EMBO/soda-nel huggingface dataset:
Nox4 regulates InsP3 receptor-dependent Ca2+ release into mitochondria to promote cell survival (2020)
Figure 3

Figure 3. Nox4 is localized at the mitochondria-associated ER membrane (MAM).

A Immunoblotting of subcellular fractions from serum-starved rat cardiomyoblasts (H9c2 cells). Input: homogenate before fractionation; Cyto: cytosol; ER: endoplasmic reticulum; Crude M: crude mitochondrial fraction, also containing MAM; Pure Mito: mitochondria; MAM: mitochondria associated-ER membranes. Cytochrome c (Cyt c) was used as a mitochondrial marker, VDAC as an outer mitochondrial membrane marker, calnexin as an ER marker, Sigma1R and FACL4 as MAM markers, and tubulin as a cytosolic marker.

B Immunoblotting of subcellular fractions from rat heart.

C Immunoblotting of subcellular fractions from rat kidney.

D Electron micrographs showing subcellular localization of Nox4 in wild-type (WT) and Nox4KO mouse hearts, and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). hiPSC-CM were depleted of Nox4 using a lentiviral shRNA (Nox4KD) or infected with a control lentivirus. FACL4 was used as a MAM marker. Arrow heads show peri-mitochondrial localization of Nox4 and FACL4, in the proximity of ER cisternae and ribosomes. M=mitochondria, ER=endoplasmic reticulum, T=T-tubule. Scale bars: 100 nm.
Nox4 regulates InsP3 receptor-dependent Ca2+ release into mitochondria to promote cell survival (2020)
Figure 4

Figure 4. In situ proximity ligation of Nox4 and MAM markers.

A Simplified photomicrographs of proximity ligation studies in WT and Nox4KO MEFs, showing cell borders, nuclei (blue) and yellow dots corresponding to co-localization of proteins. Quantification of the number of dots/cell in each condition is shown to the right. Proximity was tested for the following protein couples: FACL4/Nox4, InsP3R/Nox4, and FACL4/InsP3R. Scale bars: 10 µm. n=3/group (with >30 cells/individual experiment). Controls are shown in Appendix Figure S4B and the original unsimplified photographs in Appendix Figure S4E.

B Simplified photomicrographs of proximity ligation staining in rat cardiomyocytes transfected with Nox4 siRNA or a scrambled control (siScr), with quantification as in (A). Proximity was tested for the FACL4/Nox4 protein couple. Scale bars: 10 µm. n=3/group (with >30 cells/individual experiment). Controls and other protein couples are shown in Appendix Figure S4C and the original unsimplified photographs in Appendix Figure S4F.

C Simplified photomicrographs of proximity ligation staining in hiPSC-CM (human cardiomyocytes) transduced with Nox4 shRNA or scrambled control, with quantification as in (A). Proximity was tested for the FACL4/Nox4 protein couple. Scale bars: 10 µm. n=3/group (with >30 cells/individual experiment). Controls and other protein couples are shown in Appendix Figure S4D and the original unsimplified photographs in Appendix Figure S4G.

Data information: Data are mean ± SEM. **** P<0.0001 vs corresponding control (unpaired t-test).
Nox4 regulates InsP3 receptor-dependent Ca2+ release into mitochondria to promote cell survival (2020)
Figure 5

Figure 5. Regulation of Akt activation and InsP3R phosphorylation by Nox4.

A Immunoblotting of subcellular fractions from serum-starved cardiomyoblasts for InsP3R, Akt and PP2a, as in Fig 3A.

B Fractionation of crude mitochondrial fractions (containing mitochondria and MAM) from WT MEFs on a sucrose gradient (5-60%) and immunoblotting for relevant proteins. Fraction size: 200 µL. Representative of 3 independent experiments.

C Immunoblotting for phosphorylated and total Akt or InsP3R protein in crude mitochondrial fractions from serum-starved WT and Nox4KO MEFs. FACL4 was used as a loading control for the MAM. InsP3R was first immunopreciptated and then the precipitate was immunoblotted for total InsP3R and for the phosphorylated Akt-substrate motif RXRXX(pS/T) (p-InsP3R). Representative immunoblots are shown at the top and mean data at the bottom. Cat = PEG-catalase. Nox4KO MEFs were transfected either with active Nox4 or a Nox4P437H mutant (Mut). n=3/group.

