EMBO/soda-nel huggingface dataset:
Dead end1 is an essential partner of NANOS2 for selective binding of target RNAs in male germ cell development (2015)
Figure 3

Figure 3 | DND1 co-localizes with NANOS2 in P-bodies. A−C, Sections of male gonads from E15.5 embryos were immunostained with antibodies against DND1 (green) and DCP1a (red). D−F, Squash preparation of a male gonocyte from E16.5 embryo immunostained with antibodies against DND1 (red) and DCP1a (green). Arrowheads indicate co-localization of DND1 and DCP1a. G−I, Sections of male gonads from E15.5 embryos were immunostained with antibodies against DND1 (red) and NANOS2 (green). Arrowheads indicate co-localization of DND1 and NANOS2. J−M, NIH3T3 cells transfected with HA-tagged Dnd1 and FLAG-tagged Nanos2 were then immunostained with antibodies against DND1 (J) (magenta), NANOS2 (K) (red), and DCP1a (L) (green). N−Y, Biomolecular fluorescence complementation assay. NIH3T3 cells transfected with VENUS-C-fused Dnd1 and VENUS-N-fused Nanos2 (N−U) or Nanos2 (C61A, C96A) (V–γ) were immunostained with antibodies against DND1 (N, S, V), NANOS2 (O, W, α) or DDX6 (R, Z). Then, the signals of VENUS fusion protein were visualized (P, T, X, β). Arrowheads in Z indicate P-bodies. DNA was labeled with DAPI (blue). Scale bars: 50 m in A for A−C; 10 m in D for D–F, G for G−I, J for J−M, N for N−γ. Insets in A–C and J–γ show an enlarged vision of each picture to better depict localization of each protein. See also Fig. EV2T–Y.
Dead end1 is an essential partner of NANOS2 for selective binding of target RNAs in male germ cell development (2015)
Figure 4

Figure 4 | Conditional deletion of DND1 causes similar but not identical phenotypes to those of Nanos2-KO male gonocytes. A, Western blotting analyses of proteins in testes from E13.5 to E16.5 embryos of Dnd1flox/flox or Dnd1flox/flox_Tg(Oct4PE-CreERT2) each administered with tamoxifen at E13.5 and E15.5 embryos of Nanos2+/- or Nanos2-/- with the indicated antibodies. B–P, Sections of testes from Dnd1flox/flox (B, E, H, K, N), Dnd1flox/flox_Tg(Oct4PE-CreERT2) (C, F, I, L, O) or Nanos2-/- (D, G, J, M, P) embryos were prepared at E16.5 and then immunostained with antibodies against pH3 (B–D), STRA8 (E–G), SYCP3 (H–J), activated-Caspase3 (K–M) or LAMININ (N–P) (green). Germ cells were immunostained with TRA98 (B–D) or DAZL (N–P) (red), and DNA was labeled with DAPI (blue). Tamoxifen was administered at E13.5. Scale bars: 50 m in B for B−D and K–P, 50 m in E for E−J. See also Fig. EV3.
Dead end1 is an essential partner of NANOS2 for selective binding of target RNAs in male germ cell development (2015)
Figure 5

Figure 5 | DND1 is required for loading of the target RNAs to NANOS2-CNOT complex. A–L, Sections of testes from Dnd1flox/flox (A, B, C, G, H, I, M, N, O) and Dnd1flox/flox_Tg(Oct4PE-CreERT2) (D, E, F, J, K, L, P, Q, R) embryos were prepared at E16.5 and then immunostained with antibodies against NANOS2 (A, D) (green), DCP1a (G, J) (green), DDX6 (M, P) (green) and TRA98 (B, E, H, K, N, Q). DNA was labeled with DAPI (blue). Tamoxifen was administered at E13.5. Scale bars: 50 m in A for A−F; 50 m in G for G–L; 50 m in M for M–R. Insets show an enlarged vision of each picture to better depict localization of NANOS2, DCP1a and DDX6. S–V, Immunoprecipitation with an anti-FLAG antibody from E15.5 male gonadal extracts of wild-type and the transgenic mouse line expressing FLAG-tagged DND1 (S, T), or with an anti-NANOS2 antibody from E15.5 male gonadal extracts of Dnd1flox/flox mice with or without Oct4PE-CreERT2 (U, V). Precipitates were analyzed by western blotting (S, U) or by RT-qPCR (T, V). The RT-qPCR data are shown as average relative mRNA levels ± SEs (n=3). W, X, Western blotting analyses of proteins in NANOS2-depleted (W) or DND1-depleted (X) testis extracts from E15.5 embryos. Proteins were analyzed with the indicated antibodies.
Psoriatic skin inflammation is promoted by c-Jun/AP-1 dependent 
CCL2 and IL-23 expression in Dendritic cells (2021)
Figure 1

