EMBO/soda-nel huggingface dataset:
Phosphodiesterase 4A confers resistance to PGE2-mediated suppression in CD25+/CD54+ NK cells (2020)
Figure 5

Figure 5. PTGES affects the prognostic value of NK cells in patients with lung adenocarcinoma

A) Expression of PTGES in normal and tumor tissue.

B) Kaplan-Meier analysis of overall (left) and progression-free (right) survival in patients with lung adenocarcinoma with high PTGES (top 10%) and low PTGES (bottom 90%) expression.

C) NK cell gene signature in normal and tumor tissues based on a TCGA lung adenocarcinoma cohort and quantification of NK cell gene signature in patients with different stages of lung adenocarcinoma.

D) IL15 gene expression analysis in high and low NK gene signature (NK GS).

E) Overall survival in patients with stage I (n=269) lung adenocarcinoma with high (top 50%) and low (bottom 50%) NK cell gene expression signature expression.

F) Overall survival in patients with high (top 50%) PTGES expression in relation to high (top 50%) and low (bottom 50%) NK cell gene expression signature.

Data information: Figure A, C, D were analyzed using an unpaired parametric t test (****, p<0.0001). Log-rank (Mantel-Cox) test was used to test for significance and log-rank p-values are displayed in Kaplan-Meier analysis graph. **, p<0.01, ****, p<0.0001. LUAD: Lung adenocarcinoma. HR is calculated at 95% CI. The violin plots in Figure 5A, 5C, 5D show the median (solid line) and the 25th and 75th percentile (dashed line).
Phosphodiesterase 4A confers resistance to PGE2-mediated suppression in CD25+/CD54+ NK cells (2020)
Figure 6

Figure 6. CD54 positive NK cells infiltrate lung adenocarcinoma tumors

A) Real time image-based killing of A549 cells by NK cells isolated from two individual healthy donors at an E:T ratio of 10:1.

B) NK cells were stimulated with IL-2 or IL-15 in the presence or absence of PGE2 and then purified as CD25+/CD54+ or CD25-/CD54- cells and tested for cytotoxicity against A549 tumor cell in a 51Cr-release assay. E:T ratio = 5:1 (n=4, biological replicates). Stars indicate significant p-values comparing CD25+/CD54+ vs. CD25-/CD54-.

C) Representative image of NK cell infiltration (red) into A549 tumor spheroids at the time of addition of NK cells and at 20 hours after the addition of NK cells, scale bar: 400 μm.

D, E) Incucyte quantification of D) total and E) tumor-infiltrating CD25+/CD54+ and CD25-/CD54- NK cells. RCU, Red Calibrated Unit, (n=4, biological replicates).

F, G) Flow cytometry quantification of (F) tumor-infiltrating and (G) non-infiltrating NK cells. Results are depicted as total NK cells among CD45 positive live cells. (n=4, biological replicates).

H) 48 hours dpi zebrafish injected with A549, A549+ CD25+/CD54+, or A549+ CD25-/CD54- NK cells after 24 hours. Fluorescent image was taken by Leica epi-fluorescent stereomicroscope. Scale bar: 500 µm. The tumor injection area (perivitelline space) is depicted with a dashed line.

I) 3D view of tumor injection area 24 hours posts tumor inoculation (left). Scale bar=50 µm. The tumor injection area (perivitelline space) is depicted with a dashed line. Magnified view for tumor area (right). The A549-td tumor cells are annotated with a purple translucent mask and NK cells are color-coded based on the distance to the tumor area (near-far: blue-red). Scale bar=10 µm.

J) The distance between NK cells and tumor area in CD25-/CD54- and CD25+/CD54+ NK cell treated fish (n=5, biological replicates).

K) Frequency of tumor infiltrating NK cells in in CD25-/CD54- and CD25+/CD54+ NK cell treated fish (n=5, biological replicates).

L, M) Quantification of (L) tumor cell and (M) NK cell counts 24 hours after injection of NK cells by Imaris (n=5, biological replicates).

N) Frequency of tissue-infiltrating total NK cells (CD56+/CD3-) (left) and CD54 positive NK cells (right) among CD45 positive live cells (n=10, biological replicates).

Data information: Data were analyzed using a paired parametric t test (*, p<0.05, **, p<0.01, ***, p<0.001, ****, p<0.0001). Each circle represents an individual experiment. Error bars are depicted as standard deviation. The violin plots in Figure 6J, 6K show the median (solid line) and the 25th and 75th percentile (dashed line).

