EMBO/soda-nel huggingface dataset:
Kit ligand has a critical role in mouse yolk sac and aorta-gonad-mesonephros hematopoiesis (2018)
Figure 3

Figure 3. In the absence of Kitl, the influx and proliferation of EMPs in the early FL are reduced

A. Number of EMPs in peripheral blood (PB) of wild type and Sl/Sl midgestation embryos, calculated from the percentage of PB EMPs (Ter119- Kit+ CD41+ CD16/32+) determined by flow cytometry (Figure EV1E,F) and the total cell count (4.2±1.6x105 (wt), 3.1±1.1x105 (Sl/Sl) at E9.75; 2.6±0.8x106 (wt), 2.3±1.4x106 (Sl/Sl) at E11.5). For E9.75-E10, PB from 2 wild type (28-30sp) and 5 Sl/Sl (27-31) individual embryos was analyzed. For E11.5, PB from 1-3 embryos was pooled and 6 wild type and 4 Sl/Sl biological replicates were analyzed, with 10 wild type and 6 Sl/Sl embryos analyzed in total. Graphs show the mean (±SD) of PB EMPs per embryo equivalent (e.e.).

B. Representative confocal whole-mount immunofluorescence images of wild type and Sl/Sl FL at the onset of hematopoietic colonization. Kit+ EMPs are shown in white, CD31+ vasculature in green. Maximum intensity 3D projections from 350μm-thick Z stacks are shown. Scale bars: 100μm. Number of embryos analyzed: 3 wild type and 3 Sl/Sl for E9.75 (27-29sp), 7 wild type and 8 (Sl/Sl) for E10.5 (34-36sp).

C. Confocal whole-mount immunofluorescence analysis of proliferating hematopoietic progenitors in wild type and Sl/Sl E10.5 (34-36sp) FL. Very few CD31+ pHH3+ Kit+ proliferating hematopoietic cells (arrowheads) were detected in the Sl/Sl FL. A single 2.5μm-thick slice is shown. Scale bars: 20μm. A total of 7 wild type and 8 Sl/Sl embryos were analyzed.

D,E. Percentage of cycling Ter119- Kit+ CD41+ CD16/32+ EMPs and Ter119- Kit+ CD41- CD71+ CD44+ early erythroid (Early E) cells in the E11.5 FL, determined by flow cytometric analysis of BrdU and 7-AAD incorporation. Data are the mean (±SD) of 6 wild type and 5 Sl/Sl FLs analyzed individually over 3 independent experiments. Tail somite range: 12-17 (+/+); 12-17 (Sl/Sl).

F,G. Percentage of early apoptotic EMPs and Early E in wild type and Sl/Sl E11.5 FL, determined by flow cytometric analysis of AnnexinV staining among 7-AAD- Ter119- CD41+ Kit+ CD16/32+ EMPs and 7-AAD- Ter119- Kit+ CD41- CD71+ CD44+ Early E. Data are the mean (±SD) of 9 wild type and 7 Sl/Sl biological replicates analyzed over 3 independent experiments, with each replicate consisting of 1-3 FLs of identical genotypes. A total number of 13 wild type and 9 Sl/Sl embryos were analyzed. Tail somite range: 11-17 for both genotypes.

Data information: *P<0.05 (Unpaired two-tailed Student t-test).
Kit ligand has a critical role in mouse yolk sac and aorta-gonad-mesonephros hematopoiesis (2018)
Figure 4

Figure 4. Macrophages are depleted in Sl/Sl mutant embryos

A. Percentages of macrophage populations in wild type and Sl/Sl E14.5 brain, skin (dissected from the back), lungs and limb buds, as determined by flow cytometry. Cells were gated as Ter119- CD45+ and further gated into F4/80hi CD11blo or F4/80lo CD11bhi populations. Representative dot plots of skin analysis are shown in Appendix Figure S1A. Data are the mean (±SD) of 4 biological replicates for either genotype, with each replicate consisting of individual or pooled samples (from up to 3 embryos). A total of 8 wild type and 5 Sl/Sl embryos were analyzed over 2 independent experiments.

B. Percentage and absolute numbers of tissue macrophages per E14.5 FL. Data are mean (±SD). Number of embryos analyzed: as in (A).

