EMBO/soda-nel huggingface dataset:
Reduction of Mitochondrial Iron Alleviates Cardiac Damage During Injury (2016)
Figure 5

Figure 5. ABCB8 transgenic mice demonstrate reduced acute cellular injury and decreased left ventricular fibrosis and cardiac stress 28 days after injury.

A. Representative H&E staining of peri-infarct area demonstrated reduced cellular injury in ABCB8 TG mice after I/R. Scale bar = 115μm.

B. Representative H&E and Masson Trichrome (MT) staining in mouse heart 28 days after indicated procedure. Scale bar = 1100μm.

C. Quantification of tissue fibrosis in ABCB8 NTG and TG mice subjected to I/R. * P=0.047 with two-tailed unpaired T-test. 2-3 sections from each mice were quantified; N=4 mice for NTG and N=6 mice for TG.

D. Nppa expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.006 NTG-sham vs. NTG-I/R. * P=0.015 NTG-I/R vs. TG-I/R. N=5 mice for NTG-sham and TG-I/R, N=6 mice for TG-sham, and N=4 mice for NTG-I/R.

E. Nppb expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.001 NTG-sham vs. NTG-I/R. * P=0.004 NTG-I/R vs. TG-I/R. N=5 mice for NTG-sham and TG-I/R, N=6 mice for TG-sham, and N=4 mice for NTG-I/R.

F. Myh7 expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.002 NTG-sham vs. NTG-I/R. * P=0.011 NTG-I/R vs. TG-I/R. N=5 mice for NTG-sham and TG-I/R, N=6 mice for TG-sham, and N=4 mice for NTG-I/R. All data are expressed as mean ± SEM. N.S. = not signigicant.
Reduction of Mitochondrial Iron Alleviates Cardiac Damage During Injury (2016)
Figure 6

Figure 6. Pharmacological modulation of mitochondrial iron protects against cellular damage after I/R.

A. Mitochondrial non-heme iron in wild-type mice treated with vehicle control or indicated iron chelator for 7 days. ANOVA followed by post-hoc Tukey test was performed. * P=0.03. N=4 mice for PBS and DFO, and N=5 mice for BPD.

B. Cytosolic non-heme iron in wild-type mice treated with vehicle control or indicated iron chelator for 7 days. ANOVA followed by post-hoc Tukey test was performed. * P=5E-4 PBS vs. DFO. * P=0.01 DFO vs. BPD. P=0.06 PBS vs. DFO. N=4 mice for PBS and DFO, and N=5 mice for BPD.

C. Nuclear non-heme iron in wild-type mice treated with vehicle control or indicated iron chelator for 7 days. N=4 mice for PBS and DFO and N=5 mice for BPD.

D. Cardiac function in wild-type mice treated with vehicle control or indicated iron chelator for 7 days. N=5 mice for each group.

E. Cardiac function of chelator-treated mice after I/R. ANOVA followed by post-hoc Tukey test was performed for each time point. * P<0.0001 compared to PBS-I/R group at the same time point. # P<0.0001 compared to PBS-sham group at the same time point. Exact P-values are included in Appendix Table S3. N=5 mice PBS-sham and BPD-I/R, N=6 mice for all other groups.

F. Representative Hematoxylin & Eosin (H&E) and Masson Trichrome (MT) staining of heart sections in mice with indicated chelator treatment undergone sham or I/R. Scale bar = 1100μm. Bar graph represent quantification of tissue fibrosis. ANOVA followed by post-hoc Tukey test was performed. * P=0.048. 2-3 sections from each mice were quantified, N=4 mice for PBS, N=5 mice for DFO and N=6 mice for BPD.

G. Nppa expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.002 PBS-sham vs. PBS-I/R. * P=0.02 DFO-sham vs. DFO I/R. * P=0.007 PBS-I/R vs. BPD I/R. N=6 mice for PBS-sham and DFO-sham, N=4 mice for all other groups.

H. Nppb expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P= 0.0007 PBS-sham vs. PBS-I/R. * P= 0.014 DFO-sham vs. DFO I/R. * P= 0.003 PBS-I/R vs. BPD I/R. N=6 mice for PBS-sham and DFO-sham, N=4 mice for all other groups.