Expanded View Figure legends
Sensing of mammalian IL-17A regulates fungal adaptation and virulence (2012)
f1

Dose-dependent increase of C. albicans (SC5314) aggregation after 4-h exposure (a) and filamentation after 24-h exposure (b) to different concentrations of IL-17A in RPMI-1640 liquid medium. Cells aliquots were imaged by microscopy and magnified in the insets. Also note the increased turbidity of cell suspension at 24 h. Scale bars, 100 μm. (c) Percentages of germination of C. albicans untreated (vehicle-treated, open circle) or treated with IL-17A at 1 (black triangle) or 10 ng ml−1 (black square) for 4 or 24 h. Data are mean±s.d. P 0.05, IL-17A-treated versus vehicle-treated cells, at 4 h; P 0.01 (1 ng) and P 0.001 (10 ng) at 24 h (one-way analysis of variance Bonferroni post-test) (n=3). (d,e) IL-17A promotes biofilm formation. SEM images of C. albicans SC5314 untreated (d) or exposed to 10 ng ml−1 IL-17A (e) for 4 h and subsequently cultured onto plastic discs for 24 h. Scale bars, 10 μm. (f) IL-17A increases resistance to nitrogen starvation. Cells were first grown in YPD medium overnight and then switched to nitrogen-free SLAD medium in the presence of 10 ng ml−1 IL-17A. Viability, expressed as a percentage of colony-forming units at zero time, was measured over a 15-day period. Cells were exposed to 0.1 (black circle), 1 (black triangle) or 10 ng ml−1 (black square) IL-17A or drug vehicle (open circle) at the beginning of the cultures or, in the case of 1 ng ml−1 (open triangle), also added every two other days. Data are mean±s.d. P-values ranged from 0.05 to 0.001 for all samples treated with 10 ng IL-17A; P0.05, on days 3 and 5 for samples treated 1 ng IL-17A every two other days (one-way analysis of variance Bonferroni post-test) (n=3). (g,l) C. albicans cells are metabolically active. Untreated (g–i) or IL-17A-treated (10 ng ml−1 in RPMI-1640 liquid media for 24 h) (j–l) cells were red after staining with FUN-1 (g,j), green with Con A (h,k) and yellow in the merged images (i,l). Metabolically active cells are shown in red, and cell wall polysaccharides are shown in green. Scale bars, 5 μm. Images from one representative experiment out of two are shown.
Sensing of mammalian IL-17A regulates fungal adaptation and virulence (2012)
f2

(a,d)Venn diagram showing up or downregulated genes, (b,e) GO processes of open reading frames, (c,f) histogram of microarray data from Candida SC5314 cells exposed to IL-17A (10 ng) for 4 h (a–c) and 24 h (d–f). Text and arrows: genes involved in TOR pathway at 4 h (c) and of hyphae-induced genes at 24 h (f). (g,h) TOR inhibition in cells treated with 10 ng ml−1 IL-17A, 15 nM rapamycin or vehicle (none) by western blotting after 4-h (g) or 24-h (h) exposure. (i–n) Immunofluorescence of ATG8 in untreated (i–k) or IL-17A-treated (10 ng ml−1 for 4 h) (l–n) cells. Cells were red after TRITC–ATG8 staining (i,l), green after ConA staining (j,m), merged images (k,n). Mean values (% ATG8+ cells (merge); mean±s.d.): ATG8/none, 8.2±1.6; ATG8/IL-17A, 58.0±11.3 (P=0.002) (n=3). Scale bars, 10 μm. (o–r) Monodansilcadaverine (MDC) labelling performed on cells unexposed (o) or exposed to 1 (p) or 10 ng ml−1 (q) IL-17A for 4 h. Cells were magnified in the inset (r). Representative results from three independent experiments. The mean values (% hyphal cells with MDC-stained organelles (merge); mean±s.d.) were as follows: none, 9.5±3.2; 1 ng ml−1 IL-17A, 14.0±4.7; 10 ng ml−1 IL-17A, 56.2±18.71 (P=0.004, IL-17A 10 ng ml−1 versus none). Scale bars, 10 μm. (s,t) Quantitative real-time PCR of orfs. 19.4325, 19.130, RIC1 on cDNA from Candida SC5314 (s) or the TOR1-1/TOR1 mutant (t) strains exposed to vehicle (open column), 10 ng ml−1 IL-17A (black column) or 15 nM rapamycin (grey column) for 4 h. The actual P-values are indicated (two-tailed t-test) (n=3). (u,v) Intracellular (u) or extracellular (v) cAMP levels, vehicle (open circle), 10 ng ml−1 IL-17A (black square) or rapamycin (grey square). P0.05, IL-17A-treated versus untreated at 1 h and P0.01, IL-17A-treated versus untreated at 24 h (u), and P0.05, rapamycin-treated versus untreated at 6 h (v) (one-way analysis of variance Bonferroni post-test) (n=3). (w) orf. 19.5258 expression after 24-h Candida SC5314 exposure as in (n,o). Data are mean±s.d. (n=3). The actual P-values are indicated (two-tailed t-test).
Sensing of mammalian IL-17A regulates fungal adaptation and virulence (2012)
f3

