EMBO/soda-nel huggingface dataset:
The DNA Damage Response acts as a safeguard against harmful DNA-RNA hybrids of different origins (2019)
Figure 3

Figure 3. DNA-RNA hybrids are a source of DNA breaks in DDR-deficient cells. (A) Representative images of single-cell alkaline gel electrophoresis (comet assay) of HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). (B) Comet tail moment from single-cell alkaline gel electrophoresis (comet assay) of HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). More than 250 total cells were considered. The mean ± SEM of the median from at least three independent experiments is shown, except for RAD1, RAD17 and UBE2B (n=2). *p<0.05, **p<0.01, ***p<0.001 (one-tailed unpaired t-test). Black stars denote significant increases whereas red stars denote significant decreases.
The DNA Damage Response acts as a safeguard against harmful DNA-RNA hybrids of different origins (2019)
Figure 4

Figure 4. DDR-deficiencies lead to DNA-RNA hybrid-dependent accumulation of histone H3S10-P and H3K9me2 chromatin marks. (A) Representative images and percentage of HeLa cells with more than 5 H3S10-P foci after transfection with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). Mitotic cells were excluded for the analysis by DAPI staining. More than 300 total cells were considered. Data represent mean ± SEM from three independent experiments. (B) Representative images and H3K9me2 nuclear signal intensity of HeLa cells transfected with the indicated siRNAs and either pEGFP-C1 (RNH1-) or pEGFP-M27 (RNH1+). At least three experiments were performed. The median of each population in a representative experiment with at least 100 cells per condition is shown. Boxes and whiskers indicate 25-75 and 10-90 percentiles, respectively.

*p<0.05, **p<0.01, ***p<0.001 (one-tailed paired t-test).

Black stars denote significant increases whereas red stars denote significant decreases.
The DNA Damage Response acts as a safeguard against harmful DNA-RNA hybrids of different origins (2019)
Figure 5

Figure 5. Differential effects on replication fork progression upon depletion of DDR factors. (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). (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.
The DNA Damage Response acts as a safeguard against harmful DNA-RNA hybrids of different origins (2019)
Figure 7

Figure 7. Integrative view and model showing how the lack of DNA Damage Checkpoint and Postreplicative Repair factors can affect DNA-RNA hybrid homeostasis. (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 from Fig 2A. ***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 from Fig 3B. ***p<0.001, **p<0.01, *p<0.05 (paired t-test). Fork stalling represents the median ± SEM of all the fork asymmetry data from Fig 5B. ***p<0.001, **p<0.01, *p<0.05 (Mann-Whitney U test). Black stars denote significant increases whereas red stars denote significant decreases. (B) A model to show that spontaneous DNA-RNA hybrids impairing replication fork progression would require the 9-1-1/ATR/CHK1 for dissolution. Additionally, unrepaired DSBs (accumulated as a consequence of ATM/CHK2 depletion) and post-replicative ssDNA gaps (present in PRR-defective cells) could favor DNA-RNA hybrid accumulation without stalling replication forks.

EXPANDED VIEW FIGURE LEGENDS
Genome-wide prediction of synthetic rescue mediators of resistance to targeted and immunotherapy (2019)
Figure 6

Figure 6. (a) SR predicts resistance to PD1/PDL1 and CTLA4 blockade in patients. Cross-validation accuracy of SR-based supervised predictors in predicting resistance to PD1 (Prat et. al. & Hugo et. al.) and CTLA4 blockade (Van Allen et al.), reported in terms of the corresponding Receiver Operating (ROC) curves. Expression of the predicted rescuer genes of PD1 (CTLA4) was used to train an SVM supervised predictor of PD1 (CTLA4) blockade. For comparison, we also display the ROC curves of supervised predictors trained on the expression of all genes (shown as "All") and on the expression of genes selected randomly and controlled for the number of rescuers predicted (shown as "Random"). (b-d) The transcriptomic alterations of rescuer genes post PD1/PDL1 and CTLA4 blockade in patient tumor biopsies: Their post (vs. pre) treatment expression changes of DU/DD rescuers after anti-PD1 (b), anti-CTLA4 (c), and PD1 + CTLA4 combination therapies (d). Each panel displays the expression fold change of each predicted rescuer gene (rows) for different tumor samples (columns) and the P-value of over-all paired Wilcoxon test of the expression changes observed in paired samples. Significantly altered up/down-regulated genes are marked by (*). Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti-PD1 blockade in mice models.
A metabolic switch toward lipid use in glycolytic muscle is an early pathologic event in a mouse model of amyotrophic lateral sclerosis. (2015)
Figure 1