I. Myh7 expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.0001 PBS-sham vs. PBS-I/R. * P=0.038 DFO-sham vs. DFO I/R. * P=0.006 PBS-I/R vs. BPD I/R. N=6 mice for PBS-sham and DFO-sham, N=4 mice for all other groups. All data are expressed as mean ± SEM. N.S. = not signigicant.
Reduction of Mitochondrial Iron Alleviates Cardiac Damage During Injury (2016)
Figure 7

Figure 7. A decrease in mitochondrial iron at baseline protects against the development of cardiomyopathy in inducible cardiac-specific ABCB8 knockout mice.

A. Cardiac mitochondrial iron in ABCB8 KO and WT mice with indicated treatment harvested 4 weeks after the tamoxifen treatment. ANOVA followed by post-hoc Tukey test was performed. * P=0.02 WT-vehicle vs. WT-BPD. * P= 0.001 WT-vehicle vs. ABCB8 KO-vehicle. * P=0.004 ABCB8 KO-vehicle vs. ABCB8 KO-BPD. N=8 for WT-vehicle and N=7 for all other groups.

B. Ejection fraction in ABCB8 KO and WT mice with indicated treatment 4 weeks after gene knockout. ANOVA followed by post-hoc Tukey test was performed. * P<1E-8 WT-vehicle vs. AKBCB8 KO-vehicle. * P<1E-8 ABCB8-vehicle vs. AKBCB8 KO-BPD. N=8 for WT-vehicle and N=7 for all other groups.

C. Fractional shortening in ABCB8 KO and WT mice with indicated treatment 4 weeks after gene knockout. ANOVA followed by post-hoc Tukey test was performed. * P<1E-8 WT-vehicle vs. AKBCB8 KO-vehicle. * P<1E-8 ABCB8-vehicle vs. AKBCB8 KO-BPD. N=8 for WT-vehicle and N=7 for all other groups.

D. Nppa expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.017 WT-vehicle vs. AKBCB8 KO-vehicle. * P=0.01 ABCB8-vehicle vs. AKBCB8 KO-BPD. N=8 for WT-vehicle and N=7 for all other groups.

E. Nppb expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.001 WT-vehicle vs. AKBCB8 KO-vehicle. * P=0.0003 ABCB8-vehicle vs. AKBCB8 KO-BPD. N=8 for WT-vehicle and N=7 for all other groups.

F. Myh7 expression in mice subjected to sham or I/R procedure. ANOVA followed by post-hoc Tukey test was performed. * P=0.011 WT-vehicle vs. AKBCB8 KO-vehicle. * P=0.045 ABCB8-vehicle vs. AKBCB8 KO-BPD. N=8 for WT-vehicle and N=7 for all other groups. All data are expressed as mean ± SEM. N.S. = not signigicant.
Reduction of Mitochondrial Iron Alleviates Cardiac Damage During Injury (2016)
Figure 8

Figure 8. Modulation of mitochondrial iron influences the production of mitochondrial ROS.

A. Representative western blot demonstrating ABCB8 overexpression in H9c2 cells. EV = empty vector. N=3 independent samples for each group.

B. Complex I ROS production in mitochondria with ABCB8 downregulation with or without rotenone. ANOVA followed by post-hoc Tukey test was performed. N= 6 independent samples for each group.

C. Complex III ROS production in mitochondria with ABCB8 downregulation with or without antimycin A. ANOVA followed by post-hoc Tukey test was performed. N= 6 independent samples for each group.

D. Complex I ROS production in mitochondria with ABCB8 overexpression with or without rotenone. EV= empty vector. ANOVA followed by post-hoc Tukey test was performed. N=9 independent samples for EV-baseline and N=8 independent samples for the other groups.

E. Complex III ROS production in mitochondria with ABCB8 overexpression with or without rotenone. EV= empty vector. ANOVA followed by post-hoc Tukey test was performed. N=9 independent samples for EV-baseline and N=8 independent samples for the other groups.

F. Mitochondrial ROS in H9c2 cells with various treatments with or without oxidative stress. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=2E-8 PBS-PBS vs. PBS-H2O2. * P=2E-8 DFO- H2O2 vs. BPD-H2O2.

G. Mitochondrial membrane potential as measured by TMRE intensity in H9c2 cells treated with indicated iron chelator with or without oxidative stress. N= 6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=1E-8 PBS-PBS vs. PBS-H2O2. * P=1.8E-8 DFO-PBS vs. DFO-H2O2.