(a–g) GFP C. albicans cells were untreated (a–c) or treated (d–g) for 4 h with 10 ng ml−1 IL-17A followed by anti-IL17A-PE and examined by fluorescence microscopy (scale bars, 5 μm). Cells were green in (a,d), red upon staining with anti-IL17A-PE (b,e) and double stained in the merged images (c,f). Note the binding of IL-17A to hyphal tips (arrows and magnified in g) (n=3). Images are from one representative experiment out of three. (h,i) Binding of murine (h) or human (i) IL-17A to clinical isolates of C. albicans from human urine (yellow triangle), blood (green triangle), vagina (red triangle) and gut (blue triangle). IL-17A was added to paraformaldehyde-fixed fungal cells followed by biotinylated secondary anti-IL-17A antibody and streptavidin-horse radish peroxidase. The optical density at 450 nm was corrected by subtracting the optical density at 570 nm (normalized OD) in the enzyme-linked immunosorbent assay. P-values of samples exposed to 0.1 and 1 ng IL-17A ranged from 0.05 to 0.001 (one-way analysis of variance Bonferroni post-test). All samples exposed to 10 ng IL-17A displayed significant (P0.001, one-way analysis of variance Bonferroni post-test) OD values relative to unexposed samples (0). (j–o) Direct visualization of IL-17A on Candida freshly harvested from human vagina. Same experiment as in (a–g) on cells previously stained with Con A (j,m, green) and incubated with PE-anti-human IL-17A (k,n, red) and double stained in the merged images (l,o). Scale bars, 10 μm. Shown is one representative experiment out of three. (p–r) Cytospins of vaginal fluids from IL-17F-deficient (p), IL-17A-deficient (q) and C57BL/6 (r) mice intravaginally infected two days before with C.albicans SC5314 blastospores. Note the presence of hyphae in the vagina of IL-17F-deficient mice after May–Grünwald–Giemsa staining (scale bars, 20 μm). Shown is one representative experiment out of three. (s) Binding of IL-17A to clinical isolates of C. krusei (red square), C. glabrata (green square) and S. cerevisiae (yellow square) done as in (h,i). All samples exposed to IL-17A displayed significant (P0.001, one-way analysis of variance Bonferroni post-test) OD values (n=3). Data are mean±s.d. (n=4).
Sensing of mammalian IL-17A regulates fungal adaptation and virulence (2012)
f4

(a) Dose-dependent binding of IL-17A to fungal Crh11p (red circle) and Gas1p (light blue circle) proteins, but not to the fungal polysaccharides β-1,3-D-glucan (open circle), mannan (blue circle) and zymosan (grey circle). The lack of binding of IL-17E to Crh11p (orange circle) serves as a negative control. The optical density at 450 nm was corrected by subtracting the optical density at 570 nm (normalized OD) in the enzyme-linked immunosorbent assay. Data are mean±s.d., n=3. P0.001, Crh11p and Gas1p without versus with IL-17A (one-way analysis of variance Bonferroni post-test). (b) Co-immunoprecipitation assay of fungal Crh11p and murine IL-17A proteins. Immunoprecipitates were probed with antibodies to the corresponding antigens and secondary HRP-conjugated antibody. C, irrelevant antibody. (c) Failure of IL-17A to bind to wild-type (red square) or mutant C. albicans strains in which the three genes of the CRH family (UTR2, open light blue, CRH1, open black and CRH12, open blue square) had been deleted, and restoration of binding by CRH1 (black square) and CRH12 (blue square), but not UTR2 (light blue square), reconstitution. Data are mean±s.d. (n=3). P0.001 for red square, open light blue, black square and blue square (one-way analysis of variance Bonferroni post-test). (d,e) Cellular localization of bound IL-17A. Electron micrographs of ultrafine sections of C. albicans MKY 378 wild-type (d) or the crh11Δ mutant (e) incubated with biotinylated IL-17A and streptavidin gold-conjugated particles. Arrows indicate immunogold particles. Scale bars, 200 nm.
Sensing of mammalian IL-17A regulates fungal adaptation and virulence (2012)
f5

