EMBO/soda-nel huggingface dataset:
FAM111A Induces Nuclear Dysfunction in Disease and Viral Restriction (2020)
Figure 5

Figure 5. FAM111A affects nuclear barrier function during viral replication.

A. Representative immunofluorescence images of SV40 LT localization 72 h after transfection into U2OS cells infected with control or FAM111A shRNA. Images were taken with a Zeiss Axio Imager. LT is shown in green, and DAPI blue. Scale bar: 100 μm.

B. Quantification of LT positive cells described in A and fixed at indicated times post transfection. Five hundred or more cells were counted for each condition.

C. Quantification of RFP marker distribution upon co-transfection with SV40 LT variants into U2OS cells infected with control or FAM111A shRNA. Cells were fixed at the indicated times. Five hundred or more cells were counted for each condition. In B & C, values are mean ± s.d. of independent experiments (n = 3). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (two-tailed unpaired t-test).

D. Western blot analysis of control or FAM111A-depleted U2OS cells transfected with SV40 LT variants. Cells were collected and lysed at indicated timepoints after SV40 transfection, immunoblotted with FAM111A, LT, or PARP antibodies. Cdc2-PSTAIR was used as a loading control.

Figure EV Figure Legends
PARylation of the forkhead-associated domain protein DAWDLE regulates plant immunity (2016)
Figure 2

Figure 2. DDL is involved in plant defense.

(A) Scheme of DDL protein domains and gene structures with molecular lesions of five mutants. N-terminal NLS (nuclear localization signal) and C-terminal FHA domains are labeled. The dark grey bars indicate exons and lines in between indicate introns. The light grey bars indicate 5’ and 3’ UTR. The ddl-1, ddl-2 and ddl-3 mutants are in the Ws background, whereas ddl-6 (SALK_045025) and ddl-7 (SAIL_1281_F08) are in the Col-0 background. ddl-1, ddl-2, ddl-6 and ddl-7 are T-DNA insertional lines whereas ddl-3 is a TILLING line with a point mutation. (B) The DDL transcripts and growth phenotype of ddl-6 and ddl-7. DDL transcripts were detected in fully expanded leaves of four-week-old plants by qRT-PCR (left panel). The expression of DDL in WT Col-0 was set as 1. Plants grown under 12-hr light/12-hr dark condition for 4 weeks are shown (right panel). Bar=1cm. (C) ddl-6 and ddl-7 are more susceptible to Pst DC3000 infections. Leaves of four-week-old plants were inoculated with Pst DC3000 at OD600=5x10-4. Bacterial numbers were counted at 0 and 3 dpi. Leaf pictures were taken at 3 dpi. (D) The ddl-1 mutant in the Ws background is more susceptible to Pst DC3000 infection. (E) ddl-6 is more susceptible to Psm infection. Leaves of four-week-old plants were inoculated with Psm at OD600=5x10-4. (F) Increased bacterial growth of Pst DC3000 hrcC in ddl-6. Leaves of four-week-old plants were inoculated with Pst DC3000 hrcC at OD600=5x10-4. (G) Increased bacterial growth of Psp in ddl-6. Leaves of four-week-old plants were inoculated with Psp at OD600=5x10-4. (H) Complementation of DDL in disease resistance. Two independent T3 homozygous lines carrying pDDL::DDL-FLAG (W7 and W18) in the ddl-6 background were assayed for the susceptibility to Pst DC3000.

The data are shown as mean ± SD (n=3) with one-way ANOVA analysis and Tukey test (p<0.05). Different letters indicate significant differences. One representative result from three independent repeats is shown.
PARylation of the forkhead-associated domain protein DAWDLE regulates plant immunity (2016)
Figure 3

Figure 3. Differential PTI responses in ddl-6.