65-day-old SOD1G86R mice have improved performance during endurance exercise

A–F (A–C) Exercise performance on a treadmill apparatus using an intense exercise (incremental speed paradigm) that relies solely on anaerobic metabolism. (D–F) Exercise performance on a treadmill apparatus using a moderate exercise (constant sub-maximal speed) that relies on the recruitment of aerobic metabolism and that is representative of endurance exercise. (A, D) Kaplan–Meier curves representing values corresponding to the percentage of mice still running at a given time point. (B, E) Median values of the time that mice ran are represented in the min to max graphical representation; 15.5 min for wild-type (WT) and 14.33 min for SOD1G86R in (B); 59.33 min for WT and 65.43 min for SOD1G86R in (E). (C, F) Mean distance run ± SEM by WT and SOD1G86R mice in (C) and by WT and SOD1G86R mice in (F).

Data information: G86R versus WT, P = 0.017 in (A), **P = 0.0078 in (B), **P = 0.008 in (C) and P = 0.036 in (D) (Mantel–Cox test), **P = 0.0078 in (E), and **P = 0.0061 in (F) (Student's t-test), n = 10 and 13 for WT and SOD1G86R, respectively.
A metabolic switch toward lipid use in glycolytic muscle is an early pathologic event in a mouse model of amyotrophic lateral sclerosis. (2015)
Figure 2

Decreased grip strength, increased denervation, and increased expression of denervation and atrophy markers throughout disease progression in SOD1G86R mice

Grip test was performed to measure mouse muscle strength at different time points during disease progression. Graph represents the mean of percentage from initial force measured at 11 weeks ± SEM. At 13 weeks, *P = 0.03; at 14 weeks, P = 0.06; at 15 weeks, *P = 0.04 (n = 8 for WT and SOD1G86R, respectively, at 11–15 weeks, two-way ANOVA followed by Fisher's LSD post hoc test).

Electromyography (EMG) recordings were performed weekly during disease development, starting from 9 weeks of age. Spontaneous electrical activity was considered as positive EMG only for peak-to-peak amplitudes > 50 μV. Left: The percentage of mice presenting with positive EMG in each age group are represented. Right: Representative examples of negative (EMG−) and positive (EMG+) electromyography profiles (n = 10 for WT and SOD1G86R, respectively, at 9–13 weeks and n = 8 and 7 for WT and SOD1G86R, respectively, at 15 weeks).

Relative mRNA levels of denervation markers AChR (subunits α and γ) and MuSK were evaluated by qPCR at the indicated ages (65 and 105 days) in tibialis anterior (upper panels) and soleus (lower panels) of WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from age-matched WT (n > 5). ***P-values versus WT: < 0.0001 for AChRα, AChRγ, and MuSK in tibialis anterior; and **P-values versus WT: 0.001 for AChRα, and ***P-values versus WT: 0.0007 for AChRγ, 0.0002 for MuSK in soleus (n=7 and 8 for WT and SOD1G86R, respectively, at 65 days; n = 5 and 6 for WT and SOD1G86R, respectively, at 105 days; two-way ANOVA followed by Fisher's LSD post hoc test).