Fungi were harvested from the stomachs of C57BL/6 (a–c), IL-17F-deficient (d–f) or IL-17A-deficient (g–i) mice infected with GFP–Candida for 2 days, and stained using PE-labeled antibody to IL-17A. Cells were green in (a,d and g), red upon staining with anti-IL17A-PE (b,e and h) and double stained in the merged images (c,f and i). Scale bars, 10 μm. Images are from one representative experiment out of two. (j–l) SEM images showing fungal germination in the stomachs of C57BL/6 mice (j), filamentation in IL-17F-deficient mice (k) and neither one in IL-17A-deficient (l) mice intragastrically infected 2 days before. Scale bars, 10 μm. (a–l) Images are from one representative experiment out of two. (m–o) C57BL/6 mice were infected intragastrically with C. albicans MKY 378 strain (m) or the crh11Δ (n) and crh12Δ (o) mutants, and were monitored for fungal growth (log10 colony-forming units (CFU) at 2 (open circle) or 8 (black circle) days post infection). Data are mean±s.d. (n=3). The actual P-values are indicated (two-tailed t-test) for 2 and 8 days (m) and for 2 days (o). Fungal cells were untreated (none) or exposed to 10 ng ml−1 IL-17A for 4 h before injection.
Sensing of mammalian IL-17A regulates fungal adaptation and virulence (2012)
f6

Candida SC5314 (a), TOR1-1/TOR1(b) and atg9Δ/atg9Δ (c) mutant strains were untreated (open column) or pre-incubated with IL-17A (filled column) for 4 h before exposure to neutrophils for additional 2 h before the assessment of adhesion, phagocytosis and killing. Error bars, mean±s.d. (n=3). The actual P-values are indicated (two-tailed t-test). (d–f) IL-17A induces autophagy in vivo. Representative electron micrographs of vacuoles (v) in Candida cells purified on sucrose gradient from the stomachs of IL-17F-deficient (d), IL-17A-deficient (e) or C57BL/6 (f) mice intragastrically infected 2 days before. Arrows indicate autophagy-associated structures. Scale bars, 1 μm. (g) Fungi purified from the stomachs of mice infected as in (d–f) were subjected to RT–PCR for autophagy genes expression. C+, fungal cells grown on SPIDER solid medium. C–, no cells. (h–j) Log10 colony-forming units (CFU) at 2 days post infection with C. albicans SC5314 (wild-type) or different mutant strains in C57BL/6 (h), IL-17F-deficient (i) or IL-17A-deficient (j) mice. Data are mean±s.d. (n=3). The actual P-values are indicated (two-tailed t-test).
Sensing of mammalian IL-17A regulates fungal adaptation and virulence (2012)
f7

