EMBO/soda-nel huggingface dataset:
Phosphatidylserine clustering by the Ebola virus matrix protein is a critical step in viral budding (2022)
Figure 3

Figure 3

PS concentration, localization, and dynamics in fendiline treated HEK293 cells.

A Lipidomic analysis (LC/MS/MS) of total lipids extracted from HEK293 cells treated with the indicated concentration of fendiline (48 hours) demonstrated a significant reduction of total cellular PS levels. Values are normalized to DMSO control and are reported as mean ± s.d.; n=3 biological replicates; A one-way ANOVA was performed with multiple comparisons to the control DMSO (*p=0.012, ***p=0.0003).

B, C Analysis of PS plasma membrane localization in response to fendiline treatment in HEK293 cells. B Representative confocal images from live cell imaging of HEK293 cells expressing GFP-LactC2 and treated with fendiline for 48 hours; scale bars= 10 µm. C Effect of fendiline on PS plasma membrane localization was calculated by the ratio of GFP fluorescence at the plasma membrane intensity/intracellular intensity. Values are normalized to DMSO control and are reported as mean ± s.d.; N>15 cell per biological replicate, n=3 biological replicates; A one-way ANOVA was performed with multiple comparisons compared to the DMSO control (**p=0.0031)

D, E Analysis of PS clustering in HEK293 cells in response to fendiline treatment through N&B analysis. D Left panel: Representative images from time-lapse (30 frames) imaging of HEK293 expressing GFP-LactC2 and treated with fendiline for 48 hours; scale bar= 5 µm. Middle panel: Brightness and Intensity plots for each representative image. Right panel: Selection map correlating each pixel in the representative image to an oligomerization state (b value) (red: monomer-5mer, green: 5mer-10mer, blue: >10mer). E Average % pixels quantification from panel (d)= Percentage of GFP-LactC2 with brightness values corresponding to monomer-5mer (~1.-1.5), 5mer-10mer (~1.5-1.9) and >10mer (>1.9) over the total pixels within each image. Values are reported as mean ± s.d.; N≥9, n=3 biological replicates; A two-way ANOVA was performed with Dunnett's multiple comparisons comparted to the control DMSO % average pixels (****p<0.0001, **p=0.0043).

Data information: GFP-LactC2: phosphatidylserine sensor; N&B: Number & Brightness analysis; PM: plasma membrane; PS: phosphatidylserine.
Phosphatidylserine clustering by the Ebola virus matrix protein is a critical step in viral budding (2022)
Figure 5

Figure 5

Analysis of eVP40 and mVP40 cellular localization and oligomerization following fendiline treatment.

A-C Effect of fendiline on eVP40 and mVP40 PM localization in HEK293 cells after 48 hours of treatment. A Representative confocal images from live cell imaging experiments of HEK293 cells expressing EGFP-WT-eVP40 (top panel) and EGFP-WT-mVP40 (bottom panel) after 48 hours of fendiline treatment. scale bars= 10 µm. Effect of fendiline on mVP40 (B) and eVP40 (C) PM localization was quantified by the ratio of EGFP fluorescence intensity at the PM / total EGFP fluorescence intensity (and normalized to DMSO control). N>15 cell per biological replicate, n=3 biological replicates. Values are reported as mean ± s.d. A one-way ANOVA with multiple comparisons was performed compared to the DMSO control.

D, E Analysis of eVP40 oligomerization in HEK293 cells in response to 48 hour fendiline treatment using N&B analysis. D Left panel: Representative images from time-lapse (30 frames) of HEK293 expressing EGFP-WT-eVP40 and treated with fendiline for 48 hours. scale bar = 5 µm. Middle panel: Brightness and Intensity plots for each representative image. Right panel: Selection map correlating each pixel in the representative image to an oligomerization state (b value) (red: monomer-hexamer, green: hexamer-12mer, blue: 12mer-24mer, pink: >24mer). E Average % pixel quantification from panel (d)= % of GFP-WT-eVP40 with brightness values corresponding to monomer-hexamer (~1.-1.6), hexamer-12mer (~1.6-2.0), 12mer-24mer (2.0-3.2) and >24mer (>3.2) over the total pixels within each image. Values are reported as mean ± s.d.; N≥9 cell per biological replicate, n=3 biological replicates; A two-way ANOVA was performed with Dunnett's multiple comparisons compared to the control DMSO % average pixels (**p=0.0035).