D ROS levels indexed in MEFs using H2O2 specific HyPer probes targeted to the MAM (FACL4 HyPer) or the cytosol (Cyto HyPer). Imaging was performed after serum starvation for 12h. Representative photomicrographs of the fluorescence ratio are shown to the right and mean data for changes in fluorescence ratio presented to the left. The signal obtained using corresponding ROS-insensitive SypHer probes was used to correct for any pH-induced changes in fluorescence; the change in fluorescence ratio between HyPer and corresponding SypHer probe (ΔR) for each condition is reported. n=5 independent cell preparations/group, with at least 20 cells imaged/preparation. Scale bars: 10 µm.

Data information: Data are mean ± SEM. *** P<0.001; **** P<0.0001 among compared groups or vs control. ### P<0.001; ## P<0.01 compared to Nox4KO (1-way ANOVA).
Nox4 regulates InsP3 receptor-dependent Ca2+ release into mitochondria to promote cell survival (2020)
Figure 6

Figure 6. Functional impact of Nox4-dependent regulation of InsP3R.

A Schematic of putative mechanism underlying Nox4-mediated regulation of calcium transfer via InsP3R channels to mitochondria. Akti: Akt inhibitor; XeC: Xestospongin C (InsP3R blocker).

B Basal mitochondrial calcium levels in serum-starved WT and Nox4KO MEFs in the presence or absence of Akti (1 µmol/L) normalized versus WT MEF control. n=6/group with >20 cells imaged per experiment.

C Effect of Akti (1 µmol/L) or XeC (1 µmol/L) on histamine-induced changes in mitochondrial calcium levels in MEFs. The time course of changes in calcium levels is shown on the left and the mean data for peak mitochondrial calcium levels on the right. n=3/group with >40 cells imaged per experiment.

D Quantification of cell death in serum-starved WT and Nox4KO MEFs in the absence or presence of Akti or XeC. n=6/group.

E Cardiac troponin I (cTnI) levels as a marker of cardiomyocyte necrosis in the perfusate of isolated WT and Nox4KO hearts subjected to ischemia-reperfusion (I/R). XeC was added at a final concentration of 2 µmol/L prior to ischemia. n=6-7/group.

F Immunoblotting for the phosphorylation levels of Akt and InsP3R in crude mitochondrial fractions from WT and Nox4KO hearts after I/R. InsP3R was first immunopreciptated and then the precipitate was immunoblotted for total InsP3R and for the phosphorylated Akt-substrate motif RXRXX(pS/T) (p-InsP3R). Mean data shown to the right. n=3/group.

G Cardiac left ventricular contractile function in isolated Langendorff-perfused WT and Nox4KO hearts at baseline (Basal) and then after I/R. Hearts were treated with XeC (1 μmol/L) or vehicle control for 20 min prior to ischemia. RPP, heart rate x left ventricular pressure product; DEVP, left ventricular developed pressure. n=6-7/group.

Data information: Data are mean ± SEM. * P<0.05, ** P<0.01; **** P<0.0001 among compared groups or vs control. # P<0.05; #### P<0.0001 among different groups within the same treatment or among different treatments within the same genotype. B,G: 1-way ANOVA; C-E: 2-way ANOVA; F: unpaired t-test.
β-catenin signaling modulates the tempo of dendritic growth of adult-born hippocampal neurons (2020)
Figure 1

Fig.1 Canonical Wnt signaling activity in the adult hippocampus

A Schematic representation of the LacZ alleles of the BATGAL and Axin2LacZ/+ reporter mice for canonical Wnt signaling activity.

B Representative images showing co-expression of the stage-specific markers Nestin, Tbr2, Doublecortin (DCX), and Calbindin (all in green), with the β-Galactosidase (β-Gal, red) reporter in 8-week old BATGAL and Axin2LacZ/+ mice. Nuclei are counterstained with DAPI (in blue). Scale bar= 10µm. Insets show a 1.5x magnification of selected cells (position indicated by dashed box). Scale bar= 5µm.

C Fraction of Nestin-, Tbr2-, DCX- and Calbindin-positive cells expressing β-Gal in BATGAL and Axin2LacZ/+ animals show stage specific canonical Wnt signaling during adult hippocampal neurogenesis [BATGAL: Tbr2 vs. DCX p=0.0056, DCX vs. Calbindin p<0.0001; Axin2LacZ: Nestin vs. Tbr2 p=0.0003, Tbr2 vs. DCX p=0.0115, DCX vs. Calbindin p<0.0001] (n= 3 animals per mouse model and marker).