(A) flg22-induced callose deposition is reduced in ddl-6 and parp1,2. Leaves of four-week-old plants were inoculated with 0.5 µM flg22 for 20 hr or hrcC at OD600=0.2 for 24 hr and stained with aniline blue. Callose deposits were visualized under UV light and quantified by Image J. The data are shown as mean ± SE (n=12). Bar=100 µm. (B) The hrcC-mediated induction of some late responsive genes is compromised in the ddl-6 and parp1,2 mutants. Fully expanded leaves of four-week-old plants were hand-inoculated with hrcC (OD600=0.5), and collected at 0, 3 and 24 hpi for qRT-PCR. The data are shown as mean ± SE from three independent repeats. (C) Pst DC3000-induced PR1 and PR5 transcripts are blocked in ddl-6 and parp1,2. Fully expanded leaves of four-week-old plants were inoculated with Pst DC3000 (OD600=0.01), and collected at 0, 8 and 24 hpi for qRT-PCR. The data are shown as mean ± SE from three independent repeats. (D) flg22-induced MAPK activation in Col-0 and ddl-6. Ten-day-old seedlings were treated with 100 nM flg22 and collected at the indicted time points. MAPK activation was analyzed by an immunoblot (IB) with -pERK antibody (top panel), and the protein loading is shown by Ponceau S staining for RuBisCo (RBC) (bottom panel). (E) flg22-induced ROS in Col-0 and ddl-6. Leaf discs from four-week-old plants were assayed for ROS production upon 100 nM flg22 treatment over 30 min. The data are show as mean± SE (n=24).

The above experiments were repeated three times with similar results.
PARylation of the forkhead-associated domain protein DAWDLE regulates plant immunity (2016)
Figure 4

Figure 4. DDL directly interacts with PARP2.

(A) PARP2 and DDL association in Arabidopsis protoplasts. PARP2-HA and DDL-FLAG were transiently expressed in Col-0 protoplasts, immunoprecipitated with α-FLAG antibody (IP: -FLAG), and immunoblotted with α-HA (IB: α-HA) or α-FLAG antibody (IB: -FLAG) (top two panels). The protein inputs are shown with immunoblotting before immunoprecipitation (bottom two panels). Protoplasts were treated without or with 100 nM flg22 for 30 min. (B) PARP2 and DDL association in N. benthamiana. PARP2-FLAG and DDL-HA were transiently co-expressed in leaves of N. benthamiana. The samples were collected at 2 dpi for Co-IP assay with 100nM flg22 treatment for 1 hr. (C) PARP2 and DDL association with BiFC assay. Different combinations of constructs were transiently expressed in Arabidopsis Col-0 protoplasts and the fluorescence signals were observed with a confocal microscope. An NLS-RFP construct was co-transfected to show the nuclear signals. Bar=10 µm. (D) PARP2 and DDL interaction in vitro. GST-PARP2 protein immobilized on glutathione beads was used to pull-down HIS-SUMO-DDL-HA proteins (left panels). A PARylation reaction was performed prior to the pull-down assay (the third lane of left panels). The PARylated PARP2 and DDL were detected with -PAR antibody (right panels). (E) In vitro PARylation of DDL by PARP2. Immunoprecipitated DDL-FLAG proteins from Arabidopsis protoplasts were incubated with GST-PARP2 in a PARylation reaction containing Biotin-NAD+. The reactions in lane 4 and 5 contained 1 mM 3-AB or GST-PARG1 respectively. PARylated proteins were detected by streptavidin-HRP antibody (Strep), which recognizes Biotin-labelled PAR.

All experiments were repeated three times with similar results.
PARylation of the forkhead-associated domain protein DAWDLE regulates plant immunity (2016)
Figure 5

Figure 5. flg22 stimulates PARP1/2-dependent DDL PARylation.