Figure 1. IMQ-induced skin inflammation requires c-Jun in dendritic cells

A-C. Epidermal thickness of the back skin was analyzed at the indicated time points in c-JunΔ/Δ Mx1-Cre (A), c-JunΔ/Δ CD11c-Cre (B) and c-JunΔ/Δ K5-Cre-ERT2 mice (C) on Hematoxylin and eosin (H&E) stained sections. (n = 5-21 (A), n = 6-29 (B), n = 5-18 (C); ≥ 2 independent experiments)

H&E stained sections of back skin from indicated mice treated with IMQ for 2, 3 or 5 days. Bright-field images, Magnification: 20x, Scale bar: 100 µm.

Layers of epidermis (left) and Trans-epidermal water loss (TEWL) (right) were analyzed in the back skin of indicated mice. (Left: n = 8-14, right: n = 6-14; ≥ 2independent experiments).

F-H. Flow cytometry of total back skin after 2, 3 and 5 days of IMQ treatment. Analyzed were dermal
γδ T cells (γδ TCRint+) (F), Monocytes (CD11b+Ly6Chi) (G), and Neutrophils (CD11b+Ly6G+) (H). Graphs show immune cells as % of live, single cells. (n = 10-28 (F), n = 10-21 (G), n = 4-25 (H); ≥ 2 independent experiments).

Data information: Data are shown as mean ± SEM. P‐values were calculated by one-way ANOVA with Tukey (A- C, and E) or Bonferroni multiple comparison test (F- H). Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. See Appendix Table S3 for exact P‐values.
Psoriatic skin inflammation is promoted by c-Jun/AP-1 dependent 
CCL2 and IL-23 expression in Dendritic cells (2021)
Figure 2

Figure 2. CCL2-mediated recruitment of pDCs to IMQ-inflamed skin depends on c-Jun in DCs

Skin infiltration of pDCs (BST-2+ B220+ CD11cint CD11b-) was analyzed by flow cytometry. Graph shows immune cells as % of live, single cells. (n= 2-15; 1-3 independent experiments).

Representative flow cytometry plots of skin pDCs 12h after IMQ treatment.

Total cell number of skin pDCs defined as in (B) is shown. (n= 5-6: 2 independent experiments)

ELISA for CCL2 performed on total back skin treated with IMQ. (n= 5-14; 2 independent experiments).

BMDCs generated from c-Junfl/fl or c-JunΔ/Δ Mx1-Cre BM were stimulated with IMQ for 12h. CCL2 protein was quantified by ELISA. (n= 7-10; 3 independent experiments).

qRT-PCR detection of Ccl2 mRNA in CD11c+MHCII+ cells sorted from IMQ-treated back skin (8h).
(n = 5-12; 3 experiments).

BMDCs were generated from Tlr7-/-, c-Junfl/fl or c-JunΔ/Δ Mx1-Cre BM and stimulated with IMQ for 4h and/or pre-treated with SP600125 (JNKi, 25µM) for 1h. Ccl2 mRNA was quantified by qRT-PCR.
(n = 4-18; 2-4 experiments).

H-I. Western blot analysis of Phospho- or total JNK and c-Jun in BMDCs of indicated genotype pre-treated with DMSO (1: 1000) or SP600125 (JNKi, 25 µM) for 1h and stimulated with IMQ (5 µg/mL) for 15 min.

Flow cytometry of back skin 12h after intradermal (i.d.) injection of 1 µg rCCL2 and/or IMQ treatment. Analyzed were pDCs (BST-2+B220+CD11cintCD11b-) shown as % of live, single cells. (n = 6; 2 independent experiments).

Data information: Data are shown as mean ± SEM. P‐values were calculated by multiple t-test with the Holm-Šídák method (C) or one-way ANOVA with Bonferroni (A), Kruskal- Wallis (E) or Tukey (D, F, G, J) multiple comparison test. Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. See Appendix Table S3 for exact P‐values.
Psoriatic skin inflammation is promoted by c-Jun/AP-1 dependent 
CCL2 and IL-23 expression in Dendritic cells (2021)
Figure 4

Figure 4. c-Jun is essential in conventional type-2 DCs to control CCL2 and IL-23 expression

DC subsets (cDC1, cDC2), Macrophages (MP) and Langerhans cells (LC) were sorted from IMQ treated back skin (5h). c-Jun, Ccl2 and Il23p19 mRNA was analyzed by qRT-PCR. Sort strategy is shown in (B). (10 mice were pooled per condition, 1 experiment).