Data information: PM: plasma membrane; N&B: Number & Brightness analysis.
Phosphatidylserine clustering by the Ebola virus matrix protein is a critical step in viral budding (2022)
Figure 6

Figure 6

VLP production and morphology in HEK293 cells in the presence of fendiline.

A-D Functional budding assays assessed at 24 hours (A-B) and 48 hours (C-D) post treatment. A Representative western blot of budding assays performed at 24 hours post transfection with vehicle control (DMSO) treatment or 500 nM, 1 μM, or 5 μM fendiline treatment. VLP samples (top panel) and cell lysate samples (bottom panel) collected from HEK293 cells and immunoblotted for eVP40 expression; GAPDH served as a loading control. eVP40 detected by (Rabbit α-eVP40 and HRP-Goat α-Rabbit); GAPDH detected by mouse α-GAPDH and HRP-Sheep α-Mouse) B Quantification of relative budding index at 24 hours post vehicle or fendiline treatment. Relative budding index was determined by the western blot band density of eVP40 in the VLP fraction/(total eVP40 cell lysate + eVP40 VLP band density) and was normalized to the DMSO control. Cell lysate eVP40 band density was normalized to GAPDH band density prior to use in budding index quantification. n=3 biological replicates. Values are reported as mean ± s.d. A one-way ANOVA was performed with multiple comparisons compared to the DMSO control. C Representative western blot of budding assays performed at 48 hours post vehicle control (DMSO) treatment or 500 nM, 1 μM, or 5 μM fendiline treatment. VLP samples (top panel) and cell lysate samples (bottom panel) collected from HEK293 cells and immunoblotted for eVP40 expression; GAPDH served as a loading control. eVP40 detected by (Rabbit α-eVP40 and HRP-Goat α-Rabbit); GAPDH detected by (Mouse α-GAPDH and HRP-Sheep α-Mouse) D Quantification of relative budding index at 48 hours post fendiline treatment. Relative budding index was determined by the western blot band density of eVP40 in the VLP fraction/(total eVP40 cell lysate + eVP40 VLP band density) and was normalized to the DMSO control. Cell lysate eVP40 band density was normalized to GAPDH band density prior to use in budding index quantification. n=3 biological replicates. Values are reported as mean ± s.d. A one-way ANOVA was performed with multiple comparisons compared to the DMSO control. (*p=0.0260) E, F SEM micrographs of HEK293 cells. E Representative micrographs of mock transfected HEK293 cells harvested after 48 hours of no treatment (12,000x magnification) or DMSO treatment (10,000x magnification). F Representative micrographs of HEK293 cells expressing FLAG-eVP40 and harvested after 48 hours of no treatment (6,500x magnification), DMSO treatment (12,000x magnification), or the indicated concentration of fendiline (1 μM fendiline at 11,419x magnification and 5 μM fendiline at 15,000x magnification).

Data information: VLPs: virus-like particles; SEM: scanning electron microscopy; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; HRP: horseradish peroxidase.
Phosphatidylserine clustering by the Ebola virus matrix protein is a critical step in viral budding (2022)
Figure 7

Figure 7

Effect of fendiline on eVLP morphology and TIM-1 dependent eVLP entry.

A-C TEM analysis of eVLP morphology. A Representative transmission electron micrographs of eVLPs purified from HEK293 cells expressing FLAG-eVP40 and eGP following 48 hours of DMSO (left panel) or 5 µM fendiline treatment (right panel). B Quantification of eVLP length (µm) of DMSO-derived eVLPs (black) and fendiline-derived eVLPs (blue). N>50 VLPs per biological replicate, n=3 biological replicates. Values are reported as mean ± s.d. A two-tailed t-test was performed (**p=0.0139). C Quantification of eVLP diameter (nm) of DMSO-derived eVLPs (black) and fendiline-derived eVLPs (blue). N>50 VLPs per biological replicate, n=3 biological replicates. Values are reported as mean ± s.d. A two-tailed t-test was performed (*p=0.0430).