(A) flg22 stimulates DDL PARylation. DDL-FLAG was transiently expressed in Arabidopsis protoplasts fed with 32P-NAD+. Protoplasts were treated without or with 100 nM flg22 for 30 min. PARylated DDL was immunoprecipitated with α-FLAG affinity beads, separated on 10% SDS-PAGE and visualized by autoradiography. PAR(DDL) indicates PARylated DDL proteins. A representative result from three repeats is shown. (B-C) DDL PARylation is reduced in parp1, parp2 and parp1,2 mutants. Protoplasts isolated from parp1 or parp2 single mutants (B), or parp1,2 double mutants (C) were used for in vivo PARylation assay with 32P-NAD+. Protoplasts were treated with 100 nM flg22 for 30 min. The experiments were repeated twice with similar results.
PARylation of the forkhead-associated domain protein DAWDLE regulates plant immunity (2016)
Figure 6

Figure 6. Identification of DDL PARylation sites.

(A) DDL-N but not DDL-C associates with PARP2. FLAG-tagged DDL-N or DDL-C was co-expressed with PARP2-HA in protoplasts for Co-IP assay. Protoplasts were treated without or with 100 nM flg22 for 30 min. (B) in vivo PARylation of DDL-N or DDL-C in protoplasts fed with 32P-NAD+. PARylation was visualized with autoradiography. (C) In vitro PARylation of DDL-N or DDL-C. FLAG-tagged DDL-N or DDL-C was expressed and immunoprecipitated from Arabidopsis protoplasts and subjected to in vitro PARylation by GST-PARP2 with Biotin-NAD+. PARylated proteins were detected by streptavidin-HRP antibody. (D) List of the mass/charge values of b+ ions and y+ ions of the peptide (148AVeEALAAK156). Red mass values correspond to the identified peak in the spectrum. The modified “e” is in bold and lowercase. The mass difference between b2+ and b3+ or y6+ and y7+ indicates that Glu150 is PARylated. (E) MS/MS spectrum of a doubly charged peptide with Glu (the third AA “e” in the peptide, Glu150) PARylation. 2+ indicates doubly charged and 0 indicates loss of H2O. The mass difference between y7+ and y6+ is 144.1, which is about 15 Da bigger than the Glu residue mass of 129.04. (F) DDL12E mutants substantially diminish its PARylation in vivo. In vivo PARylation of DDL12E1 or DDL12E2 was performed using Arabidopsis protoplasts fed with 32P-NAD+. (G) Reduced association of DDL12E1 with PARP2 in protoplasts. Protoplasts were treated with 100 nm flg22 for 30 min before Co-IP assay. The above experiments except MS analysis were repeated twice with similar results.
PARylation of the forkhead-associated domain protein DAWDLE regulates plant immunity (2016)
Figure 7

Figure 7. DDL PARylation is required for plant innate immunity.

(A) Phenotype of plants complemented with WT DDL or DDL12E1. Four-week-old plants of two independent transgenic lines of pDDL::DDL-FLAG (W7 and W18) or pDDL::DDL12E1–FLAG (E11 and E15) were photographed. Bar=1cm. The expression of transgene was determined by an α-FLAG immunoblot (bottom). (B) Susceptibility of complementation lines to Pst DC3000 infections. Four-week-old transgenic plants were inoculated with Pst DC3000 (OD600=5x10-4) and the bacterial number was determined at 0 and 3 dpi. (C) PR1 and PR5 induction in transgenic lines. Leaves of four-week-old plants were inoculated with Pst DC3000 (OD600=0.01), and collected at 0, and 24 hpi for qRT-PCR analysis. The data in B and C are shown as mean ± SD (n=3) from three independent repeats with one-way ANOVA analysis and Tukey test (p<0.05). Different letters indicate significant differences. (D) flg22-induced callose deposition in transgenic lines. Leaves of four-week-old plants were inoculated with 500 nM flg22, and callose deposits were stained with aniline blue at 20 hpi. Number of callose deposits was quantified with Image J (mean ± SE, n=10). All experiments were repeated three times with similar results.
A human kinase yeast array to identify kinases modulating phospho-dependent protein interactions (2022)
Figure 2

Figure 2: The yeast growth phenotype is a readout for the kinase phosphotransferase activity.