Sort strategy for the separation of MPs (CD45+Lin-CD64+CD172a+CD11b+), and the DC (CD45+Lin-CD64-CD11c+MHCII+) subsets cDC1 (XCR1+CD11b- DC), cDC2 (XCR1-CD11b+CD24-/intCD326-) and LC (XCR1-CD11b+CD24+CD326+) in IMQ-treated (5h) murine back skin.

qRT-PCR detection of c-Jun, Ccl2 and Il23p19 in MPs and cDC2 sorted as described in (B) from indicated mice. (MPs: n= 7-9; 3 independent experiments, cDC2: n = 5-10; 2-3 independent experiments and back skin from 2 mice was pooled).

Sort strategy for CD11c expressing MPs (CD45+Lin-CD11cint/lowMHCII+CD64+CD11b+CD172a+) is shown. Murine skin was treated for 5h with IMQ.

Expression of indicated targets was analyzed by qRT-PCR in cells sorted as described in (D). (n= 3; Back skin from 2 mice was pooled).

Data information: Data are shown as mean ± SEM. P‐values were calculated by unpaired, two-tailed t-test (C, E). Statistical significance: ns > 0.05, *P < 0.05. See Appendix Table S3 for exact P‐values.
Psoriatic skin inflammation is promoted by c-Jun/AP-1 dependent 
CCL2 and IL-23 expression in Dendritic cells (2021)
Figure 5

Figure 5. Blocking JNK/c-Jun signaling ameliorates psoriasis-like skin inflammation

Experimental design for blocking JNK/c-Jun signaling in the IMQ-induced skin inflammation model.
c-Jun fl/fl, c-JunΔ/ΔCD11c-Cre, Tlr7-/- and Tlr7-/- c-JunΔ/ΔCD11c-Cre mice were treated with IMQ and JNK inhibitor SP600125 (JNKi, 15 mg/kg, i.p.) or vehicle for 5 consecutive days.

B-E. After 5 days of treatment TEWL was measured on the back skin (B), epidermal thickness was analyzed on H&E stained sections (C), epidermal Ki-67+ cells (D) and the ratio of Ratio of K5 to K10 positive areas (E) were analyzed by immunofluorescence as described in Fig EV3 (B,C). (n = 4-15; 5 independent experiments).

F-H. Quantification of immune cell populations as defined in Fig EV3 (E, F). Results are given as percentage of live, single cells. (n = 4-12; 3-4 independent experiments).

Experimental design for blocking JNK/c-Jun signaling in c-Jun/JunB Δ/Δ K5-Cre-ERT2 mice. Psoriasis-like disease was induced by injection of Tamoxifen (i.p., 5 days). Treatment with JNK Inhibitor (15 mg/kg, every other day, i.p.) started on day 14 for 2 weeks.

Representative images of ear, tail and paw of c-Jun/JunB Δ/Δ K5-Cre-ERT2 at the start (Day14) and end (Day 28) of treatment with JNK Inhibitor or vehicle.

H&E stained sections of mouse ear from indicated mice. Bright-field images, Scale bar: 250 µm, Magnification: 10x.

Psoriasis-like phenotype was scored from 0-4 for ear, tail, paw and snout. Shown is the cumulative score at therapy start (Day 14) and end (Day 28). Non-responders are c-Jun/JunB Δ/Δ K5-Cre-ERT2 mice treated with JNK Inhibitor, which showed disease-progression. (n = 2-8, 2 independent experiments).

Representative t-SNE- plots (n = 2-3) show the cutaneous immune cell phenotype at therapy end (Day 28). Numbers adjacent to gates give cell populations frequency among live, single, CD45+ cells. t-SNE Legend-Plot labels the populations defined by t-SNE- algorithm.

Dermal γδ T cells (γδ TCRint+), Monocytes (CD11b+Ly6Chi), and Neutrophils (CD11b+Ly6G+) were analyzed by flow cytometry in mouse ear at the end of treatment (Day 28). Graphs show immune cells as % of live, single cells. (n = 2-8, 2 independent experiments).