(a–f) GFP Aspergillus conidia, incubated overnight at room temperature, were untreated (a–c) or exposed (d–f) to 10 ng ml−1 IL-17A for 4 h followed by anti-IL-17A-PE. Cells were green in (a,b), red upon staining with anti-IL17A-PE (b,e) and double stained in the merged images (c,f). Scale bars, 10 μm. (g,h) IL-17A promotes biofilm formation in A. fumigatus AF293 strain. SEM images refer to cells untreated (g) or pretreated (h) with 10 ng ml−1 IL-17A for 4 h and subsequently cultured onto plastic discs for 24 h. Scale bars, 20 μm. (i–k) IL-17A induces autophagy in Aspergillus. Representative electron micrographs of vacuoles (v) in A. fumigatus AF293 unexposed (k) or exposed to 10 ng ml−1 IL-17A (j) or 15 nM rapamycin (i) for 4 h before TEM. Scale bars, 500 nm. (l) IL-17A binds the Aspergillus Crf1p protein. Dose-dependent binding of IL-17A to Crf1p (red circle), Gel1p (light blue circle) or the Aspergillus-derived polysaccharides β-1,3-D-glucan (blue circle), α-1,3-D-glucan (open circle) and galactomannan (grey circle). The optical density at 450 nm was corrected by subtracting the optical density at 570 nm (normalized OD) in the enzyme-linked immunosorbent assay (ELISA) on microtitre-coated plates. Data are mean±s.d. (n=5). P-values ranged from 0.01 to 0.001 for IL-17A-treated versus untreated (0) Crf1p (one-way analysis of variance, Bonferroni post-test). (m) ELISA assay showing failure of IL-17A to bind the Aspergillus mutant crf1Δ (blue square) as opposed to the wild-type strain AF293 (red square). Data are mean±s.d. (n =5). P-values ranged from 0.05 to 0.01, wild-type versus mutant strain exposed to 1 or 10 ng ml−1 IL-17A (one-way analysis of variance, Bonferroni post-test). (n–q) IL-17A promotes filamentation in the mutant crf1Δ (o) but not the wild-type AF293 (q) strain after 24-h exposure in liquid culture. Scale bars, 500 μm. (r,s) C57BL/6 mice were infected intranasally with the Aspergillus crf1Δ mutant (r) or the AF293 (s) strain and monitored for fungal growth (log10 colony-forming units (CFU)±s.d.) in the lungs 2 days later. Fungal cells were exposed or not (none) to 10 ng ml−1 IL-17A for 4 h, extensively washed and injected. Data are mean±s.d. (n=4). The actual P-values are indicated (two-tailed t-test).
Role of Angiomotin-Like2 mono-ubiquitination on YAP inhibition (2015)
Figure 1

Figure 1. USP9X is required for YAP activity.

A. 293T cells were co-transfected with CMV-renilla, 8X-TBS (eight repeats of the TEAD-binding-sequence) luciferase reporter, Flag-YAP, HA-TEAD2 and wild-type (WT) or catalytically inactive (CS) USP9X-V5. One day after transfection, luciferase activity was measured and normalized with respect to renilla activity. The value for Flag-YAP/HA-TEAD-transfected cells (2nd column) was adjusted to 1. Shown below is a representative Western blot showing that the expression levels were comparable between samples (n=4). Error bars indicate the S.E.M. (*p< 0.05, **p < 0.01; paired Student’s t-test).

B. 293T cells were transfected with control or USP9X-targeting siRNAs. One day after siRNA transfection, the cells were co-transfected with CMV-renilla, 8X-TBS luciferase, Flag-YAP and HA-TEAD2. Reporter activity was quantified as described in (A) (n=4). Error bars indicate the S.E.M. (***p < 0.001; paired Student’s t-test).

C. RPE or MCF10A cells were transfected with the indicated siRNAs for 24 hours and then re-seeded to either sparse or dense culture conditions. At 48 hours after siRNA transfection, the cells were harvested and cell extracts were analyzed by Western blotting for the indicated proteins. L.E., long exposure, S.E., short exposure.

D. Cells were treated as described in (C), the indicated mRNAs were analyzed with RT-qPCR, and the results were normalized with respect to the β-actin mRNA (n=4). Error bars indicate the S.E.M. (*p< 0.05, **p < 0.01; paired Student’s t-test).

E. RPE cells were transfected with the indicated siRNAs for 24 hours, and then co-transfected with CMV-renilla and 8X-TBS luciferase. One day after the latter transfection, the cells were re-seeded to either sparse or dense conditions, and reporter activity was measured at 24 hours after re-seeding (n=3). Error bars indicate the S.E.M. (**p < 0.01; paired Student’s t-test).
Role of Angiomotin-Like2 mono-ubiquitination on YAP inhibition (2015)
Figure 2

Figure 2. USP9X regulates YAP localization.

A. Representative anti-YAP immunofluorescence images of sparsely cultured RPE cells transfected with the indicated siRNAs. Green, YAP. Blue, DAPI. Scale bar, 10 μm.