D, E Fluorescence based DiI TIM-1 dependent entry assay. D Representative confocal images from the DiI-entry assay comparing entry of eVLPs produced from DMSO (top panel) and fendiline-treated HEK293 cells (bottom panel) into target cells (HEK293 cells transiently expressing increasing amounts of TIM-1; 0.0 µg, 0.5 µg, 1.0 µg). A stack of 10 frames was acquired for each image. DiI (initially red) was recolored to yellow for easier observation in print; blue (Hoechst 3342 stain); scale bar = 10 µm. E Quantification of eVLP entry was performed by calculating the total number of DiI punctate / the total number of DiI-positive cells. Three images from each z-stack were quantified. N=9 cell per replicate, n=3 biological replicates. Values are reported as mean ± s.d. A one-way ANOVA was performed with multiple comparisons against the 0.0 µg TIM-1 condition for both DMSO- and fendiline derived eVLPs.(****p<0.0001; **p=0.0093).

Data information: eVLP: entry-competent virus-like particles; TEM: transmission electron microscopy; TIM-1: t-cell immunoglobulin receptor-1; eVLPs: entry-competent VLPs; eGP: Ebola virus glycoprotein; DiI: 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate.
NSMCE2 suppresses cancer and aging in mice independently of its SUMO ligase activity (2015)
Figure 1

NSMCE2 colocalizes with BRCA1 at sites different to DSB

Immunohistochemistry of NSMCE2 on mouse testis. The inset illustrates the accumulation of NSMCE2 on the XY body. Scale bar, 50 μm.
Immunofluorescence (IF) of NSMCE2 and SCP3 on isolated mouse spermatocytes. Threads of NSMCE2 can be detected on regions of weak SCP3 signal, indicative of asynapsis (arrowheads). Scale bar, 2.5 μm.
Colocalization of NSMCE2 and BRCA1 at the XY body of mouse spermatocytes.
Representative image of NSMCE2 and BRCA1 foci in control and MMS‐treated MEFs. Scale bar, 2.5 μm.
Quantification of NSMCE2 and BRCA1 foci in wild‐type MEFs exposed to IR (10 Gy; 1‐h recovery), MMS (1 mM for 1 h; 24‐h recovery), and PARP inhibitor (1 μM for 1 h; 24‐h recovery). Data indicate mean values and are representative of three independent experiments. Error bars indicate SD.
Representative image of IR‐ (10 Gy; 1‐h recovery) and MMS‐ (1 mM for 1‐h; 24‐h recovery) induced NSMCE2 and γH2AX foci in MEFs. Scale bar, 2.5 μm.
NSMCE2 suppresses cancer and aging in mice independently of its SUMO ligase activity (2015)
Figure 2

NSMCE2 is essential for early embryonic development

Scheme illustrating the Nsmce2 genetrap allele (Nsmce2GT) used in this study.
Comparison of the wt and mutant NSMCE2 protein. Note that the trapped protein lacks most of the C‐terminal region including the SUMO ligase domain.
Brightfield microscopy of 2.5 dpc embryos obtained from crosses of Nsmce2GT/+ mice. The right panel is of the same 2 embryos shown on the left panel, 36 h after being cultured in vitro. The arrow indicates embryos that showed asymmetric cell sizes when isolated, which degenerated upon culture. These embryos were identified as Nsmce2GT/GT through genotyping.
Examples of the three genotypes found at Mendelian frequencies (see Appendix Table S1) on 2.5 dpc embryos.
Examples of the type of nuclei found on Nsmce2GT/GT and Nsmce2+ (+/+ or +/GT) 2.5 dpc embryos, 12 h after in vitro culture. Mutant embryos were identified due to the absence of NSMCE2 signal (green). DAPI was used to stain DNA (blue). White arrows indicate examples of cells with condensed chromosomes or irregular shapes, which are hardly seen in wild type embryos.
NSMCE2 suppresses cancer and aging in mice independently of its SUMO ligase activity (2015)
Figure 3