Data information: Data are shown as mean ± SEM. P‐values were calculated by paired, two-tailed t-test (L) and one-way ANOVA with Tukey (B-H) or Bonferroni multiple comparison test (N). Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. See Appendix Table S3 for exact P‐values.
Psoriatic skin inflammation is promoted by c-Jun/AP-1 dependent 
CCL2 and IL-23 expression in Dendritic cells (2021)
Figure 6

Figure 6. c-Jun, CCL2 and IL-23 are co-expressed in type-2/inflammatory DCs in psoriatic lesions

Gene expression values of Cd207, Cd1a, Cd1c, Cd14, Xcr1 in healthy (n= 38), non-lesional (n= 27) and lesional (n= 28) skin. Expression values were obtained from the GEO Data Set [GSE121212].Box and whiskers plot: Central band shows median, box extends from the 25th to 75th percentiles and whiskers go down to the smallest (min) and up to the largest (max) value.

Immunofluorescence of c-Jun (green), CD1a or CD1c or CD14 (red) and DAPI in psoriatic lesions. Shown is a representative image (Magnification 25x, Scale Bar: 100µm) with an Inset (Magnification 63x, Scale Bar: 20 µm, Deconvoluted). Arrows indicate c-Jun+ DCs. Asterisk highlights a DC with prominent c-Jun expression as shown enlarged in 2 white-framed windows (DC Marker (red) + c-Jun (green, left) or DAPI (blue, right)).

Quantification of c-Jun+ DCs (CD1a, CD1c or CD14) in psoriatic lesions as shown in (B). Four randomly chosen fields on one section were analyzed for each sample. (n = 2 patient samples/condition). Box and whiskers plot: Central band shows median, box extends from the 25th to 75th percentiles and whiskers go down to the smallest (min) and up to the largest (max) value.

D-E. Representative immunofluorescence of c-Jun (green), CD1a (red), CCL2 (D) or Il23p19 (E) (white) and DAPI in psoriatic lesions. Arrows indicate triple-positive cells and an asterisk highlights a representative one that is shown enlarged in a white-framed inlet. Magnification: 40x. Scale bar: 50 µm. (n = 2 patient samples).

Data information: Data are shown as mean ± SEM. P‐values were calculated by one-way ANOVA with Tukey multiple comparison test (A, C). Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. See Appendix Table S3 for exact P‐values.
Psoriatic skin inflammation is promoted by c-Jun/AP-1 dependent 
CCL2 and IL-23 expression in Dendritic cells (2021)
Figure 7

Figure 7. JNK/AP-1 Inhibitors repress CCL2 and IL-23 expression in human mo-DCs

Western blot analyses of Phospho- or total JNK and c-Jun in human mo-DCs pre-treated with DMSO (1: 1000) or SP600125 (JNKi, 25 µM) for 1h and stimulated with R848 (10 µg/mL) for 15 min.

B-C Human mo-DCs pre-treated with JNKi (SP600125, 25µM) (B) or AP-1 Inhibitor (T-5224, 20 µM) (C) for 1h were stimulated with R848 (10 µg/ml). CCL2 and IL-23 were analyzed by ELISA. (n = 5-11; 2-3 independent experiments).

Representative flow cytometry plots (left) and histograms (right) of human mo-DCs stimulated with LL-37-RNA-40 for 24h are shown. Inhibitors (JNKi; 25 µM or AP-1 Inhibitor; 20 µM) were given 1h before stimulation. Plots shown are pre-gated on live, single, CD1a+ cells.

E-F Human moDCs described in (D) were analyzed for mean fluorescence intensity of CD80 or CD86 by flow cytometry (E) and CCL2 or IL-23 protein by ELISA (F). (n = 7; 2 independent experiments).

Data information: Data are shown as mean ± SEM. P‐values were calculated by paired, two-tailed t-test (B, C, E and F). Statistical significance: ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. See Appendix Table S3 for exact P‐values.

Expanded View Figure Legends
TIMELESS-TIPIN and UBXN-3 promote replisome disassembly during replication termination in C. elegans (2021)
Figure 1

Figure 1

An RNAi screen for candidate E2 enzymes that contribute to CMG-MCM-7 ubiquitylation during DNA replication termination in C. elegans.

(A) Model for the priming and elongation of ubiquitin chains on substrates of cullin ubiquitin ligases in metazoa. See text for details.