A. Statistical comparison of the number of yeast growth phenotypes between kinase group and control protein group. In each strain, across all conditions, the percentage of kinases that cause a phenotype upon exogenous expression is significantly higher than the percentage of control proteins, than cause a growth phenotype upon overexpression. Data are from a single large-scale growth measurement. The boxes extend from 1st to 3rd quartile with the central band representing the median. The whiskers extend to the furthest point up to 1.5 times the interquartile range away from the nearest quartile, with any further points marked as outliers. P-values are calculated using the Mann-Whitney test.

B. Growth comparisons of selected kinases with the kinase‑dead mutant match. 24h growth curves (OD595) from liquid culture of yeast strains (W303) expressing human kinase in the presence or absence of 3.2% (v/v) DMSO. Upper panel: BUB1: BUB1 wild type, BUB1-KD: BUB1(K821M); upper middle panel: NEK6: NEK6 wild type, NEK6-KD: NEK6(K74M&K75M); lower middle panel: PKCα-ac: PKCα(A25E), PKCα-KD: PKCα(K368R); lower panel: Control with empty plasmid. Error bars represent SD from three biological replicates.

C. Human protein kinases phosphorylate large sets of yeast proteins. Upper panel: In total, 27 human protein kinases showed activity towards yeast proteins visible on western blots through enhanced reactivity with phospho-motif antibodies on yeast proteins in comparison to other human kinases, kinase‑dead mutant versions or vector control samples. Commercially available pS/T-antibodies with relatively low sequence specificities were chosen to enable detection of phosphorylation of the yeast proteome by human kinases. Lower panel: Examples of western blots with equal amount of whole yeast cell lysate from strains expressing human kinases loaded. When developed with the indicated phospho-motif recognizing antibodies, immunoreactive bands indicated phosphorylation of yeast proteins.
A human kinase yeast array to identify kinases modulating phospho-dependent protein interactions (2022)
Figure 5

Figure 5: Identification of human S/T kinases modulating the AAR2-PRP8 protein interaction

A. Schematic of phospho-dependent interaction of the spliceosomal AAR2 and PRP8. AAR2, a spliceosomal assembly protein, and PRP8 from a stable complex, that dissociates upon phosphorylation of AAR2. PPR8 is then free to bind the spliceosomal interaction partner protein BRR2 (Weber et al, 2013; Galej et al, 2013). This regulatory interaction switch is thought to be conserved between yeast and human. A protein kinase that can modulate the dissociation of AAR2 from PRP8 is unknown.

B. Superposition of the yeast Aar2 (orange, PDB entry 3SBT; (Weber et al, 2011)) 3D structure and a homology model of human AAR2 derived from HHpred (marine), based on the yeast Aar2p structure (Santos et al, 2015). The inset on the right shows a close-up view of the C-terminal domain, where serines 253 (yeast) and 284 (human) are displayed as sticks. Residues 154-170 and 318-356 of yeast Aar2 as well as residues 1-18, 170-201 and 359-384 of human AAR2 are not shown for clarity.

C. Y2H interaction experiment with human AAR2 (FL) as bait and PRP8 (aa1755-2335) as prey. Growth on selective media is abolished when AAR2 carries a S284E phospho-mimicry amino acid substitution. Mutation of second candidate phospho-site, AAR2(T356), does not influence the AAR2-PRP8 protein interaction.

D. Interaction of AAR2 and PRP8 in human cells. HA‑tagged PRP8 (aa1755-2335) was co-expressed in HEK293 cells with either wildtype AAR2, AAR2(S284A) or AAR2(S284E) mutant versions. Immunoprecipitation of PRP8 led to a co-precipitation of wild typeAAR2 and phospho-null AAR2(S284A). In contrast, the phospho-mimicry AAR2(S284E) version did not bind PRP8.

E. Results of the phospho-Y2H screen to identify kinases modulating the interaction of AAR2 and PRP8. Growth on selective media (SD5), of strains carrying AAR2 bait and PRP8 prey plasmids is reduced through co-expression of either SGK2 or CK2α1(CSNK2A1). Growth reduction is visible in a 1:10 dilution series on selective agar (SD5) in comparison to the control media (SD3). Ctrl: non-related kinases.