D Detection of the corrected total cell fluorescence (CTCF) of the β-Gal reporter in Nestin-, Tbr2-, DCX- and Calbindin-positive cells in BATGAL mice corroborate stage specific activity pattern of canonical Wnt signaling during lineage progression [Nestin vs. Tbr2 p <0.0001, Tbr2 vs. DCX p=0.0029, DCX vs. Calbindin p<0.0001] (Nestin: n= 236 cells, Tbr2: n= 134 cells, DCX: n= 200 cells, Calbindin: n= 300 cells).

E BrdU pulse chase scheme. BrdU was injected intraperitoneally (i.p.) three times: i) every two hours for 30-minute time point [0 days post injection (dpi)] ii) every 24 hours for all other time points. Animals were sacrificed 30-minutes after the final BrdU injection

F Quantification of BrdU+ cells expressing β-Gal reveals biphasic activity of canonical Wnt signaling during adult hippocampal neurogenesis [0 vs. 7dpi p=0.0036, 7 vs. 28dpi p=0.0115, 7 vs. 42dpi p=0.0007] (0dpi time-point; n= 4 animals), 3dpi (n= 3 animals), 7dpi (n= 3 animals), 14dpi (n= 4 animals), 28dpi (n= 6 animals) and 42dpi (n= 6 animals).

G Total cell fluorescence of β-Gal in BrdU+ cells indicate a biphasic pattern in canonical Wnt signaling strength during lineage progression [0 vs. 3dpi p<0.0001, 7 vs. 14dpi p<0.0001, 14 vs. 28dpi p<0.0001, 28 vs. 42dpi p=0.0333] (0dpi n= 194 cells, 3dpi n= 245 cells, 7dpi n= 224 cells, 14dpi n= 72 cells, 28dpi n= 103 cells and 42dpi n= 114 cells)

Data information: Data represented as mean ± SEM. Significance was determined using Kruskal-Wallis test followed by Dunn´s multiple comparisons test, significance levels are displayed in GP style [p<0.0332 (*), p<0.0021 (**) and p<0.0001 (****)].
β-catenin signaling modulates the tempo of dendritic growth of adult-born hippocampal neurons (2020)
Figure 2

Fig. 2 Loss of canonical Wnt signaling and sustained canonical Wnt signaling impair dendritogenesis of adult-born neurons

A Experimental scheme of retroviral injection paradigm. Adult mice were stereotactically co-injected with the MMLV CAG-dnLEF-IRES-GFP (dnLEF) and CAG-RFP (control) and were sacrificed 17dpi.

B Representative images of transduced adult-born neurons at 17dpi. CAG-dnLEF-IRES-GFP (dnLEF, green) and CAG-RFP (control, red) double transduced cells (arrows) and CAG-RFP single transduced cells (arrowheads). Scale bar= 20 µm

C Representative reconstructions of control and dnLEF neurons. Scale bar= 20 µm

D Analysis of morphology showed a reduction in dendritic length in dnLEF transduced neurons [p<0.0001], while the number of branch points remained comparable [p=0.7914]. Sholl analysis displayed a reduction in dendritic complexity in dnLEF transduced neurons and indicated decreased growth of terminal dendritic branches [p<0.0001] (control: n= 20 cells from 3 animals, dnLEF: n= 38 cells from 6 animals).

E DnLEF neurons displayed basal dendrites [p<0.0001] (control: n= 20 cells from 3 animals, dnLEF: n= 38 cells from 6 animals).

F Schematic representation of the conditional alleles of the Ctnnb1(ex3)fl (β-catex3) mouse model and the retroviral paradigm used to analyze canonical Wnt signaling gain of function in neural progenitors. Control animals harbor the wildtype allele for Ctnnb1.

G Representative images depicting CAG-GFP-IRES-Cre transduced adult-born neurons in control and β-catex3 mice at 17dpi. Scale bar= 20µm

H Representative reconstructions of control and β-catex3 neurons. Scale bar= 20µm

I Quantification showed decreased dendritic length of β-catex3 neurons [p<0.0001]; no difference was apparent in number of branch points [p=0.3256]. Sholl analysis displayed a less complex dendritic tree of β-catex3 neurons [p<0.0001] (control: n= 20 cells from 5 animals, β-catex3: n= 20 cells from 5 animals).