(B) Family tree for the E2 ubiquitin conjugating enzymes encoded by the C. elegans genome. For each of the indicated groups, a single plasmid was generated to express RNAi to the component genes (see Materials and Methods).

(C) Summary of RNAi screen to detect synthetic lethality, upon combining ubxn-3 RNAi with a plasmid expressing RNAi to lrr-1 or to one of the groups of E2 enzymes indicated in (B).

(D) Summary of synthetic lethality data for the screen described in (C). Worms were fed on the indicated proportions of bacteria expressing RNAi to ubxn-3, lrr-1, E2 enzyme groups G1-G6, or else containing empty vector as indicated. The data represent the means and standard deviations from three biological replicates.

(E, F) Synthetic lethality resulting from the combination of RNAi to E2 group 5 and RNAi to ubxn-3 was deconvolved in similar experiments to those in (D), using plasmids expressing RNAi to individual E2 enzymes, or pairs of E2 enzymes, as indicated.

(G) GFP-psf-1 worms were fed on bacteria containing a single plasmid expressing the indicated RNAi treatments, before preparation of embryonic cell extracts and isolation of GFP-PSF-1 by immunoprecipitation. The indicated factors were monitored by immunoblotting.

(H) The presence of GFP-PSF-1 on mitotic chromatin (indicated by white arrows) was monitored by spinning disk confocal microscopy (see Materials and Methods), in GFP-psf-1 mCherry-Histone H2B worms that were fed on bacteria containing a single plasmid expressing the indicated RNAi treatments ('Control' = empty vector). NEB = nuclear envelope breakdown. The scalebars correspond to 5 µm.

(I) Analogous experiments to those in (D, E) to deconvolve the synthetic lethality induced by RNAi to ubxn-3 in combination with E2 group 6.
TIMELESS-TIPIN and UBXN-3 promote replisome disassembly during replication termination in C. elegans (2021)
Figure 2

Figure 2

In vitro reconstitution of replisome-specific ubiquitylation of CMG-MCM-7 by CUL-2LRR-1 and an array of associated ubiquitylation enzymes.

(A) Purified C. elegans proteins (see Materials and Methods).

(B) Ubiquitylation of CMG-MCM-7 was reconstituted with the indicated factors as described in Materials and Methods. 'Neddylation' indicates addition of the C. elegans ULA-1_RFL-1 E1 enzyme, the UBC-12 E2 enzyme, the DCN-1 E3 enzyme and NED-8.

(C) Equivalent reactions to those in (B) but using lysine-free (K0) ubiquitin.

(D) Similar reactions comparing wild type ubiquitin to the indicated ubiquitin mutants.

(E) Reactions containing CMG and either CUL-2LRR-1 or CUL-2VHL-1 were performed in the presence or absence of other replisome factors as indicated. CMG was then isolated by immunoprecipitation of SLD-5 and the indicated factors were monitored by immunoblotting.
TIMELESS-TIPIN and UBXN-3 promote replisome disassembly during replication termination in C. elegans (2021)
Figure 3

Figure 3

TIMELESS-TIPIN stimulates the priming and elongation of ubiquitin chains on CMG-MCM-7 by stabilising the association of CUL-2LRR-1 with the C. elegans replisome.

(A-C) Reconstituted CMG-MCM-7 ubiquitylation reactions were performed as above in the presence of the indicated factors. 'Neddylation' indicates addition of the C. elegans ULA-1_RFL-1 E1 enzyme, the UBC-12 E2 enzyme, the DCN-1 E3 enzyme and NED-8.

(D) The indicated factors were assembled in the absence of ubiquitin and then incubated as for (A) before isolation of CMG via immunoprecipitation of SLD-5. The association of CUL-2 with the CMG-replisome was monitored by immunoblotting.

(E, F) In reactions analogous to those in (A), ubiquitylation of the indicated factors was monitored by immunoblotting. Reactions were performed in sets of three as indicated (1 = dropout of CUL-2LRR-1; 2 = wt Ubiquitin; 3 = lysine-free or K0 ubiquitin).
TIMELESS-TIPIN and UBXN-3 promote replisome disassembly during replication termination in C. elegans (2021)
Figure 4

Figure 4

UBXN-3 stimulates disassembly of ubiquitylated CMG by CDC-48_UFD-1_NPL-4.

(A) CDC-48, UFD-1_NPL-4 (U_N) and UBXN-3 (full-length and UBXN-3-∆UBX) were purified as described in Materials and Methods and analysed by coomassie blue staining of SDS-PAGE gels.