Relative mRNA levels of muscle atrophy markers MuRF1 and Atg-1 were measured by qPCR at the indicated ages (65 and 105 days) in tibialis anterior (upper panels) and soleus (lower panels). Graphs represent mean fold change ± SEM from age-matched WT. ***P-values versus WT: < 0.0001 for MuRF1 and Atg-1 in tibialis anterior; and **P-values versus WT: 0.007 for MuRF1 and P = 0.006 for Atg-1 in soleus (n = 7 and 8 for WT and SOD1G86R, respectively, at 65 days, n = 5 and 6 for WT and SOD1G86R, respectively, at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test).
A metabolic switch toward lipid use in glycolytic muscle is an early pathologic event in a mouse model of amyotrophic lateral sclerosis. (2015)
Figure 3

Phosphofructokinase 1 is inhibited in glycolytic muscle and glucose is rerouted toward glycogen stores

Enzymatic activity of phosphofructokinase in whole tibialis anterior muscle cytosolic homogenates is expressed as mean fold change ± SEM from age-matched WT at 65 days of age *P = 0.016 (WT n = 6, SOD1G86R
n = 7) and 105 days of age ***P < 0.0001 (n = 5/genotype), two-way ANOVA followed by Fisher's LSD post hoc test. Data shown are representative of two independent experiments having similar results.

Pyruvate was measured in whole tibialis anterior muscle tissue homogenates. The mean fold change ± SEM compared to age-matched WT are represented with *P = 0.019 at 65 days of age (n = 5/genotype) and *P = 0.041 at 105 days of age (n = 4/genotype), two-way ANOVA followed by Fisher's LSD post hoc test. Data shown are representative of two independent experiments having similar results.

Left: Representative Western blot showing glycogen synthase and phosphorylated glycogen synthase levels. Right: Quantification represents the mean ratio of optical densities of glycogen synthase/P-glycogen synthase ± SEM. At 65 days of age n = 6/genotype, *P = 0.0162, and at 105 days of age n = 5/genotype, *P = 0.0163, two-way ANOVA followed by Fisher's LSD post hoc test.

Left: Representative microphotographs of PAS staining from WT and SOD1G86R
tibialis anterior cross-sections at 65 and 105 days of age showing glycogen-negative (light pink) and glycogen-positive (dark pink) fibers. Scale bar: 200 μm. Right: Quantification of glycogen-negative (−) and glycogen-positive (+) fibers at 65 and 105 days of age. For (−) fibers: **P-values versus WT: 0.0026 at 65 days and 0.0074 at 105 days, for (+) fibers: ##P-values versus WT: 0.0025 at 65 days and 0.0073 at 105 days (n = 5/genotype at 65 days and n = 4/genotype at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test).
A metabolic switch toward lipid use in glycolytic muscle is an early pathologic event in a mouse model of amyotrophic lateral sclerosis. (2015)
Figure 4

Altered fuel preference in glycolytic muscle of 65-day-old asymptomatic SOD1G86R mice

Relative mRNA levels of Pdk4 and Pdk2 evaluated by qPCR at the indicated ages in tibialis anterior of WT and SOD1G86R mice. Graphs represent mean fold change ± from age-matched WT. Pdk4: **P-values versus WT: 0.014 at 65 days and 0.018 at 105 days; Pdk2: ***P-value versus WT < 0.0001 at 105 days. n = 7 and 8 for WT and SOD1G86R, respectively, at 65 days; n = 5/genotype at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test.

Relative mRNA levels of genes involved in lipid handling evaluated by qPCR at the indicated ages in tibialis anterior of WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from age-matched WT. P-values versus WT: Lpl **P = 0.0009, Cd36 *P = 0.051, Acsf2 ***P < 0.0001 at 65 days, **P = 0.0059 at 105 days and Cpt-1β **P = 0.01 at 65 days and *P = 0.02 at 105 days (n = 6 and 7 for WT and SOD1G86R, respectively, at 65 days; n = 10 and 8 for WT and SOD1G86R, respectively, at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test).

Relative levels of (left) ATP and (right) NADH and NAD+ measured in total tibialis anterior homogenates of WT and SOD1G86R mice. Left: Graphs represent mean fold change ± SEM in ATP from age-matched WT. **P = 0.002 (n = 9 and 12 for WT and SOD1G86R, respectively, at 65 days; n = 10/genotype at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test). Right: The amounts of NADH and NAD+ relative to protein content in whole tibialis anterior homogenates are represented as mean ± SEM. *P = 0.027 and **P = 0.001 (n = 5, Student's t-test).
A metabolic switch toward lipid use in glycolytic muscle is an early pathologic event in a mouse model of amyotrophic lateral sclerosis. (2015)
Figure 5

