Control GST GST-RAP. α2-mg. 170 kda. b-actin. 42 kda LRP-1
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1 % of max Supplementary Figure 1 Control GST GST-RP 17 kda α2-mg 42 kda b-actin Gate: CD11c+ (DCs) Gate: F4/8+ (Mfs) IgG Cd11cCre + Lrp1 fl/fl LRP-1 Supplementary figure 1. () MDCs were pretreated with μm chloroquine to prevent degradation of internalized proteins and then incubated with methylamine-activated α2-macroglobulin (α2-mg) for 3 min in the presence of vehicle control (PS), GST (1 μg/ml), or GST-RP (1 μg/ml). The internalized α2-mg was detected by immunoblot using an antibody against α2-mg (top blot). β-actin was used as a loading control (bottom blot). () nalysis of expression levels of LRP-1 in splenic DCs (left panel, CD11c+ gate) or splenic macrophages (right panel, F4/8+ gate) in (blue line) and Cd11cCre + Lrp1 fl/fl mice (DC-LRP1 KO; red line), respectively. The green line represents cells stained with an IgG control antibody.
2 % C uptake by splenic Mφs % C uptake by splenic Mφs Supplementary Figure CD xl KO Supplementary figure 2. In-vivo efferocytosis by splenic macrophages. Similar to Figure 2, except the FCS analysis was gated on F4/8 + cells to quantify uptake of PKH67- labeled Cs by splenic macrophages. LRP-1 was inhibited by i.v. injection of GST-RP (2 mg/mouse), and Tim-3 was inhibited by i.p. injection of a neutralizing anti-tim-3 antibody (RMT3-23, 1 μg/mouse). n=3 mice per group., p<.;, no significant difference.
3 Mean fluorescence intensity % efferocytosis 3 3 Supplementary Figure # # # xl -/- xl -/- xl -/- xl -/- IgG + RP + Tim3 nb Scarf1 nb IgG Scarf1 nb 2 1 IgG Scarf1 nb 1 2 Supplementary Figure 3. () MDCs from or xl -/- mice were treated with. µg/ml IgG or neutralizing antibody (nb) against Tim3 or Scarf1 (Proteintech) for 3 min prior to addition of PKH67- labeled Cs. Efferocytosis was measured 2 h later by microscopic analysis., p<.; #, p,. as compared with its appropriate control;, no significant difference. () Related to Fig. 3, positive control for Scarf1 nb in human splenic DCs. Human splenic DCs were treated with. µg/ml IgG or Scarf1 nb (. μg/ml) (R&D systems) for 3 min prior to addition of DiI-labeled acetylated low-density lipoprotein (DiI-c-LDL, 1 μg/ml). The uptake of DiI-c-LDL was measured by microscopic analysis (red staining); the right panels show the phase images. The quantified data of c-ldl uptake measured by analysis of mean fluorescence intensity is shown in the bar graph. n=3., p<..
4 Count % efferocytosis Supplementary Figure 4 MDCs CD11c+ gate Cd11cCre + Lrp1 fl/fl CD11c+ gate xl -/- xl LRP-1 Supplementary Figure 4. () MDCs from Lrp1 fl/fl or Cd11cCre + Lrp1 fl/fl mice were incubated with PKH67-labeled Cs for 2 h at 37 C with or without neutralizing antibodies against CD36 and αv integrin. Efferocytosis was quantified as the percentage of MDCs with internalized Cs., p<.;, no significant difference. () The histogram plots are representative data of expression levels of xl (left panel) and LRP-1 (right panel) in splenic DCs (CD11c + gate) of the indicated groups of mice. n=3 mice per group.
5 % of max Count Count Supplementary Figure IP: LRP-1 LRP1-46 (donor) xl-647 (acceptor) xl-46 (donor) LRP-647 (acceptor) 84 kda LRP-1 Control Control Lrp fl/fl 14 kda xl xl -/- CD11cCre + Lrp fl/fl FRET channel (sensitized emission) C Control LC-46 LC-46+ HC-647 Control LC-46 LC-46+ HC-647 Senstized emission Donor fluorescence quenching Supplementary figure. () Immunoprecipitation of LRP-1 from whole cell lysates of MDCs obtained from or xl -/- mice followed by Western blotting to detect LRP-1 (top panel) or xl (bottom panel). () Flow cytometric analysis of FRET (sensitized emission) between LRP-1 and xl, using either LRP-1 as donor (LRP-1-46) in splenic DCs (CD11c + gate) of and xl -/- mice (left panel), or using xl as donor (xl-46) in and Cd11cCre + Lrp1f l/fl mice (right panel). IgG conjugated to lexa fluor 46 was used as control (orange line). (C) FCS-FRET analysis between MHC-I heavy and light chain was used as a positive control. Shown are sensitized emission (left panel) and donor fluorescence quenching (right panel) in DCs stained with an antibody against MHC-I light chain conjugated to a donor fluorophore and antibody against MHC-I heavy chain conjugated to an acceptor fluorophore.