J The number of basal dendrites was increased in β-catex3 neurons [p=0.0003] (control: n= 20 cells from 5 animals, β-catex3: n= 20 cells from 5 animals).

Data information: Data represented as mean ± SEM, significance was determined using two-way ANOVA for Sholl analysis and two-tailed Mann-Whitney U Test for all other analyses, significance levels were displayed in GP style [p<0.0002 (***) and p<0.0001 (****)].
β-catenin signaling modulates the tempo of dendritic growth of adult-born hippocampal neurons (2020)
Figure 3

Fig. 3 Enhanced canonical Wnt signaling activity in immature neurons of young adult mice modulates dendritic growth and spine formation

A Experimental paradigm: Tamoxifen (TAM) was applied i.p. every 12 hours for five days to control and β-catex3 iDCX mice and animals were analyzed 3 and 13 days post Tamoxifen (dpT).

B Representative reconstructions of control and β-catex3 iDCX neurons at 3dpT. Scale bar= 20µm.

C Analysis of morphology showed increased dendritic length in β-catex3 iDCX neurons at 3dpT [p=0.0320]. The number of branch points was unaltered [p=0.0651]. Sholl analysis indicated increased growth of terminal dendrite branches in neurons of β-catex3 iDCX mice [p<0.0001] (control: n = 25 cells from 9 animals; β-catex3 iDCX: n = 25 cells from 8 animals).

D Analysis of morphology showed no difference in dendritic length [p=0.1029] and branch point number [p=0.3726] between experimental groups at 13dpT. Sholl analysis indicated a decrease in length of terminal dendrites in neurons of β-catex3 iDCX mice [p<0.01] (control: n= 23 cells from 9 animals, β-catex3 iDCX: n= 26 cells from 8 animals).

E Spine densities were comparable between experimental groups at 3dpT [iML p=0.1512, oML p=0.3841]. 13dpT β-catex3 iDCX neurons showed significantly increased spine density in the inner molecular layer [iML p<0.0001 , oML p=0.0402] (3dpT: control: n= 20 cells from 3 animals, β-catex3 iDCX: n= 20 cells from 4 animals; 13dpT control: n= 20 cells from 5 animals, β-catex3 iDCX: n= 20 cells from 7 animals). Representative images showing dendritic segments in the inner molecular layer of the DG at 13dpT. Scale bars= 1µm.

F Representative images of recombined (GFP+) neurons co-expressing Prox1 (grey) as a marker for neuronal fate and the stage specific markers DCX (red) for immature neurons and Calbindin (red) for mature neurons. Arrows and arrowheads indicate marker negative and marker positive cells, respectively. Scale bar = 10µm.

G Fraction of DCX expressing neurons is slightly reduced in β-catex3 iDCX at 3dpT [p=0.0338], while fraction of Calbindin expressing cells is slightly increased in β-catex3 iDCX at 3dpT [p=0.0345]. At 13dpT no difference is visible in stage specific marker expression [DCX p=0.8564, Calbindin p=0.0841] (3dpT: control: n= 12 animals, β-catex3 iDCX: n= 12 animals, 13dpT: control: n= 10 animals, β-catex3 iDCX: n= 14 animals).
β-catenin signaling modulates the tempo of dendritic growth of adult-born hippocampal neurons (2020)
Figure 4

Fig. 4: Analysis of birthdated immature neurons in young adult mice confirms modulation of dendritic growth and spine formation by canonical Wnt signaling activity

A Experimental scheme for birthdating of adult-born neurons and recombination. For morphology analysis, CAG-RFP was stereotactically injected into the DG (C-F), for marker expression analysis, BrdU was injected i.p. every 24 hours for three days (B). Tamoxifen was applied i.p. every 12 hours for five days from day 10 to day 14. Mice were sacrificed 3 days post Tamoxifen (dpT) and 13dpT, which corresponds to 17dpi and 27dpi, respectively.

B Expression of DCX and Calbindin in BrdU+ neurons was comparable between experimental groups at 3dpT and 13dpT (control: n= 5 animals, β-catex3 iDCX: n= 5 animals).

C Representative reconstructions of control and β-catex3 iDCX neurons expressing RFP at 3dpT. Scale bar= 20µm.