Transcription factors, PGC-1α, and citrate synthase are differentially regulated in SOD1G86R mice

A–D Relative mRNA levels of (A) Pparß/δ, (B) Foxo1, (C) citrate synthase, and (D) Pgc-1α were evaluated by qPCR at the indicated ages in tibialis anterior of WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from age-matched WT. P-values versus WT: Pparβ/δ **P = 0.0016 at 65 days; Foxo1 ***P = 0.0001 at 105 days, citrate synthase ***P < 0.0001 at 105 days, Pgc-1α *P = 0.0104 at 105 days (n = 7 and 8 for WT and SOD1G86R, respectively, at 65 days; n = 5/genotype at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test).

E Representative Western blot of PGC-1α and respective actin that was used for normalization. PGC-1α protein levels in tibialis anterior muscle tissue are represented as mean fold change ± SEM from age-matched WT. **P-values versus WT: 0.02 (n = 6/genotype at 65 days; n = 5/genotype at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test).

F Relative mRNA levels of ERRα, Mfn2, and Nrf1 were evaluated by qPCR at the indicated ages in tibialis anterior of WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from age-matched WT. P-values versus WT: ERRα ***P < 0.0001, Mfn2 **P = 0.002 and Nrf1 **P = 0.009 (n = 6–7 and 7–8 for WT and SOD1G86R, respectively, at 65 days; n = 5 and 6 for WT and SOD1G86R, respectively, at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test).
A metabolic switch toward lipid use in glycolytic muscle is an early pathologic event in a mouse model of amyotrophic lateral sclerosis. (2015)
Figure 6

Altered mitochondrial function in glycolytic muscle of SOD1G86R mice

Mitochondrial DNA (mtDNA) was quantified using qPCR. Relative mtDNA levels are expressed as the ratio between mitochondrial-encoded gene Cox1 and the nuclear-encoded gene cyclophilin A. Graphs represent mean fold change ± SEM from age-matched WT for tibialis anterior (left panel) and soleus (right panel) of WT and SOD1G86R mice. ***P < 0.0001 (n = 5 and 4 for WT and SOD1G86R, respectively, at 65 days; n = 6/genotype at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test).

Relative mRNA levels of Gpx1 were measured by qPCR at the indicated ages in tibialis anterior (left panel) and soleus (right panel) of WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from age-matched WT. ***P < 0.0001 (n = 7 and 8 for WT and SOD1G86R, respectively, at 65 days; n = 9 and 8 for WT and SOD1G86R, respectively, at 105 days, two-way ANOVA followed by Fisher's LSD post hoc test).
A metabolic switch toward lipid use in glycolytic muscle is an early pathologic event in a mouse model of amyotrophic lateral sclerosis. (2015)
Figure 7

DCA treatment had beneficial effects on metabolism and mitochondrial function of SOD1G86R mice

A–I Relative mRNA levels of (A) Pdk4, (B) Pparβ/δ, (C) Foxo1, (D) Pfk1, (E) Acsf2, (F) citrate synthase, (G) PGC-1α, (H) Mfn2, and (I) Gpx1 were evaluated by qPCR in tibialis anterior of control (CT) or DCA-treated (DCA) WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from CT WT group. P-values versus WT: Pdk4 ***P = 0.002 and ##P = 0.0039, Pparβ/δ
#P = 0.0178, Foxo1 **P = 0.0033 and ##P = 0.0038, Pfk1 ***P = 0.0002 and ##P = 0.0080, Acsf2 *P = 0.0437, citrate synthase
###P = 0.0004, Pgc-1α **P = 0.0084 and ###P = 0.0009, Mfn2 *P = 0.0272, $P = 0.0145 and ###P = 0.0002 and Gpx1 ***P < 0.0001 and ###P < 0.0001 (n = 9/genotype in CT groups, n = 9 and 8 for WT and SOD1G86R, respectively, in DCA group, two-way ANOVA followed by Fisher's LSD post hoc test).
A metabolic switch toward lipid use in glycolytic muscle is an early pathologic event in a mouse model of amyotrophic lateral sclerosis. (2015)
Figure 8

DCA treatment had protective effects on muscle strength and prevented the expression denervation markers

Grip strength is represented as mean of percent from T0 for each experimental group ± SEM. ##P = 0.0045 and ***P = 0.0003 (n = 9/genotype in CT groups, n = 9 and 8 for WT and SOD1G86R, respectively, in DCA group, two-way ANOVA followed by Fisher's LSD post hoc test).