C. Representative flow cytometry analysis of macrophages in wild type and Sl/Sl E10.5 AGM+VU. Cells were gated as shown, with percentages in individual gates as indicated. Data are the mean (±SD) of 4 wild type (32-35 sp) or 3 Sl/Sl (34-35 sp) biological replicates, analyzed over 2 independent experiments. Embryos were analyzed individually.

D. Quantification of flow cytometry analysis in (C), showing percentage of F4/80+CD11b+ macrophages within live cells. E11.5 data are the mean (±SD) of 4 wild type (10-13 tail somites) or 3 Sl/Sl (10-13 tail somites) biological replicates, analyzed over 3 independent experiments. Embryos were analyzed individually.

E. Confocal whole-mount immunofluorescence analysis of macrophages in wild type and Sl/Sl E10.5 AGM+VU. 3D panels show maximum intensity projections from 400μm-thick Z stacks. Slice panels show single 2.5μm-thick Z slices of clusters in the dorsal aorta (da). A total of 4 wild type (31-34 sp) and 3 Sl/Sl (31-33 sp) embryos were analyzed. Scale bars: 50μm (3D), 10μm (slice).

F. Quantification of whole-mount immunofluorescence in (E), showing absolute numbers (mean ± SD) of F4/80+ macrophages within hematopoietic clusters in the dorsal aorta.

Data information: *P<0.05 (Unpaired two-tailed Student t-test).
Kit ligand has a critical role in mouse yolk sac and aorta-gonad-mesonephros hematopoiesis (2018)
Figure 8

Figure 8. Kitl is required for Akt and Erk phosphorylation in the AGM and YS

A, B. Confocal whole-mount immunofluorescence analysis of phosphorylated Akt (pAkt) (A) and phosphorylated p44/42 MAPK (pErk1/2) (B) in Kit+ cells of wild type (32-36sp) and Sl/Sl (31-35sp) E10.5 dorsal aorta, yolk sac and fetal liver. Images are 2.5μm-thick single longitudinal Z slices; dorsal up (DA). Arrowheads point to Kit+ cells with varying levels of pAkt and pErk1/2 expression. A total of 2 wild type and 2 Sl/Sl embryos for each staining were analyzed. Scale bars: 20μm.

C, D. Quantification of the immunofluorescence in (A, B), showing mean fluorescence intensity (MFI) ratio of pAkt and p44/42 MAPK (pErk1/2) within wild type (+/+) and Sl/Sl CD31+ Kit+ dorsal aorta (DA), yolk sac (YS) and fetal liver (FL) hematopoietic cells. MFI values were normalized to background in the adjacent ventral mesenchyme for DA, to erythrocytes in the YS or stroma in the FL. Additional quantification was performed on sections from 2 wild type and 2 Sl/Sl embryos of similar age range. MFI measurements were taken using ImageJ. Number of cells measured for pAkt: 100 hematopoietic cells and 73 background (+/+); 79 hematopoietic and 52 background (Sl/Sl) in the AGM; 127 hematopoietic cells and 95 background (+/+); 110 hematopoietic and 82 background (Sl/Sl) in the YS; 102 hematopoietic cells and 78 background (+/+); 78 hematopoietic and 83 background (Sl/Sl) in the FL. Number of cells measured for pErk1/2: 94 hematopoietic cells and 82 background (+/+); 114 hematopoietic and 91 background (Sl/Sl) in the AGM; 174 hematopoietic cells and 109 background (+/+); 123 hematopoietic and 82 background (Sl/Sl) in the YS; 100 hematopoietic cells and 70 background (+/+); 63 hematopoietic and 57 background (Sl/Sl) in the FL. N=4 (+/+), 4 (Sl/Sl) embryos analyzed for DA and YS; N=3 (+/+), 2 (Sl/Sl) for FL. Error bars represent SD.

E. Multiplex Fluidigm qRT-PCR analysis of pre-HSC type I (Ter119- VE-Cadherin+ Kit+ CD41+ CD45-), pre-HSC type II/HSC (Ter119- VE-Cadherin+ Kit+ CD41+ CD45+), YS and FL EMP (Ter119- Kit+ CD41+ CD16/32+), FL Early E (Ter119- Kit+ CD41-) and peripheral blood LMPP (Lin- CD19- B220- CD45+ Kit+ Flt3+ IL7Rα+), isolated from E11.5 (11-17 tail somites) wild type and Sl/Sl embryos. 4 biological replicates for wild type (9 total embryos) and 5 biological replicates (9 total embryos) for Sl/Sl were analyzed. Data are mean (±SD).