H. The levels of lipid peroxidation products in ABCB8 KO mice with or without chelator treatment. N=4 mice for WT-vehicle, N=6 mice for ABCB8 KO-vehicle, and N=5 mice for all other groups. ANOVA followed by post-hoc Tukey test was performed. * P=0.004 WT-vehicle vs. ABCB8 KO-vehicle. * P=1E-6 ABCB8 KO-vehicle vs. ABCB8 KO-BPD.

I. Lipid peroxidation products in ABCB8 TG and NTG mice two days after I/R. N=6 mice for NTG-sham, and N=4 mice for the other groups. ANOVA followed by post-hoc Tukey test was performed. * P=0.024 NTG-sham vs. NTG-I/R. * P=0.016 NTG-I/R vs. TG-I/R. All data are expressed as mean ± SEM. N.S. = not signigicant.
Reduction of Mitochondrial Iron Alleviates Cardiac Damage During Injury (2016)
Figure 9

Figure 9. Modulation of mitochondrial iron preserves the activity of mitochondrial complexes after oxidative stress.

A. Mitochondrial aconitase activity in H9c2 cells with or without ABCB8 overexpression and treated with or without H2O2. N=4 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.0004 EV-PBS vs. EV-H2O2. * P=0.0001 EV-H2O2 vs. ABCB8-H2O2.

B. Complex I activity in H9c2 cells with or without ABCB8 overexpression and treated with or without H2O2. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.0005 EV-PBS vs. EV-H2O2. * P=0.00001 EV-H2O2 vs. ABCB8-H2O2.

C. Complex II activity in H9c2 cells with or without ABCB8 overexpression and treated with or without H2O2. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.001 EV-PBS vs. EV-H2O2. * P=0.001 EV-H2O2 vs. ABCB8-H2O2.

D. Complex IV activity in H9c2 cells with or without ABCB8 overexpression and treated with or without H2O2. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.0008 EV-PBS vs. EV-H2O2. * P=0.0006 EV-H2O2 vs. ABCB8-H2O2.

E. Mitochondrial aconitase activity in H9c2 cells treated with indicated iron chelators with or without H2O2. N=4 independent samples in each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.036 PBS-PBS vs. PBS-H2O2. * P=0.0047 DFO-PBS vs. DFO-H2O2. * P=0.035 PBS- H2O2 vs. BPD-H2O2.

F. Complex I activity in H9c2 cells treated with indicated iron chelators with or without H2O2. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.0004 PBS-PBS vs. PBS-H2O2. * P=0.0003 DFO-PBS vs. DFO-H2O2. * P<0.0001 PBS- H2O2 vs. BPD-H2O2.

G. Complex II activity in H9c2 cells treated with indicated iron chelators with or without H2O2. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.0001 PBS-PBS vs. PBS-H2O2. * P<0.0001 DFO-PBS vs. DFO-H2O2. * P<0.0001 PBS- H2O2 vs. BPD-H2O2.

H. Complex IV activity in H9c2 cells treated with indicated iron chelators with or without H2O2. N=6 independent samples for each group. ANOVA followed by post-hoc Tukey test was performed. * P=0.02 PBS-PBS vs. PBS-H2O2. * P=0.0009 DFO-PBS vs. DFO-H2O2. * P=0.006 PBS- H2O2 vs. BPD-H2O2. All data are expressed as mean ± SEM.
Negative regulation of Type I IFN signaling by phosphorylation of STAT2 on T387 (2016)
Figure 1

Figure 1. Identification of novel phosphorylated residues in STAT2.

A. MS/MS analysis of peptides corresponding to amino acids 380-399 of STAT2. 293T cells expressing flag-tagged STAT2 were used.

B. Abundance of T387 phosphorylated STAT2 in 293T cells expressing flag-tagged STAT2.

C. Location of T387 in STAT2.

D. Conservation of T387.
Negative regulation of Type I IFN signaling by phosphorylation of STAT2 on T387 (2016)
Figure 2

Figure 2. Threonine phosphorylation of T387 STAT2 negatively regulates type I IFN signaling.

A. U6A cells expressing wild-type or T387A STAT2 were treated with IFN-β (100 IU/ml). Cells were harvested after 4 h and total RNAs were analyzed by real-time PCR. Values are the means ± SD from three independent experiments.

