2 UBE2D1. Time-dependent (0, 10, 20, 40, 80, 120 min) CHN-1 auto-Ub was performed as indicated using wild-type ubiquitin. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. Below, a graph representing the nmol of ubiquitylated CHN-1 vs. time for CHN-1 alone (black) or CHN-1+UFD-2 (cyan). Plotted data are the mean of three technical replicates. Error bars represent the standard error of measurement (SEM); statistical and significance was determined using Pearson correlation coefficients which define the statistical relation between two continuous variables [CHN-1 vs Time, CHN-1+UFD-2 vs Time, and CHN-1 vs CHN-1+UFD-2 with increasing time]. (*P < 0.05; **P < 0.01; ***P < 0.001)

B) CHN-1 auto-Ub was performed in the presence of UFD-2 with the increasing molar concentration as indicated. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. Right, signal quantification of the unmodified CHN-1 (yellow), ubiquitylated fraction <70 kDa (cyan) and >70 kDa (magenta), plotted as a percentage of different CHN-1 species present in the indicated condition. Plotted data are the mean from the three technical replicates. Error bars represent the SEM; statistical significance was determined using a two-way ANOVA test. (**P < 0.01)

C) Auto-Ub was performed as indicated using recombinant CHN-1 and UFD-2P951A, UBE2D1 E2, UbWT, UbNoK, or Ub with substitutions of lysines 29, 48, and 63 to arginines (Ub3KTR). Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies.

D) Surface plasmon resonance (SPR) sensorgrams of the interaction between linear di-Ub (M1- linear from UbiQ) and C. elegans UFD-2 (magenta) or S. cerevisiae Ufd2p (cyan). Y-axis: response unit (U) value. X-axis: molar concentration of linear di-Ub.

E) CHN-1 auto-Ub was performed in the presence of recombinant C. elegans UFD-2 and S. cerevisiae Ufd-2p and UBE2D1. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies.

Data information: Representative immunoblots for at least three independent experiments are shown in the panels.
A Heterotypic Assembly Mechanism Regulates CHIP E3 Ligase Activity (2022)
Figure 2

Figure 2: UFD-2 stabilizes CHN-1 U-box dimer.

A) HDX-MS was used to analyze changes in the structural dynamics of residues within CHN-1 when complexed with UFD-2. The model diagram represents regions of retarded (red) and enhanced (blue) exchange in CHN-1. Right, schematics showing CHN-1 domain organization and the rate of deuterium exchange (coloured box: blue, light red, medium red, dark red) in the different domains upon interaction with UFD-2.

B) CHN-1 auto-Ub was performed as indicated using increasing molar concentrations (0.5, 1, 2, 3, 4 µM) of UBE2D1 without and with a complexing equimolar concentration (1:1) of recombinant CHN-1 and UFD-2P951A (1.5 µM CHN-1 with 1.5 µM of recombinant UFD-2P951A). Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. Right, a graph representing the nmol of ubiquitylated CHN-1 vs. UBE2D1 (µM) for CHN-1 alone (black) or CHN-1+UFD-2P951A (cyan). Plotted data are the mean from the three technical replicates. Error bars represent the SEM; statistical significance was determined using Pearson correlation coefficients which define the statistical relation between two continuous variables [nmol of ubiquitylated CHN-1 vs UBE2D1(µM) in the presence of CHN-1 or CHN-1+UFD-2P951A and CHN-1 vs CHN-1+UFD-2P951A with increasing UBE2D1(µM)]. (*P < 0.05; ****P < 0.0001)

C) Discharging assay of Ub-charged UBE2D1 was carried out in the presence of CHN-1 or CHN-1-UFD-2P951A. The experimental sample was run together with the control with and without a reducing agent (50 mM DTT). The reaction was stopped after 30 min by boiling the samples in 1x SDS sample buffer. Proteins were resolved via SDS-PAGE and immunoblotted with anti-UBE2D1 antibodies. Below, quantification of uncharged UBE2D1 plotted as the uncharged UBE2D1 fraction vs. UBE2D1-Ub (µM). The graph representing the UBE2D1 fraction vs. UBE2D1-Ub (µM) for CHN-1 alone (black) or CHN-1+UFD-2P951A (cyan) and Plotted data are the mean of three technical replicates. Significance was determined using Pearson correlation coefficients which define the statistical relation between two continuous variables [UBE2D1 vs UBE2D1-Ub(µM) in the presene of CHN-1 or CHN-1+UFD-2P951A and CHN-1 vs CHN-1+UFD-2P951A with increasing UBE2D1-Ub(µM)]. (P < 0.05; ***P < 0.001; ****P < 0.0001)

D) CHN-1 auto-Ub was performed as indicated in the presence of Ube2W-Ub or Ube2W-UbFLAG without and with equimolar concentration of recombinant CHN-1 and UFD-2P951A. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. For each sample, the quantified relative signal after probing the blot using anti-CHN-1 antibodies is shown as a ratio above the respective signal. Below, a schematic of the CHN-1-UbFLAG (cyan), CHN-1-Ub (magenta), and unmodified CHN-1 signal (black) presented as the ratio and the signal fold change between CHN-1 and CHN-1-UFD-2P951A.

Data information: Representative immunoblots for at least three independent experiments are shown in the panels.

Source data for HDX-MS measurements are available in the Table EV1.