D Quantification of dendritic length [p=0.0003], branch points [p=0.0433], and Sholl analysis [p<0.0001] showed a higher dendritic complexity of β-catex3 iDCX neurons at 3dpT (control: n= 22 cells from 4 animals, β-catex3 iDCX: n= 19 cells from 5 animals).

E Representative reconstructions of control and β-catex3 iDCX neurons expressing RFP at 13dpT. Scale bar= 20µm.

F Quantification of dendritic length [p<0.0001], and branch points [p=0.0495], and Sholl analysis [p<0.0001] showed a lower dendritic complexity of β-catex3 iDCX neurons at 13dpT (control: n= 20 cells from 4 animals, β-catex3 iDCX: n= 19 cells from 5 animals).

Data information: Data represented as mean ± SEM, significance was determined using two-way ANOVA for Sholl analysis and two-tailed Mann-Whitney U Test for all other analyses, significance levels are displayed in GP style [p<0.0332 (*), p<0.0021 (**) and p<0.0002 (***), p<0.0001 (****)].
β-catenin signaling modulates the tempo of dendritic growth of adult-born hippocampal neurons (2020)
Figure 5

Fig. 5 Canonical Wnt signaling activity is decreasing with increasing age

A Representative overview images and quantification of the β-Galactosidase reporter signal in the DG of 8-week old, 24-week old and 36-week old BATGAL mice. Note the decrease in reporter signal with increasing age (n= 4 animals per age, 200-300 cells per animal) [8 weeks vs 24 weeks p=0.0268, 8 weeks vs 36 weeks p=0.0268]. Scale bar= 100µm.

B Representative images of BrdU+ cells (red) expressing DCX (grey) or β-Galactosidase (green) at 14 days post BrdU injection (dpBrdU), 28dpBrdU and 42dpBrdu in 8-week old and 24-week old BATGAL mice. Scale bar= 10µm.

C Percentage of BrdU+ cells expressing DCX in 8-week old and 24-week old BATGAL mice (14dpi: n= 3 animals, 28dpi: n= 3 animals, 42dpi: n= 3 animals). Note the prolonged expression of DCX in 24-week old mice [28dpBrdU p=0.0155].

D Percentage of BrdU+ cells expressing the β-Galactosidase reporter in 8-week old and 24-week old BATGAL mice (14dpi: n= 3 animals, 28dpi: n= 3 animals, 42dpi: n= 3 animals). Note the reduced reporter expression in 24-week old mice [28dpBrdU p=0.0117, 42dpBrdU p=0.0034].

Data information: Data represented as mean ± SEM, significance was determined using two-way ANOVA followed by Sidak´s multiple comparison analysis, significance levels are displayed in GP style [p<0.0332 (*) and p<0.0021 (**)].
β-catenin signaling modulates the tempo of dendritic growth of adult-born hippocampal neurons (2020)
Figure 6

Fig. 6 Precocious activation of canonical Wnt signaling in middle-aged mice rescues aging phenotype

A Experimental scheme to analyze the impact of β-catenin signaling activity on adult-born neuron development in middle-aged mice. Tamoxifen (TAM) was applied i.p. every 12 hours for five days. Animals were analyzed 3 and 13 days post Tamoxifen (dpT).

B Representative reconstructions of control and β-catex3 iDCX neurons in 24-week old mice at 3dpT. Scale bar = 20µm.

C Dendritic length [p<0.0001] and number of branch points [p<0.0001] was increased in 24-week-old β-catex3 iDCX compared to 24-week-old controls at 3dpT (control: n= 21 cells from 5 animals, β-catex3 iDCX: n= 20 cells from 6 animals). At 13dpT dendritic length [p=0.5788] and number of branch points [p=0.4978] were comparable (control: n= 21 cells from 5 animals, β-catex3 iDCX: n= 20 cells from 8 animals).

D Representative images showing dendritic segment of the inner molecular layer of the DG in 24-week old mice at 13dpT. Quantification of the number of spines showed increased spine density in inner molecular and outer molecular layer in 24-week old β-catex3 iDCX mice at 13dpT [iML p=0.0047, oML p<0.0001] (control n=19 cells from 4 animals, β-catex3 iDCX: n= 20 cells from 4 animals). Scale bars = 1µm.

E Representative images of recombined (GFP+, green) neurons in 24-week old animals at 13dpT co-expressing Prox1 (grey) as a marker for neuronal fate and the stage specific markers DCX (red) for immature neurons and Calbindin (red) for mature neurons. Arrows and arrowheads indicate marker negative and marker positive cells, respectively. Scale bar= 10µm.