B. U6A cells expressing wild-type, T387A, or T387D STAT2 were treated with IFN-β (100 IU/ml). Cells were harvested after 4 h and total RNAs were analyzed by real-time PCR. Values are the means ± SD from three independent experiments.

C, D. U6A cells expressing wild-type or T387A STAT2 were treated with IFN-β for 0, 4, 8, or 24 h. Total RNAs were analyzed by using an Illumina HumanHT-12 v4 Expression BeadChip array. The average signal for each probe was used to determine expression levels. Genes with average signals below 25 and detection P values greater than 0.01 in the untreated or treated cells were excluded from the analysis. Inductions of less than 2-fold were not scored. The Venn diagram shows the numbers of genes induced by 1.2 fold or more in U6A cells expressing T387A STAT2, relative to untreated control cells. The numbers in the table are fold changes calculated from the ratios T387A STAT2 treated / untreated and wild-type STAT2 treated / untreated.
Negative regulation of Type I IFN signaling by phosphorylation of STAT2 on T387 (2016)
Figure 3

Figure 3. Phosphorylation of T387 of STAT2 inhibits the antiviral and growth suppressive activities of IFN-β

A. HME cells expressing wild-type, T387A, or T387D STAT2 were seeded in 96-well plates at 8000 cells/well. The cells were exposed to vesicular stomatitis virus (VSV) for 2 h, with or without pre-treatment with IFN-β (100 IU/ml). After 20 h, the amounts of the VSV-M and STAT2 proteins were analyzed by the Western method.

B, C. HME cells expressing wild-type, T387A, or T387D STAT2 were seeded in 96-well plates at 8000 cells/well. On the second day, the cells were treated with IFN-β (10 IU/ml) for 2 h. The cells were then exposed to different multiplicities of infection of VSV for 2 h, followed by fresh media containing 10 IU/ml of IFN-β. The cells were analyzed by staining with crystal violet (B) or the MTT assay (C) after 48 h.

D. U6A cells expressing wild-type, T387A, or T387D STAT2 were placed into 96 well plates (2000 cells/well) and the cells were treated with IFN-β (1000 IU/ml) for 96 h. Cell survival was analyzed by the MTT assay. Each experiment was carried out two independent times, with results similar to the representative examples that are shown.
Negative regulation of Type I IFN signaling by phosphorylation of STAT2 on T387 (2016)
Figure 4

Figure 4. Kinetics of STAT2 T387 phosphorylation in response to IFN-β, and down regulation by CDK inhibitors.

A. An immuno-precipitation was performed using whole cell lysates from cells expressing Flag-tagged wild-type STAT2, and samples were analyzed by the Western method. The numbers in the chart are the proportions of T387 phosphorylated STAT2 in each cell type, calculated in comparison with 293T cells.

B. HME cells expressing wild-type STAT2 were treated with IFN-β (100 IU/ml). Whole cell lysates harvested at the times indicated were analyzed by the Western method;

C. HME cells expressing wild-type STAT2 were seeded in complete medium. On the second day, the cells were re-fed with medium lacking supplements. On the third day, the cells were treated with each supplement for 4 h, and total lysates were analyzed by the Western method. 1. EGF, 2. Insulin, 3. Hydrocortisone, 4. GA-1000, 5. BPE.

D. HME cells were pretreated with hydrocortisone (0.04mg/ml and 0.4mg/ml) for 3 h and then treated with IFN-β (100 IU/ml). Cells were harvested after 4 h and total RNAs were analyzed by real-time PCR. Values are the means ± SD from three independent experiments.

E. Consensus sequences for CDK-catalyzed phosphorylation, compared to residues 383-391 of STAT2.

F. HCC827 and LnCAP cells expressing wild-type STAT2 were treated with CDK inhibitors (1 µM) for 4 h and whole-cell lysates were analyzed by the Western method. 1. Flavopiridol, 2. AT7519, 3. Dinaciclib.
Negative regulation of Type I IFN signaling by phosphorylation of STAT2 on T387 (2016)
Figure 5

Figure 5. T387 phosphorylation negatively regulates the binding of ISGF3 to DNA.