Data information: *P<0.05 (Unpaired two-tailed Student t-test).

EXPANDED VIEW FIGURE LEGENDS
Nanobodies dismantle post-pyroptotic ASC specks and counteract inflammation in vivo (2022)
Figure 1

Figure 1: VHHASC blocks the extracellular pro-inflammatory function of ASC specks.

(A) Mouse IL-1β (mIL-1β) concentrations in cell-free supernatants of LPS-primed BMDMs (200 ng ml−1, for 2h), that were stimulated with in vitro-generated human ASC specks (200 µg ml−1, O/N). ASC specks were pre-incubated with 200 µg ml−1 of VHHASC, its mutated variant, mutVHHASC or 50 µg ml−1 of polyclonal ASC antibody (a-ASC pAb, AL177) for 15 min at RT. Data is pooled from at least two independent experiments, each represented by a different symbol. Data is displayed as floating bars with the max/min values and mean (thicker band).

(B) Immunoblot analysis of pro- (p31) and cleaved (p17) IL-1β, and pro- (p45) and cleaved (p20) caspase-1 in cytosolic fractions of ASC deficient (Pycard-/-) immortalized murine macrophages that were primed with LPS (200 ng ml−1, for 3h). Fractions were incubated with recombinant ASC specks in the presence of 2, 20 or 200 µg ml−1 of VHHASC or mutVHHASC. One representative of three independent experiments is shown.

(C) Quantitative analysis of band densitometry of three independent experiments as shown in B. Data represents the fold change from all conditions (lanes 1- 9) .vs Pycard−/− lysates incubated with ASC specks alone (lane 2). ns, p > 0.05; *, p<0.05; **, p<0.005; ***, p<0.0002, One-way ANOVA, multiple comparison (Dunnet test). Data is displayed as floating bars with the max/min values and mean (thicker band).
Nanobodies dismantle post-pyroptotic ASC specks and counteract inflammation in vivo (2022)
Figure 2

Figure 2: VHHASC disrupts the "prionoid" activities and disassembles ASC specks.

(A) Time-lapse confocal imaging of the in vitro nucleation of soluble ASC-mTurquoise (red) by ASC-TagGFP (ASC-GFP) specks (green), that were left untreated (None), or pre-incubated with VHHASC or mutVHHASC (200 µg ml−1 for 15 min). Scale bars: 10 μm.

(B) Median fluorescence intensity (MFI) of mTurquoise graphed over time showing its polymerization seeded by ASC-TagGFP specks. Each line shows the mTurquoise MFI around a seeding ASC-TagGFP speck. Data is one representative of three independent experiments.

(C) WES capillary electrophoresis and immunoblotting of DSS cross-linked in-vitro-generated human ASC-mTurquoise (ASC) specks that were incubated for 1h (RT) with 2, 20 or 200 µg ml−1 of VHHASC, mutVHHASC or with 20 µg ml−1 of a polyclonal anti-ASC Ab (a-ASC pAb). Data is from one representative of four independent experiments.
Nanobodies dismantle post-pyroptotic ASC specks and counteract inflammation in vivo (2022)
Figure 3

Figure 3: VHHASC and VHHmASC prevent IL-1β release and ASC speck formation induced by PFO, but not nigericin in primary human and mouse cells.

A-D (A-B) Human IL-1β (hIL-1β) concentrations in cell-free supernatants of LPS-primed (10 ng ml−1, 150 min) primary human macrophages that were left untreated, or pre-incubated with VHHASC or mutVHHASC (100 µg ml−1), CRID3 (50 µM) or VX-765 (50 µM) for 30 min before stimulation with (A) nigericin (10 µM), or (B) PFO (30 ng ml−1) for 2h. (C-D) Mouse IL-1β (mIL-1β) concentrations in cell-free supernatants of LPS-primed mouse BMDMs (200 ng ml−1, 150 min), incubated with VHHs, CRID3 or VX765, before activation with nigericin (10 µM), or PFO (250 ng ml−1). Data is combined from two independent experiments, each performed with two donors (A-B) or mice (C-D), represented with individual symbols (4 donors or mice in total). Data is displayed as floating bars with the max/min values and mean (thicker band).