B. The nuclear YAP intensities of the cells shown in (A) were quantified using the Image J software. At least 70 cells from five or six random fields were analyzed. Quantified values were normalized by adjusting the average of “siControl Dense cells” to 1. We performed two independent experiments and obtained similar results. Data from one representative experiment are presented. The red bar indicates the average value. S, Sparse. D, Dense (***p < 0.001; paired Student’s t-test).

C. Sparsely cultured RPE or MCF10A cells were transfected with the indicated siRNAs, fractionated into cytosolic and nuclear extracts, and analyzed by Western blotting for the indicated proteins.

D. Representative anti-YAP immunofluorescence images of densely cultured RPE cells transfected with the indicated plasmids. Green, GFP or V5. Red, YAP. Blue, DAPI. Scale bar, 10 μm.

E. The nuclear YAP intensities of transfected cells in (D) were quantified using the Image J software. At least 70 cells from five to six random fields were analyzed. Quantified values were normalized by adjusting the average of “EGFP transfected cells” to 1. Similar results were obtained from two independent experiments. Data from one representative experiment are presented. The red bar indicates the average value. (***p < 0.001; paired Student’s t-test).

F. Densely cultured stable RPE clones integrated with empty vector (Vec), USP9X-V5 WT or CS mutant were fractionated into cytosolic and nuclear extracts, and analyzed by Western blotting for the indicated proteins.

G. Densely cultured RPE cells transduced with CRISPR-SAM targeting USP9X were fractionated into cytosolic and nuclear extracts, and analyzed by Western blotting for the indicated proteins.
Role of Angiomotin-Like2 mono-ubiquitination on YAP inhibition (2015)
Figure 3

Figure 3. USP9X regulates YAP phosphorylation through LATS kinase.

A. RPE cells were analyzed by Western blotting for the indicated proteins.

B. Indicated stable RPE clones in densely cultured conditions were analyzed by Western blotting for the indicated proteins.

C. Densely cultured RPE cells transduced with CRISPR-SAM targeting USP9X were analyzed by Western blotting for the indicated proteins.

D. RPE or MCF10A cells expressing the control or USP9X shRNA were transfected with the LATS1/2 siRNA as indicated. Cells were re-seeded to low density and harvested after one day.

E. RPE cells transduced with Flag-YAP WT or Flag-YAP 5SA were transfected with the indicated siRNAs. Cells were re-seeded to low density and harvested after one day.

F. RT-qPCR of CTGF mRNAs in the cells described in (E) (n=3). Error bars indicate the S.E.M. (*p< 0.05, paired Student’s t-test).
Role of Angiomotin-Like2 mono-ubiquitination on YAP inhibition (2015)
Figure 4

Figure 4. AMOTL2 is downstream of USP9X.

A. RPE or MCF10A cells were transfected with the indicated combinations of siRNAs and re-seeded to low density, and cell extracts were analyzed by Western blotting for the indicated proteins.

B. The cells described in (A) were analyzed by RT-qPCR for the indicated target genes (n=4). Error bars indicate the S.E.M. (*p< 0.05, **p < 0.01; paired Student’s t-test).

C. Sparsely cultured RPE cells transfected with the indicated siRNAs were immunoprecipitated with an anti-AMOTL2 antibody and analyzed by Western blotting for the indicated proteins. WCL, whole cell lysates.
Role of Angiomotin-Like2 mono-ubiquitination on YAP inhibition (2015)
Figure 5

Figure 5. AMOTL2 is a substrate of USP9X.

A. 293T cells were transfected with the indicated siRNAs, cultured for 24 hours, and then transfected with the indicated DNAs. 48 hours after siRNA transfection, the cells were harvested and ubiquitination of Flag-AMOTL2 was examined. C, control siRNA.

B. 293T cells were transfected with the indicated combinations of DNAs, cultured for 24 hours, and subjected to in vivo ubiquitination assays.

C. RPE cells transduced with vector (control) or Flag-AMOTL2 were seeded under sparse (S) or dense (D) conditions, and an in vivo ubiquitination assay was performed.

D. RPE cells transduced with Flag-AMOTL2 were transfected with a 1:1 mixture of the two USP9X siRNAs. One day after siRNA transfection, the cells were re-seeded to the sparse condition and an in vivo ubiquitination assay was performed after one day.

E. 293T cells were transfected with the indicated combinations of DNAs, and a co-immunoprecipitation assay was performed.

F. Sparsely cultured RPE or MCF10A cells were immunoprecipitated with an anti-AMOTL2 antibody.