NSMCE2 SUMO ligase activity is largely dispensable in mice

Scheme illustrating the Nsmce2‐mutant allele (Nsmce2SD) used in this study, which was generated through introducing the mutations on exon 6 (see ).
Aminoacid and nucleotide sequences of the Nsmce2‐mutant allele. The SUMO ligase‐inactivating mutation used C185S; H187A (SD), which is the same originally described in yeast (Andrews et al, ) and which has been used as a SUMOylation‐deficient model in multiple studies. The nucleotide sequence derives directly from sequencing DNA from Nsmce2SD/SD mice.
Immunoprecipitation of SMC5 (and IgG, as a control) from a 48‐h culture of B lymphocytes of wt and Nsmce2SD/SD animals. NSMCE2 and SMC6 levels on the immunoprecipitates were analyzed by WB. The image of the Ponceau staining is shown as a loading control.
IF of NSMCE2 foci in MMS‐treated Nsmce2+/+ and Nsmce2SD/SD MEFs. Scale bar, 2.5 μm.
Representative picture of 6‐month‐old Nsmce2+/+ and Nsmce2SD/SD littermates.
Kaplan–Meyer survival curves of Nsmce2+/+ and Nsmce2SD/SD mice. The P‐value was calculated with the Mantel–Cox long‐rank test.
NSMCE2 suppresses cancer and aging in mice independently of its SUMO ligase activity (2015)
Figure 4

NSMCE2 is a haploinsufficient tumor suppressor

A Kaplan–Meyer survival curves of Nsmce2+/+ and Nsmce2+/GT mice. The P‐value was calculated with the Mantel–Cox long‐rank test. ***P 0.001.
B Tumor incidence found on Nsmce2+/+ and Nsmce2+/GT mice at the time of death (i.e. moribund mice).
C NSMCE2 levels evaluated by Western blot in 2 independent pairs of Nsmce2+/+ and Nsmce2+/GT MEFs. β‐actin was used as a loading control. Numbers shown bellow indicate the relative amounts of NSMCE2 in each sample.
D Sister chromatid exchange (SCE) events found per metaphase on Nsmce2+/+ and Nsmce2+/GT MEFs. ***P 0.001.
E, F Relative percentages of micronuclei (E) and polynucleated cells (F) on cultures of Nsmce2+/+ and Nsmce2+/GT MEFs. Data are representative of three independent experiments. Error bars indicate SD. ***P 0.001.
NSMCE2 suppresses cancer and aging in mice independently of its SUMO ligase activity (2015)
Figure 5

NSMCE2 suppresses recombination and micronuclei formation

Scheme illustrating the Nsmce2 conditional allele (Nsmce2lox) used in this study. A cross with Flp recombinase‐expressing mice eliminated the FRT‐PGK‐neo‐FRT cassette present on the initial allele (Nsmce2lox‐neo) (see ). In the conditional allele, exon 3 is flanked by loxP sites allowing for its deletion in the presence of Cre recombinase.
NSMCE2 levels assessed by WB in UQ.CreERT2 transgenic Nsmce2+/+ and Nsmce2lox/lox littermate MEFs, 24 h after exposure to 4‐OHT. Tubulin levels are shown as a loading control.
Clonogenic assay on immortalized UQ.CreERT2/Nsmce2lox/lox MEFs. The picture shows the colonies detected after 10 days of growth in the presence or absence of 4‐OHT, upon staining with crystal violet.
NSMCE2 and BRCA1 foci in MMS‐treated (1 mM for 1 h; 24‐h recovery) UQ.CreERT2/Nsmce2lox/lox MEFs, 48 h after being treated (or not) with 4‐OHT. Scale bar, 5 μm.
SCE events per metaphase on UQ.CreERT2/Nsmce2lox/lox MEFs grown in the presence of 4‐OHT for 72 h. ***P 0.001.
Example of SCE events (arrows) found on Nsmce2‐deleted MEFs.
SCE events at telomeres on UQ.CreERT2/Nsmce2lox/lox MEFs grown in the presence of 4‐OHT for 72 h, as detected by CO‐FISH (see ). Data are representative of three independent experiments. Error bars indicate SD. *P 0.05.
Telomere lengths of UQ.CreERT2 Nsmce2lox/lox MEFs treated or not with 4‐OHT for 72 h.
Example of the intensity of telomere signals (yellow) used for (H).
Percentage of cells with micronuclei found on cultures of UQ.CreERT2/Nsmce2lox/lox MEFs treated or not with 4‐OHT for 72 h. Data are representative of three independent experiments. Error bars indicate SD. ***P 0.001.
NSMCE2 suppresses cancer and aging in mice independently of its SUMO ligase activity (2015)
Figure 6