E, F (E) Epifluorescence microscopy imaging and (F) quantification of ASC speck formation in BMDMs from ASC-mCitrine (Green) transgenic mice. Cells were primed with LPS (200 ng ml−1, 150 min), pre-treated with VX-765 (50 µM, 30 min), then treated with VHHASC, VHHmASC (100 µg ml−1) or CRID3 (50 µM) for another 30 min before stimulation with nigericin (top), or PFO (bottom) for 2h and finally fixed with 4% PFA and nuclei stained with DRAQ5 (Blue). Scale bars: 100 μm. Images in (E) are representative of one out of three independent experiments that were quantified in F. Data is displayed as floating bars with the max/min values and mean (thicker band).

Data information: **, p<0.005; ***, p<0.0002; ****, p<0.0001, One-way ANOVA, multiple comparison (Tukey test).
Nanobodies dismantle post-pyroptotic ASC specks and counteract inflammation in vivo (2022)
Figure 4

Figure 4: Contribution of GSDMD in the effect of VHHASC on the release of IL-1β and cell death induced by PFO or nigericin.

THP-1 cells expressing a Dox-inducible CRISPR-Cas9 cassette targeting GSDMD gene were treated with 1 µg ml-1 Dox for one or two cycles of 72 h (1x, or 2x respectively). As control, cells were left untreated and cultured in parallel (-).

(A) Immunoblot analysis of GSDMD expression following the indicated course of Dox treatment and PMA-differentiation, where indicated. Data is from one representative of two independent experiments.

(B) IL-1β concentration or percentage of LDH released into cell-free supernatants of PMA-differentiated THP-1 cells that were treated with VHHASC (200 µg ml-1) or CRID3 (25 µM) for 30 min prior to stimulation with nigericin (10 µM, left panels) or PFO (30 ng ml-1, right panels) for 2h. Data is average of experimental duplicates from three independent experiments, each represented by a different symbol. ns, p > 0.05; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001, Two-way ANOVA, multiple comparison (Tukey test). Data is displayed as floating bars with the max/min values and mean (thicker band).

(C) Confocal microscopy imaging of live PMA-differentiated and nigericin-treated (10 µM) THP-1 cells expressing human ASC-GFP (green) in the presence of AlexaFluor647-labeled VHHASC (VHHASC-AF647, 10 µg ml-1, cyan) in the medium. Cells were either left untreated or incubated with VX-765 (50 µM) for 1h prior to nigericin stimulation. Nuclei were stained with Hoechst 34580 (magenta). Scale bar: 50 µm. Data is from one experiment out of two independent experiments.
Nanobodies dismantle post-pyroptotic ASC specks and counteract inflammation in vivo (2022)
Figure 5

Figure 5: Pyroptosis allows VHHASC to access the cytosol and target ASC specks.

(A) Wide-field fluorescence microscopy imaging of live PMA-differentiated and nigericin- (10 µM, top) or PFO-activated (25 ng ml-1, bottom) THP-1 expressing human ASC-GFP. Cells were incubated with VHHASC (200 µg ml-1) alone or in the presence of the caspase-1 inhibitor VX-765 (50 µM). Nuclei were stained with DRAQ5 (blue). Cells were imaged live with a CellDiscoverer 7 microscope. For each condition, a total of 8 positions within 2 wells (2x4 images/well) were imaged. Data is maximal intensity projections from Z-stacks. Scale bars: 50 µm. See also Movie EV2.

(B) Graphic representation of the percent of specking cells over time. Maximal intensity projections from Z-stacks were generated for each image set before the number of cells and specks per field were calculated using CellProfiler. (A) represents images from one experiment out of three independent experiments of which percentage of specking cells data is represented in (B) with each line representing one field-of-view.

C, D (C) GFP+ specks detected in culture supernatants by flow cytometry and (D) immunoblotting analysis of ASC-GFP oligomeric state (DSS cross-linked) of extracellular specks recovered from supernatants of cells treated similarly to (A-B). Data in (C) is from two independent experiments (each represented by a different symbol) and in (D) is from one out of at least two independent experiments. Data is displayed as floating bars with the max/min values and mean (thicker band).