Relative mRNA levels of muscular atrophy markers Murf1 and Atg-1 were measured by qPCR in tibialis anterior of control (CT) or DCA-treated (DCA) WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from CT WT group. **P = 0.0079 and ***P = 0.0018 (n = 9/genotype in CT groups, n = 9 and 8 for WT and SOD1G86R, respectively, in DCA group, two-way ANOVA followed by Fisher's LSD post hoc test).

Relative mRNA levels of denervation markers AChRα, AChRγ, and MuSK were measured by qPCR in tibialis anterior of control (CT) or DCA-treated (DCA) WT and SOD1G86R mice. Graphs represent mean fold change ± SEM from CT WT group. **P = 0.015 for AChRα, #P = 0.028 and **P = 0.074 for AChRγ, #P = 0.042 and **P = 0.0029 for MuSK (n = 9/genotype in CT groups, n = 9 and 8 for WT and SOD1G86R, respectively, in DCA group, two-way ANOVA followed by Fisher's LSD post hoc test).
KSHV‐TK is a tyrosine kinase that disrupts focal adhesions and induces Rho‐mediated cell contraction (2014)
Figure 1

KSHV‐TK induces cell contraction and blebbing

Live cell imaging reveals that expression of GFP‐KSHV‐TK, but not GFP induces contraction of HeLa cells (see Supplementary Movies S1 and S2). The time from the start of imaging is indicated above each panel, and the scale bars represent 18 μm. Quantification of GFP‐KSHV‐TK‐induced cell contraction. The graphs show cell area (μm2) of cells expressing the indicated protein, and error bars represent SEM from three independent experiments in which n = 224 for GFP and n = 203 for GFP‐KSHV‐TK. ***P 0.001.
Phase images taken from Supplementary Movie S3 illustrating GFP‐KSHV‐TK induced blebbing of contracted cells. The time from the start of imaging is indicated above each panel, and the scale bars represent 18 μm.
Scanning electron micrographs of HeLa cells expressing GFP or GFP‐KSHV‐TK.
Immunofluorescence images illustrating the association of GFP‐KSHV‐TK, but not GFP, with actin stress fibres. Scale bar, 10 μm.
KSHV‐TK is a tyrosine kinase that disrupts focal adhesions and induces Rho‐mediated cell contraction (2014)
Figure 2

KSHV‐TK induces disruption of the integrity of focal adhesions

Co‐expression of GFP‐RhoN19 but not GFP inhibits RFP‐KSHV‐TK‐induced cell contraction. Graphs represent cell area (μm2) of KSHV‐TK, RhoN19 or KSHV‐TK and RhoN19 expressing cells. Error bars represent SEM from 3 independent experiments, n = 204 for KSHV‐TK, n = 223 for RhoN19 and n = 189 for KSHV‐TK and RhoN19.
GFP‐KSHV‐TK‐induced cell contraction does not occur in cells treated with the ROCK and myosin II inhibitors Y‐27632 and blebbistatin, respectively. Error bars represent SEM from three independent experiments, n = 213 for DMSO, n = 189 for Y‐27632 and n = 245. ***P 0.001.
Immunoblot analysis of rhotekin pull‐down (P.D.) assays reveals that GFP‐KSHV‐TK increases the level of GTP‐bound RhoA (GTP‐RhoA). Actin is included as a loading control, and the input lysates are indicated.
GFP‐KSHV‐TK but not GFP induces the loss of phosphotyrosine, phospho‐FAK Y925 and phospho‐paxillin Y31 epitopes from focal adhesions.