F Quantification of marker expression. The fraction of Calbindin expressing cells was increased in the recombined population in β-catex3 iDCX at 3dpT [p=0.0081] and 13dpT [p=0.0259] (3dpT: control: n= 4 animals, β-catex3 iDCX: n= 9 animals, 13dpT: control: n= 8 animals, β-catex3 iDCX: n= 10 animals).
β-catenin signaling modulates the tempo of dendritic growth of adult-born hippocampal neurons (2020)
Figure 7

Fig. 7 Analysis of birthdated immature neurons in middle aged mice confirms modulation of dendritic growth and spine formation by canonical Wnt signaling activity

A Birthdating of adult-born neurons prior to recombination was conducted in 24-week old mice. For marker expression analysis, BrdU was injected i.p. every 24 hours for three days (B and C); for morphology analysis, CAG-RFP was stereotactically injected into the DG (D-G). Tamoxifen was applied i.p. every 12 hours for five days. Mice were sacrificed 3dpT and 13dpT.

B Representative images of recombined (GFP+, green) BrdU+ (grey) neurons in 24-week old control and β-catex3 iDCX animals at 13dpT expressing the stage specific markers DCX (red) for immature neurons and Calbindin (red) for mature neurons. Arrows and arrowheads indicate marker negative and marker positive cells, respectively. Scale bar= 10µm.

C Quantification of DCX and Calbindin expression in BrdU+ recombined cells. At 13dpT, labeled neurons in β-catex3 iDCX animals show a shift towards a more mature marker profile [DCX p=0.0238, Calbindin p=0.0714] (3dpT: control: n= 4 animals, β-catex3 iDCX: n= 8 animals; 13dpT control: n= 5 animals, β-catex3 iDCX: n= 5 animals).

D Representative reconstructions of RFP-birthdated control and β-catex3 iDCX neurons in 24-week old mice at 3dpT. Scale bar= 20µm.

E Quantification of dendritic length [p=0.0007], branch points [p=0.3909], and Sholl analysis [p=0.0001] showed increased complexity of birthdated β-catex3 iDCX cells at 3dpT (control: n= 20 cells from 4 animals, β-catex3 iDCX: n= 19 cells from 5 animals).

F Representative reconstructions of RFP-birthdated control and β-catex3 iDCX neurons in 24-week old mice at 13dpT. Scale bar= 20µm.

G Quantification of dendritic length [p=0.1857], branch points [p=0.1331], and Sholl analysis [p=0.1501] showed no difference between β-catex3 iDCX and control cells at 13dpT (control: n= 18 cells from 4 animals, β-catex3 iDCX: n= 20 cells from 4 animals)

Data information: Data represented as mean ± SEM, significance was determined using two-way ANOVA for Sholl analysis and two-tailed Mann-Whitney U Test for all other analyses, significance levels are displayed in GP style [p<0.0332 (*), p<0.0021 (**) and p<0.0002 (***), p<0.0001 (****)].

Expanded view figure legend
Crystal structure of bacterial cytotoxic necrotizing factor CNFY reveals molecular building blocks for intoxication  (2020)
Figure 2

Figure 2. Synthesis, secretion, and host cell binding of N- and C-terminal deletion variants of CNFY.

A Schematic overview of marker-tagged CNFY deletion variants.

B 3xFlag-tagged CNFY deletion variants were expressed in Y. pseudotuberculosis YP147 (ΔcnfY) from plasmids under control of their own promoter and were detected in whole cell extracts using an anti-Flag antibody.

C To test secretion of the CNFY variants, full-length CNFY and different N- and C-terminally deleted variants fused to beta-lactamase (TEM) were expressed in Y. pseudotuberculosis YP147 (ΔcnfY). Beta-lactamase activity in the culture supernatant was subsequently measured using nitrocefin as substrate. The data represent the mean ± SD of three independent experiments, carried out in triplicates.

D HEp-2 cells remained untreated or were incubated with 20 µg/ml of whole cell extract of Y. pseudotuberculosis expressing full-length CNF1, CNFY or the N- or C-terminally deleted toxin variants at 37°C for 4 h. The cells were thoroughly washed, pelleted, lysed and the toxin variants bound to the cells were identified by western blotting using an anti-Flag antibody.