(E) Mouse BMDMs expressing ASC-mCitrin and primed with LPS (200 ng ml-1) for 3h were pre-treated with VHHmASC (200 µg ml-1) alone or in the presence of the caspase-1 inhibitor VX-765 (50 µM) for 30 min. Cells were then treated with a mixture of LFn-MxiH and PA (Mx/PA, 100 ng ml-1/1 µg ml-1) or PFO (250 ng ml-1) and imaged as in (A) for 3h. Data is a graphic representation of specking cells over time calculated as in (B). Data is from one experiment out of three independent experiments. See also Movie EV6.
BRCA1 and CtIP promote alternative non‐homologous end‐joining at uncapped telomeres (2014)
Figure 1

The DNA damage response at telomeres uncapped through TRF2 depletion does not require BRCA1 or CtIP

Immortalized Brca1F/− MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. SMC1 and tubulin were used as loading controls. *non‐specific band.
Cells treated as in (A) were fixed 48 h after selection and stained with an anti‐53BP1 antibody (green). Telomeres were visualized with a Cy3‐conjugated (CCCTAA)3‐PNA probe (red). Yellow arrowheads point to 53BP1 foci that co‐localize with telomeres.
Quantification of TIFs in cells treated as in (B). A minimum of 200 nuclei were scored for each sample. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05; NS, P > 0.05.
BRCA1 and CtIP promote alternative non‐homologous end‐joining at uncapped telomeres (2014)
Figure 2

BRCA1 and CtIP are required for telomeric end‐to‐end fusions induced by TRF2 inactivation

Immortalized Brca1F/− MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h. Cells were arrested in mitosis with colcemid and mitotic chromosomes were processed for CO‐FISH analysis. Metaphase chromosome spreads were stained with Cy3‐conjugated leading strand telomeric PNA probe (red) and FITC‐conjugated lagging strand telomeric PNA probe (green). DNA was counter‐stained with DAPI (blue).
The frequency of chromosome‐type telomeric fusions in cells treated as in (A) was quantified as a percentage of total number of chromosomes (illustrated in Supplementary Fig S2A). A minimum of 2,000 chromosomes were scored for each treatment. Error bars represent SD of at least two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05; **P ≤ 0.01; NS, P > 0.05.
BRCA1 and CtIP promote alternative non‐homologous end‐joining at uncapped telomeres (2014)
Figure 3

BRCA1 and CtIP mediate Ku‐ and LIG4‐independent telomere fusions

A Immortalized Brca1F/− MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h. Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe. The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion. A minimum of 2,000 chromosomes were scored for each sample. Error bars represent SD of three independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. NS, P > 0.05.
B Immortalized Brca1F/C61G MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase, followed by selection with puromycin for 72 h. The frequency of end‐to‐end chromosome‐type fusions was analysed as in (A).
C–F MEFs of the indicated genotypes were infected with retroviruses expressing TRF2 and/or CtIP shRNAs, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. GAPDH was used as a loading control. *non‐specific band. Cells treated as in (C) and (E) were arrested in mitosis with colcemid, and mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe (D, F). The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion. Error bars represent SD of two independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. *P ≤ 0.05.
BRCA1 and CtIP promote alternative non‐homologous end‐joining at uncapped telomeres (2014)
Figure 4

BRCA1 and CtIP promote LIG3‐ and PARP1‐dependent end‐joining of telomeres uncapped by TRF2 inactivation