F. Interaction of AAR2 and PRP8 in human cells in the presences of SGK and CK2α1 kinases. Experiment as in D. Co-expression of wild type SGK2 and CK2α1 reduces the amount of AAR2 co-immunoprecipitated with RPR8.
A human kinase yeast array to identify kinases modulating phospho-dependent protein interactions (2022)
Figure 6

Figure 6: Identification of human S/T kinases modulating the ERα-14‑3‑3 phosphorylation‑dependent interaction

A. Schematic of phospho-dependent interaction of the estrogen receptor ERα with 14‑3‑3β. Phosphorylation of ERα promotes its interaction with 14‑3‑3 proteins. This interaction removes ERα from the E2-stimulated ERα dimerization and consequently inhibits ERα-DNA interactions and gene expression. A protein kinase that can modulate the binding of ERα to 14‑3‑3 proteins is unknown.

B. Results of the phospho-Y2H screen to identify kinases modulating the interaction of the ERα with 14‑3‑3β. The interaction of ERα (prey) with 14‑3‑3β (bait) is promoted by the co-expression of wildtype kinases of PAK5, CDK17, BUB1 and NEK6, visible by the yeast growth on selective media in comparison to the control (three biological replicas are shown). The coexpression of the kinase‑dead mutants of PAK5 and CDK17 also promoted the interaction of ERα and 14‑3‑3β. In contrast the kinase‑dead mutant versions of BUB1(K821M) and NEK6(K74M&K75M) did not promote the Y2H interaction.

C. Phosphorylation of ERα in mammalian cells. ERα was co-expressed with the indicated wildtype kinases or kinase‑dead versions in HEK293 cells and immunoprecipitated. Phosphorylation status of ERα was analyzed with an in-gel phosphostain (Pro-Q® Diamond phosphoprotein gel stain, Invitrogen), relative to the whole protein stain (SYPRO®Ruby protein gel stain, Invitrogen).

D. Interaction of ERα and 14‑3‑3β in a GST pull-down assay. GST‑tagged 14‑3‑3β was expressed in E. coli and immobilized on beads. PA‑tagged ERα was co-expressed with either wildtype PAK5 or NEK6, the kinase‑dead (KD) version or without a kinase in HEK293 cells. Immobilized 14‑3‑3β was used to pull PA‑tagged ERα from the HEK lysates. Co-precipitation of ERα was successful in the presence of wild kinases, but not in their absence or with kinase‑dead mutant versions.

E. Effect of NEK6 co-expression on ERα gene activation. Relative luciferase activity of ERα in the presence and absence of 1 nM E2 (17β-estradiol) was measured. A dose‑dependent reduction (ng plasmid transfected) in E2-Erα‑dependent luciferase expression was observed in HEK293T, MCF-7 and US-OS cancer cell lines. This effect was not observed with the NEK6(K74M&K75M) kinase‑dead version. Graphs represent ≥ 5 biological replicates, each derived from averaging 6 technical replicates. Error bars indicate SD across the biological replicates.
Defining the proximal interaction networks of Arf GTPases reveals a mechanism for the regulation of PLD1 and PI4KB (2022)
Figure 3

Fig. 3 PLD1 is an ARL11 and ARL14 interacting protein

(A) PLD1 is enriched by ARL11 and ARL14 miniTurboID. A graph showing proteins that are enriched with ARL11 or ARL14 compared to the vector. Result is presented as the mean of two biologically independent samples.

(B) Interaction between PLD1 and ARL11/14 is stronger than with ARF1/6, RhoA. Flag-tagged PLD1 was co-expressed with HA-tagged GTP-bound mutants of small GTPases in HEK293A cells as indicated. PLD1 was immunoprecipitated with Flag beads, and the co-precipitated small GTPases were detected with an HA antibody. The result is a representative of three biologically independent experiments.