(B) Ubiquitylation and disassembly of the C. elegans CMG helicase was reconstituted in vitro as indicated (see Materials and Methods for further details).

(C) CMG was isolated by immunoprecipitation of SLD-5, after ubiquitylation in the presence of TIM-1_TIPN-1, before incubation with the indicated factors. 'Neddylation' indicates addition of the C. elegans ULA-1_RFL-1 E1 enzyme, the UBC-12 E2 enzyme, the DCN-1 E3 enzyme and NED-8. CMG disassembly was monitored by release of CDC-45 or MCM-2-7 proteins into the supernatant, whilst SLD-5 and other GINS subunits remained on the beads. Asterisks indicate non-specific bands in the anti-MCM-7 and CDC-45 immunoblots.

(D) Equivalent reactions to (C) involving ubiquityation of CMG in the absence of TIM-1_TIPN-1. For (C) and (D), the bands corresponding to MCM-7 conjugated to 1-5 ubiquitins are indicated in lane 4.
TIMELESS-TIPIN and UBXN-3 promote replisome disassembly during replication termination in C. elegans (2021)
Figure 5

Figure 5

The TIMELESS-TIPIN complex is required in C. elegans early embryos for efficient CMG helicase ubiquitylation and the association of CUL-2LRR-1 with the post-termination replisome.

(A) TAP-psf-1 worms were fed on bacteria containing a single plasmid expressing the indicated RNAi treatments, before preparation of embryonic cell extracts and isolation of TAP-PSF-1 by immunoprecipitation. The bound material was released from beads by cleavage of the TAP tag with TEV protease, before detection of the indicated factors by immunoblotting.

(B) Similar experiment comparing the impact of RNAi inactivation of lrr-1, tim-1 and tipn-1 upon CMG-MCM-7 ubiquitylation and the association of CUL-2LRR-1 with the worm replisome. Asterisks indicate non-specific bands in the immunoblots.
TIMELESS-TIPIN and UBXN-3 promote replisome disassembly during replication termination in C. elegans (2021)
Figure 6

Figure 6

Co-depletion of TIMELESS and UBXN-3 impairs CMG disassembly during DNA replication termination in C. elegans and causes loss of viability.

(A) Embryonic viability was monitored as described in Materials and Methods, after feeding worms on the indicated proportions of bacteria expressing RNAi ('% RNAi') to the denoted genes, mixed with bacteria containing empty vector (to make a total of 100%).

(B) The presence of GFP-PSF-1 on mitotic chromatin (indicated by white arrows) was monitored by spinning disk confocal microscopy (see Materials and Methods), in embryos derived from worms that were fed on bacteria containing a single plasmid expressing the indicated RNAi ('Control' = empty vector). The scalebars correspond to 5 µm.

(C) Analogous experiment to that in (A), but with worms fed on the indicated proportions of bacteria expressing ubxn-3 RNAi, in combination with 20% bacteria expressing tim-1 RNAi specific for tim-1, plus the required remainder of bacteria containing empty vector (to make a total of 100%).

Data information: The data in (A) and (C) represent the means and standard deviations from three biological replicates.

Expanded View Figure legends
Human IRGM regulates autophagy and cell-autonomous immunity functions through mitochondria (2010)
figf1

(a) Subcellular compartments from U937 cell extracts were separated by sedimentation velocity on discontinuous sucrose gradient and probed for the indicated proteins by immunoblotting. Numbers represent fractions from the gradient (1, top; 5, bottom). (b) Immunoblot analysis of cellular compartments after purification of mitochondria by Qproteome Mitochondria Isolation Kit. (c) Immunoblot analysis of KDEL and IRGM distribution in different cellular fractions after purification of mitochondria as in b. (d) Intracellular localization of IRGM analysed by confocal microscopy. Cells were stained with antibodies against the indicated proteins or vitally with MitoTracker Red (MTR) where indicated. Images on the right are merged from images on the left. A–L; endogenous IRGM localization in HeLa cells relative to calnexin, MTR, cytochrome c and COX IV. M–O; IRGM localization in primary human macrophages relative to mitochondria visualized with cytochrome c antibody. Scale bars, 5 μm. (e) Analysis of IRGM (green) and COX IV (red) intensity as a function of distance along the line shown in the inset. Image in inset corresponds to panels J–L in d. Uncropped images of blots are shown in Supplementary Information, Fig. S8.