Impact of NSMCE2 deletion in embryos and adult mice

Intrauterine dwarfism observed upon NSMCE2 deletion on 17.5 dpc embryos. Pregnant females were treated at 14.5 dpc for 3 days with intraperitoneal injections of 4‐OHT. The image represents the difference in size observed between Nsmce2+/+ and Nsmce2lox/lox embryos carrying the UQ.CreERT2 transgene (Nsmce2+/+ and Nsmce2lox/lox, from now on).
Representative picture of Nsmce2+/+ and Nsmce2lox/lox animals fed for 40 weeks with a 4‐OHT‐containing diet (see also Movies EV1 and EV2).
Images illustrating the presence of altered pigmentation problems that arise on Nsmce2lox/lox mice.
Percentage of fat against total body mass on Nsmce2+/+ and Nsmce2lox/lox animals. ***P 0.001.
White blood cell counts (WBC) from Nsmce2+/+ and Nsmce2lox/lox animals. ***P 0.001.
Percentage of γH2AX‐positive micronuclei found on the intestinal epithelia of Nsmce2+/+ and Nsmce2lox/lox animals (see Appendix Fig S11). Data are representative of three independent experiments. Error bars indicate SD. *P 0.05.
NSMCE2 suppresses cancer and aging in mice independently of its SUMO ligase activity (2015)
Figure 7

NSMCE2 is dispensable for DNA replication

A WB illustrating the depletion of NSMCE2 in B cells isolated from 2‐month‐old CD19+/Cre animals of the indicated genotypes, 48 h after being stimulated in vitro with lipopolysaccharide (LPS). Tubulin was used as a loading control.
B Representative FACS analysis from the B‐cell cultures mentioned in (A). DNA content was measured with propidium iodide (PI). Note the presence of cells with > 4n DNA content in NSMCE2‐deleted cells.
C, D Frequency of origin firing (C) and fork rate (D) were analyzed by DNA fiber analyses (see ) from fibers prepared from B‐cell cultures explained in (A). No significant differences were observed between Nsmce2+/+ and Nsmce2lox/lox B cells. Data are representative of two independent experiments. Error bars indicate SD. Between 200 and 300 tracks were measured to estimate fork rate and 300–500 for the calculation of origin firing frequencies.
E Representative images of the DNA fibers used for the analysis explained in (C, D). CldU (red) and IdU (green) channels are shown.
NSMCE2 suppresses cancer and aging in mice independently of its SUMO ligase activity (2015)
Figure 8

Synthetic lethality between NSMCE2 and BLM in B cells

Representative pictures of the spleens from 2‐month‐old CD19+/Cre animals of the indicated genotypes.
Numbers of B and T cells from 2‐month‐old CD19+/Cre animals of the indicated genotypes evaluated by flow cytometry (B220: B‐cell marker; CD3: T‐cell marker).
SCE events per metaphase on B cells obtained from CD19+/Cre animals of the indicated genotypes, 72 h after being stimulated in vitro with LPS. **P 0.01, ***P 0.001.
Analysis of forward (FSC) and side (SSC) scatter parameters of B cells obtained from CD19+/Cre animals of the indicated genotypes, 48 h after being stimulated in vitro with LPS. The emergence of bigger cells is noticeable on cells lacking both BLM and NSMCE2.
Representative images of B‐cell cultures of the indicated genotypes 48 h after being stimulated in vitro with LPS. Insets provide a magnified view of an individual cell (arrowhead) for better comparison of the observed differences in cell size. Scale bar, 100 μm.
Examples of the types of nuclei found on Blm and Nsmce2 double‐mutant B cells (compared with wild‐type B cells), in cells obtained from (E). DAPI was used to stain DNA. Whereas wt nuclei are regular in size, double‐mutant cells invariably presented enlarged, multilobulated, and irregular nuclei. Scale bar, 2.5 μm.
Tyrosine kinase inhibition increases functional parkin-Beclin-1 interaction and enhances amyloid clearance and cognitive performance. (2013)
Figure 1

Tyrosine Kinase inhibition restores parkin-Beclin-1 interaction

Graph represents brain levels of TKIs over a 24 hr period after IP injection with Imatinib, Nilotinib, Bosutinib and DMSO (N = 7). [Correction added after publication on 4 July 2013: The y axis of the graph shown in Fig. 1A has been corrected from “concentration (mM)” to “concentration (nM)”]

WB in Tg-APP total brain lysates on 4–12% NuPAGE SDS gel (Invitrogen) show total Abl (top blot) T412 Abl (2nd blot), soluble parkin level (3rd blot), and LC3 (4th blot) relative to MAP-2 (N = 9).