Immortalized MEFs were infected with retroviruses expressing the indicated shRNAs, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. SMC1 was used as a loading control. *non‐specific band.
Quantification of the frequency of end‐to‐end chromosome‐type fusions of cells treated as in (A) represented as a percentage of fusions observed after TRF2 depletion. A minimum of 2,000 chromosomes were scored for each sample. Error bars represent SD of three independent experiments. The P‐value was calculated using an unpaired two‐tailed t‐test. NS, P > 0.05.
Immortalized Brca1F/− MEFs were infected with retroviruses expressing the indicated shRNAs and/or Cre recombinase. Mitotic chromosomes isolated 48 h later were fixed and stained with a Cy3‐conjugated (CCCTAA)3‐PNA probe. The frequency of end‐to‐end chromosome‐type fusions is represented as a percentage of fusions observed after TRF2 depletion. A minimum of 2,000 chromosomes were scored for each sample. Error bars represent SD of three independent experiments. P‐values were calculated using an unpaired two‐tailed t‐test. NS, P > 0.05.
Immortalized Brca1F/− MEFs were infected with retroviruses expressing TRF2 shRNAs and/or Cre recombinase, together with a lentivirus expressing PARP1 shRNA, followed by selection with puromycin for 72 h. Cell extracts were prepared 48 h later and analysed by Western blotting as indicated. SMC1 was used as a loading control. *non‐specific band.
Quantification of the frequency of end‐to‐end chromosome‐type fusions of cells treated as in (D) represented as a percentage of fusions observed after TRF2 depletion. A minimum of 1,500 chromosomes were scored for each sample. Error bars represent SD of three independent experiments. The P‐value was calculated using an unpaired two‐tailed t‐test. NS, P > 0.05.
Dead end1 is an essential partner of NANOS2 for selective binding of target RNAs in male germ cell development (2015)
Figure 1

Figure 1 | DND1 interacts directly with NANOS2 via its zinc finger motif. A, B, Western blotting analysis of proteins co-precipitated with anti-FLAG antibody from testis extracts of E15.5 wild-type embryos and transgenic embryos expressing FLAG-tagged NANOS2 with or without RNase (A) or from extracts of HeLa cells transfected with FLAG-tagged DND1 and with or without HA-tagged NANOS2 (B). Precipitates were analyzed with the indicated antibodies. C, GST pull-down assay using E. coli extracts expressing GST, GST-fused NANOS2, or NANOS3 mixed with MBP-tagged LacZ or DND1. Precipitates were analyzed by Coomassie Brilliant Blue (CBB) staining or western blotting with an anti-MBP antibody. An arrowhead indicates MBP-DND1 co-precipitated with GST-tagged NANOS2 or NANOS3. See also Fig. EV3A. D, E, Western blotting analyses of proteins co-precipitated with anti-FLAG antibody from extracts of HeLa cells transfected with FLAG-tagged NANOS2, NANOS2 (ΔN10), NANOS2 (ΔC10), NANOS2 (C61A, C96A) or NANOS3, and HA-tagged DND1 (D) or with FLAG-tagged NANOS2, NANOS2 (C61A), NANOS2 (C96A) or NANOS2 (C61A, C96A), and HA-tagged DND1 (E). Precipitates were analyzed with the indicated antibodies. F, GST pull-down assay using E. coli extracts expressing GST-fused DND1 mixed with MBP-tagged LacZ, NANOS2, NANOS2 (C61A, C96A) or NANOS2 (60-114). Arrowheads indicate MBP-tagged NANOS2 (lane 2) or NANOS2 (60-114) (lane 4).
Dead end1 is an essential partner of NANOS2 for selective binding of target RNAs in male germ cell development (2015)
Figure 2

Figure 2 | Relationship between NANOS and PUMILIO. A, B, Western blotting analyses of proteins co-precipitated with anti-FLAG antibody from extracts of HeLa cells transfected with HA-tagged NANOS1, NANOS2 or NANOS3, and FLAG-tagged DND1 (A) or with FLAG-tagged PUMILIO1 or PUMILIO2, and HA-tagged NANOS1, NANOS2 or NANOS3 with or without MYC-tagged DND1 (B). C, Characterization of antibodies against PUMILIO1 and PUMILIO2. Western blotting analyses of Flag-tagged PUMILIO1 and PUMILIO2 in HeLa cells transfected with FLAG-tagged PUMILIO1 or PUMILIO2 using antibodies against FLAG, PUMILIO1 or PUMILIO2. Note that the antibodies against PUMILIO1 specifically recognized FLAG-tagged PUMILIO1 while the anti-PUMILIO2 antibody could detect both Flag-tagged PUMILIO1 and PUMILIO2 but less actively. D, FLAG-tagged NANOS2 was immunoprecipitated with an anti-FLAG antibody from E15.5 testicular extracts from transgenic mice expressing FLAG-tagged NANOS2. Precipitates were analyzed by western blotting with the indicated antibodies. Note that co-precipitation of both PUMILIO1 and PUMILIO2 were not detected even though both CNOT1 and DND1 were clearly co-precipitated.