A. U6A cells expressing wild-type or T387A STAT2 were treated with IFN-β (100 IU/ml) for 30 min or were untreated. The cells were washed with PBS and the media was replaced with fresh media containing staurosporine (500 nM). Whole-cell lysates were harvested and analyzed by the Western method;

B. U6A cells expressing wild-type or T387A STAT2 were treated with IFN-β (100 IU/ml) for 4 h or were untreated. Whole-cell lysates were used for immunoprecipitations of IRF9 and Flag-STAT2.

C. U2A cells expressing wild-type or T387A STAT2 were treated with IFN-β (100 IU/ml) for 4 h or were untreated. Whole-cell lysates were used for immunoprecipitation of Flag-STAT2.

D. U3A cells expressing wild-type or T387A STAT2 were treated with IFN-β (100 IU/ml) for 4 h or were untreated. Whole-cell lysates were used for immunoprecipitation of Flag-tagged STAT2.

E. EMSAs using an ISRE probe. Extracts from U6A cells expressing wild-type or T387A STAT2 treated with IFN-β for 1 h were analyzed. The position of ISGF3 is labeled.
Zika virus alters centrosome organization to suppress the innate immune response (2022)
Figure 1

Figure 1: ZIKV infection induces the formation of supernumerary Centrin foci in neural precursor cells

(A) Neural precursors derived from human iPS cells (iNPCs) were mock or ZIKV-infected and co-stained for Centrin (green), Sox2 (blue) and flavivirus envelope (red). All cells were fixed 16 hours post infection (hpi).

(B) Mock and ZIKV-infected U87 and H4 cells in S phase co-stained for Centrin (green), flavivirus envelope (red) and DNA (blue).

(C) Quantification of the percentage of mock or ZIKV-infected iNPC, U87 or H4 cells in S phase with greater than four Centrin foci. Bars represent the mean ± SD normalized to the control. Asterisk denotes p<0.005 (Student's t test). Three biological replicates are represented.

(D) Neural precursor cells mock or ZIKV-infected and co-stained for Centrin (green), flavivirus envelope (red) and SOX2 (blue).

(E) Mock or ZIKV-infected iNPC and U87 cells in S phase co-stained for Centrin (red) and the centriole distal end component, CP110 (green).

(F) Mock or ZIKV-infected iNPC and U87 cells co-stained for Centrin (red) and the pericentriolar material proteins γ-tubulin (green)

(G) Transmission electron micrographs of centrioles from mock or ZIKV-infected U87 cells.

Data information: Scale bars indicate 100nm. Arrows indicate electron dense particles.
Zika virus alters centrosome organization to suppress the innate immune response (2022)
Figure 2

Figure 2: ZIKV induces supernumary Centrin foci in a CEP63-dependent manner.

(A) Mock or ZIKV-infected U87 cells in S phase co-stained for Centrin (red) and the centrosomal microcephaly-associated protein, CEP63 (green).

(B) The fluorescence intensity ± s.d. of CEP63 at the centrosome (0.5μm around the Centrin signal) and around the centrosome (1μm surrounding the Centrin signal minus the centrosomal CEP63) were quantified in mock and ZIKV infected U87 cells. For fluorescence quantifications 8 cells were analyzed per experiment (n=3). Asterisk denotes p<0.005 (Student's t test).

(C) Mock and ZIKV-infected neuronal progenitors (NPC) isolated from fetal brain cortex and iPSC derived neural precursor cells (iNPC) co-stained for Centrin (red), CEP63 (green) and SOX2 (blue).

(D) Immunoblot analysis of lysate from mock or ZIKV-infected U87 cells probed for CEP63 and ZIKV NS3. Actin served as a loading control. Asterisk denotes specific band for CEP63.

(E) S-phase mock and ZIKV infected control and Cep63gt/gt MEFs co-stained for Centrin (green) and flavivirus envelope (red).

(F) Quantification of the percentage of mock or ZIKV-infected Het and Cep63gt/gt MEFs in S phase with greater than four Centrin foci. Bars represent the mean ± SD normalized to the control. Asterisk denotes p<0.005 (Student's t test). Three biological replicates are represented.

Data information: Scale bars indicate 5μm for all images. All cells were fixed 16 hours post infection (hpi),
Zika virus alters centrosome organization to suppress the innate immune response (2022)
Figure 3

Figure 3: ZIKV NS3 binds the microcephaly-associated protein CEP63 and accumulates it at the centrosome

(A) 16hpi mock or ZIKV-infected iNPC cells were co-stained for Centrin (red) and NS3 (green).