Quantitative ELISA showing soluble (STEN extract) and insoluble (4 M urea extract) mouse parkin in Tg-APP (N = 9). Parkin−/− brains were used as a specificity control.

(D) Blots represent immunoprecipitated Beclin-1 in mice probed with parkin antibody, and (E) control IgG in parallel with immunoprecipitates, (F) immunoprecipitated Beclin-1 probed with parkin and the reverse experiment in post-mortem human AD cortex analyzed on 4–12% SDS-NuPAGE gel.

In situ proximity ligation assay (PLA) shows endogenous parkin-Beclin-1 complexes in (G) WT C57BL/6 mice (N = 5) and (H) parkin−/− as control.

PLA in Tg-APP mice IP injected once daily for 3 weeks with (I) DMSO (J) 5 mg/kg Bosutinib and (K) 10 mg/kg Nilotinib (N = 5).

PLA in human post-mortem brains in the (L) cortex of a normal subject and (M) cortex of an AD patient; the hippocampus of (N) a normal subject and (O) an AD patient; the caudate of (P) a normal subject and (Q) an AD patient.

Graph represents human Aβ1–42 ELISA in rat B35 neuroblastoma cells transfected with human cDNA Aβ1–42 (or LacZ) or Beclin-1 shRNA for 24 hr, and then treated with 1 μM Bosutinib for an additional 24 hr (N = 12). *Significantly different to control or as indicated, Mean ± SEM, ANOVA with NeumannKeuls multiple comparison.
Tyrosine kinase inhibition increases functional parkin-Beclin-1 interaction and enhances amyloid clearance and cognitive performance. (2013)
Figure 2

Bosutinib decreases Aβ levels and eliminates plaques in AD models

Staining of 20 μm brain sections shows plaque formation with 6E10 antibody and DAB in the brain in different (A,B) Tg-APP + DMSO and (C) thioflavin-S staining in Tg-APP treated with DMSO (N = 7). (D,E) Tg-APP treated with 5 mg/kg Bosutinib for 3-weeks and (F) thioflavin-s staining.

Staining of 20 μm thick brain sections shows (G) parkin, (H) Aβ1–42 and (I) merged figure in cortex of Tg-APP mice after 3 weeks of DMSO treatment, and (J) parkin, (K) Aβ1–42 and (L) merged figure in cortex of Tg-APP mice after 3 weeks of 5 mg/kg Bosutinib treatment. (M) Parkin, (N) Aβ1–42 and (O) merged figure in hippocampus of Tg-APP mice after 3 weeks of DMSO treatment, and (P) parkin, (Q) Aβ1–42 and (R) merged figure in cortex of Tg-APP mice after 3 weeks of Bosutinib treatment (N = 7).

(S) Graphs represent quantification of amyloid plaques in Tg-APP with and without Bosutinib. WB in Tg-APP total brain lysates on 4–12% NuPAGE SDS gel (Invitrogen) showing (T) levels of BACE1 (1st blot), ADAM-10 (2nd blot) and presinilin-1 (3rd blot) relative to actin and (U) total APP (top blot), CTFs (2nd blot) and phospho-tyrosine (3rd blot) relative to actin (N = 9).
Tyrosine kinase inhibition increases functional parkin-Beclin-1 interaction and enhances amyloid clearance and cognitive performance. (2013)
Figure 3

Chronic treatment with TKI alters brain amyloid level*Significantly different to control, Mean ± SEM, ANOVA with Neumann Keuls multiple comparison, p < 0.05.

Graphs represent ELISA of human Aβ1–42 in (A) brain levels and (B) blood Aβ1–42 levels in 8-month old Tg-APP mice injected I.P. every other day for 6 weeks with Bosutinib or Nilotinib (N = 10).

ELISA of human soluble and insoluble brain (C) Aβ1–42 and (D) Aβ1-40 levels as well as (E) mouse p-Tau levels in 8-month old Tg-APP mice injected IP daily for 3 weeks with 5 mg/kg Bosutinib (N = 9).

WB in Tg-APP total brain lysates on 4–12% NuPAGE SDS gel (Invitrogen) show total Tau (1st blot), serine 396 p-Tau (2nd blot), threonine 231 (AT180, 3rd blot) and serine 199 p-Tau (AT8, 4th blot) relative to actin (N = 7).