Data information: All scale bars represent 20 μm.
KSHV‐TK is a tyrosine kinase that disrupts focal adhesions and induces Rho‐mediated cell contraction (2014)
Figure 3

Virally expressed KSHV‐TK induces cell contraction and loss of focal adhesions

A schematic illustrating the BAC strategy used to generate a recombinant MuHV‐4 virus (MuHV‐4 [g‐KSHV‐TK]) in which the endogenous MuHV‐4 TK is exchanged for GFP‐KSHV‐TK.
Immunoblot analysis of BHK cells infected with MuHV‐4 and MuHV‐4 [g‐KSHV‐TK] or uninfected cells using antibodies against phosphotyrosine (p‐Tyr), GFP, MuHV‐4 TK, ORF17 (capsid) and glycoprotein N.
Immunofluorescence analysis of MuHV‐4‐ and MuHV‐4 [g‐KSHV‐TK]‐infected cells with the indicated antibodies reveals GFP‐KSHV‐TK expressed in the context of lytic infection (MuHV‐4 [g‐KSHV‐TK]) induces the loss of phospho‐FAK Y925 and paxillin Y31 from focal adhesions. White arrowheads indicate examples of infected cells expressing GFP‐KSHV‐TK or GFP (indicating WT MuHV‐4 infection, BAC+ virus).
Immunofluorescence analysis reveals that GFP‐KSHV‐TK expressed in the context of lytic infection (MuHV‐4 [g‐KSHV‐TK]) does not alter focal adhesion integrity (paxillin) in the presence of the ROCK inhibitor Y‐27632.
Immunofluorescence analysis of paxillin in the lytically induced rKSHV.219/Vero cell line in the presence or absence of Y‐27632.

Data information: All scale bars represent 20 μm.
KSHV‐TK is a tyrosine kinase that disrupts focal adhesions and induces Rho‐mediated cell contraction (2014)
Figure 4

The kinase activity of KSHV‐TK is required to disrupt focal adhesions

Cellular localisation and immunoblot analysis of phosphotyrosine (upper panel) and GFP (lower panel) in cells expressing wild‐type or kinase‐dead GFP‐KSHV‐TK.
Scanning electron micrographs of HeLa cells expressing GFP or GFP‐KSHV‐TK‐DEAD.
Immunofluorescence analysis of HeLa cells expressing GFP‐KSHV‐TK‐DEAD (white arrowheads) with the indicated phospho antibodies.
Immunofluorescence images of the localisation of FAK and zyxin in HeLa cells expressing GFP‐KSHV‐TK‐DEAD (white arrowheads).
Purified recombinant GST‐EBV‐TK, GST‐MuHV‐4 and GST‐KSHV‐TK immunoblotted for phosphotyrosine and GST following a sequential dephosphorylation and kinase reaction.
Purified recombinant GST‐KSHV‐TK and GST‐KSHV‐TK‐KINASE‐DEAD immunoblotted for phosphotyrosine and GST.
Immunoblot analysis demonstrating that the N‐terminus of KSHV‐TK is only auto‐phosphorylated by the kinase domain in cis.

Data information: All scale bars represent 20 μm.
KSHV‐TK is a tyrosine kinase that disrupts focal adhesions and induces Rho‐mediated cell contraction (2014)
Figure 5

The activity of KSHV‐TK depends on the phosphorylation of tyrosines 65 and 85

A Immunoblot analysis of N‐terminal deletions of KSHV‐TK demonstrates that the first 100 amino acids are required for tyrosine phosphorylation.
B Immunoblot analysis reveals that mutation of tyrosines 65, 85 and 120 ablates tyrosine phosphorylation of the KSHV‐TK.
C–E Immunofluorescence analysis with the indicated antibodies reveals that mutation of tyrosines 65, 85 and 120 to phenylalanine abrogates the ability of GFP‐KSHV‐TK to induce loss of phosphotyrosine epitopes, phospho‐paxillin and phospho‐FAK from focal adhesions.
F Immunoblot analysis of level of GTP‐bound and total RhoA in cells expressing the indicated GFP‐tagged KSHV‐TK proteins.
G Immunofluorescence analysis reveals that localisation of paxillin at focal adhesions is unaffected by KSHV‐TK‐Y3F expressed in the context of lytic infection.

Data information: Scale bars represent 20 μm.