(C) ARL11/14 co-localize with PLD1. Flag-tagged PLD1 was co-expressed with HA-tagged GTP-bound mutants of small GTPases in HEK293A cells as indicated. After 24 h, cells were fixed with 4% paraformaldehyde, followed by incubation with anti-Flag and anti-HA primary antibodies. The Flag and HA-antibodies were detected by anti-mouse and anti-rabbit secondary antibodies, respectively. DAPI (blue) was used for DNA staining. Scale bars: 10 μm. The result is a representative of two biologically independent experiments.

(D) Mander's overlap coefficient of (C). Mander's overlap coefficient calculated using JACoP plugin in ImageJ. Mean± Standard Deviation (SD); n= 6 technically independent samples.

(E) PLD1 preferentially binds to the GTP-form, not the GDP-form, of ARL11. Flag-tagged PLD1 was co-expressed with HA-tagged ARL11Q67L (GTP-form) or ARL11T26N (GDP-form) mutant in HEK293A cells. The result is a representative of two biologically independent experiments.

(F) The subcellular co-localization of ARL11 with PLD1 is GTP-dependent. HA-tagged GTP-bound ARL11Q67L, wild-type ARL11 or GDP-bound ARL11T26N was co-expressed with Flag-tagged PLD1. After 24 h, cells were fixed, followed by incubation with anti-Flag and anti-HA primary antibodies. DAPI (blue) was used for DNA staining. Scale bars: 10 μm. The result is a representative of three biologically independent experiments.

(G) Mander's overlap coefficient of (F). Mander's overlap coefficient calculated using JACoP plugin in ImageJ. Mean± SD; One way ANOVA; n= 6 technically independent samples; *P < 0.05.
Defining the proximal interaction networks of Arf GTPases reveals a mechanism for the regulation of PLD1 and PI4KB (2022)
Figure 4

Fig. 4 The loop region of PLD1 is required for interaction with and activation by ARL11

(A) Schematic representation of the full-length PLD1 and various truncations. ∆N, deletion of the N-terminal PX and PH lipid-binding domain (1-329aa); ∆M, deletion of the central catalytic domain (330-967aa); ∆C, deletion of the C-terminal domain (968-1074aa); ∆HDK1, deletion of the HDK1 domain (330-500aa); ∆Loop, deletion of the Loop region (501-642aa); ∆HKD2, deletion of the HDK2 domain (643-967aa). " + " denotes positive interaction with ARL11, and " - " denotes no interaction.

(B) The loop region of PLD1 is required for the interaction between ARL11 and PLD1. Flag-tagged full-length or truncations of PLD1 was co-expressed with HA-tagged ARL11Q67L, PLD1 was immunoprecipitated with Flag beads, and ARL11 that associated with it was detected with an HA antibody. The result is a representative of two biologically independent experiments.

(C) Deletion of the loop region disrupts the PLD1 and ARL11 co-localization. Flag-tagged full-length or ∆Loop truncation of PLD1 was co-expressed with HA-tagged ARL11Q67L. After 24 h, cells were fixed with 4% paraformaldehyde, followed by incubation with anti-Flag and anti-HA primary antibodies. DAPI (blue) was used for DNA staining. Scale bars: 10 μm. The result is a representative of three biologically independent experiments.

(D) Mander's overlap coefficient of (J). Mander's overlap coefficient calculated using JACoP plugin in ImageJ. Mean± SD; two-tailed Student's t-test; n= 6 technically independent samples; *P < 0.05.

(E) ARL11 and ARL14 stimulation of PLD1 activity is dependent on the loop region. The in vitro activity assay was performed by using PLD1 protein of catalytic domain (PLD1 ∆N) with or without the loop region. Details were described in the method part. Data are presented as mean± SD of three biologically independent samples. One way ANOVA, n.s denotes not significant, *P < 0.05.

(F) A model of PLD1 activation by GTPases. In contrast to ARF1/6 and RHOA, ARL11/14 stimulate PLD1 activity in a manner dependent on the loop region.