(B) Mock or ZIKV-infected U87 cell lysates were immunoprecipitated for CEP63 or c-Myc, which served as a negative control for this and other immunoprecipitations (Control). Precipitated proteins were immunoblotted for CEP63 and NS3. Asterisk denotes specific band.

(C) We expressed Myc-tagged Brazilian Zika NS3 in 293T/17 cells and immunoprecipitated c-Myc. Precipitated proteins were probed for the Myc tag of NS3 and endogenous CEP63. The centriole distal end component, CP110 served as a negative control. Asterisk denotes specific band for CEP63.

(D) Mock and ZIKV-infected control and Cep63gt/gt MEFs were co-stained for Centrin (red) and ZIKV NS3 (green).

(E) Schematic representation of Myc-NS2B-NS3 being proteolytically cleaved by itself to generate Myc-NS2B and NS3 proteins. Immunoblot of HeLa cells transfected with Myc-NS2B-NS3 probed with antibodies to Myc and NS3. Actin served as a loading control.

(F) Mock or Myc-NS2B-NS3 expressing HeLa cells co-stained for c-Myc (red) and Centrin (green).

(G) HeLa cells transfected with Control or Myc-tagged Brazilian ZIKV NS3 (Br NS3) co-stained for Myc (red) and Centrin (green).

(H) Quantification of the percentage of control and NS3 expressing HeLa cells with greater than four Centrin foci. Bars represent the mean ± SD normalized to the control. For all quantifications at least 100 cells were counted per experiment (biological replicates=3). p<0.005 (paired Student's t test) for the comparison of NS3 to mock transfected cells was denoted with asterisk.

(I) Control or Myc-tagged Brazilian ZIKV NS3 (Br NS3) transfected HeLa cells were co-stained for Myc (red) and MCPH protein CEP152 or CEP63.

(J) Control or Myc-tagged Ugandan NS3 (Ug NS3) expressing HeLa cells were co-stained for Myc (red) and Centrin (green) or CEP63 (green).

Data information: Scale bars indicate 5μm for all images.
Zika virus alters centrosome organization to suppress the innate immune response (2022)
Figure 4

Figure 4: ZIKV degrades TBK1 to dampen the innate immune response.

(A) HeLa cell lysates were immunoprecipitated with antibodies to CEP63 and TBK1. Precipitated proteins were immunoblotted for CEP63 and TBK1.

(B) Mock and ZIKV-infected U87 cells were co-stained for γ-tubulin (red) and p-TBK1 (green).

(C) Lysate from mock or ZIKV-infected U87 cells at 16 hpi were immunoblotted with antibodies to NS3, TBK1, and p-TBK1. Actin served as a loading control.

(D) RT-ddPCR of IFNβ transcript at the indicated time points in mock and ZIKV-infected H4 cells. Bars represent the mean ± SD normalized to the control. Asterisk denotes p<0.005 (Student's t test). Three biological replicates are represented.

(E) Control and Cep63gt/gt MEFs were co-stained with antibodies to γ-tubulin (red) and p-Tbk1 (green).

(F) Immunoblot analysis of control and Cep63gt/gt MEFs probed with antibodies to Tbk1 and p-Tbk1. Actin served as a loading control.

(G) HeLa cell lysate was immunoprecipitated with antibodies to CEP63, DTX4 or c-Myc (negative control). Precipitated proteins were blotted with antibodies to CEP63 and DTX4.

(H) Mock and ZIKV-infected U87 cells co-stained for Centrin (red) and DTX4 (green).

(I) Total cell lysate from mock and ZIKV-transfected U87 cells 16 hpi were analyzed by western blot using antibodies to ZIKV NS3 and DTX4. Actin served as a loading control.

(J) SC and CEP63 siRNA treated HeLa cells were co-stained for Centrin (red) and DTX4 (green).

(K) TBK1 or c-Myc (negative control) were immunoprecipitated from Control or Cep63 mutant MEFs. Precipitating proteins were probed with antibodies to TBK1 and K48-linked ubiquitin.

(L) 16hpi Mock and ZIKV-infected U87 cell lysate was immunoprecipitated for TBK1 or c-Myc. The immunoprecipitations were probed using antibodies to TBK1 and K-48-linked ubiquitin.

Data information: Scale bars indicate 5μm for all images.