6 CD11c hi cells (% total splenocytes) CD8α+ cells (% CD11c hi splenocytes) % C uptake % of max % of max Supplementary Figure 6 IgG Ranbp9 -/- Ranbp9 -/- Ranbp9 -/- RanP9 xl LRP-1 C D RanP9 KO E Mφ Supplementary figure 6. () Flow cytometric analysis of RanP9 expression in splenic DCs (CD11c + gate) of chimeric mice generated by transplantation of (blue line) or Ranbp9 -/- (green line) fetal liver cells. The data are representative of 3 independent experiments. () nalysis of cell surface expression of xl and LRP-1 in CD11c hi -gated splenocytes derived from mice transplanted with fetal liver from (blue line) or Ranbp9 -/- (red line) embryos. (C-D) The bar graphs show the percent CD11c hi cells of total splenocytes and the percent CD8α + of total CD11c hi cells in and Ranbp9 -/- mice. (n=3; n.s, no significant difference). (E) The bar graph shows quantified data of splenic macrophage efferocytosis in or Ranbp9 -/- chimeric mice 3 h post-injection of PKH67-labeled Cs. n=4 mice per group., no significant difference.
7 % efferocytosis Supplementary Figure 7 3 MDC Con sigulp xl -/- Con xl -/- sigulp Supplementary figure 7. MDCs from or xl -/- mice were transfected with negative control scrambled RN (Con) or sirn against GULP1 (sigulp1). Efferocytosis was assayed 48 h later by incubating the cells with PKH67-labeled Cs for 2 h at 37 C. Efferocytosis was quantified as the percentage of MDCs with internalized Cs., p<.;, no significant difference.
8 + HSV1 xl -/- + HSV1 Lrp1 fl/fl + HSV1 Cd11cCre + Lrp1 fl/fl + HSV1 Ranbp9 -/- + HSV1 drenal weight (mg) Supplementary Figure 8 Gate: CD8+CFSE+ xl -/ Lrp1 fl Cd11cCre + Lrp1 2 fl Ranbp9 -/ xl -/- Lrp1 fl/fl Cd11cCre + Lrp1 fl/fl Ranbp9 -/- Supplementary figure 8. () CFSE dye-dilution analysis of CD8 + -gated gt-i.1 transgenic T cells in the indicated groups of mice 4 days post-infection with HSV-1. n=3 mice per group. () Weights of adrenal glands of the indicated groups of mice 4 days post-infection with HSV-1. n=3 mice per group., p<.;, no significant difference.
9 Pfu (x1 )/ml cell extract Pfu (x1 )/ml cell extract Supplementary Figure 9 MDC Primary adrenal cells Supplementary figure 9. MDCs (left panel) or primary adrenal cells (right panel) from, xl -/-, or Cd11cCre + Lrp1 fl/fl mice were infected with HSV-1 at an MOI of 2 pfu/cell. On day 2 postinfection, the cells were lysed, and viral titers were determined by quantifying plaque forming units in Vero cells., p<.;, no significant difference (n=3).
10 % C uptake by splenic DCs in vivo % un-divided gt-i.1 T cells Supplementary Figure DC-LRP1 KO Supplementary figure 1. () xl -/- or Cd11cCre + Lrp1 fl/fl mice were injected with PKH67-labeled apoptotic HSV-1 infected primary adrenal cells (3x1 6 cells/mouse) and then splenic DCs were assayed for efferocytosis. () Lrp1 fl/fl or Cd11cCre + Lrp1 fl/fl mice were injected with CFSElabeled CD8 + gt-i.1 Tg T cells (1 6 cells/mouse) and UV-inactivated HSV-1 killed primary adrenal cells (1 6 cells/mouse). On day 4 post-injection, the splenocytes were analyzed by flow cytometry for CFSE dye dilution on the CD8 + CFSE + -gated population., p<. (n=3 mice per group).
11 HSV-1 pfu (x1 3 )/adrenal IFN-α (pg/ml) Supplementary Figure xl KO xl -/- Day 7 Day Supplementary Figure 11. () Viral titer in adrenal glands of and xl -/- mice on day 7 and 1 post-infection with HSV-1 (4x1 pfu/mouse). n=3 mice per group. () ELIS measurement of IFN-α levels in serum of or xl -/- mice on day 4 post-infection with HSV-1 (4x1 pfu/mouse). n=3 mice per group., p<.;, no significant difference.
Supplementary Figure 1. BMS enhances human T cell activation in vitro in a
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Supplemental Figure 1
Supplemental Figure 1 1a 1c PD-1 MFI fold change 6 5 4 3 2 1 IL-1α IL-2 IL-4 IL-6 IL-1 IL-12 IL-13 IL-15 IL-17 IL-18 IL-21 IL-23 IFN-α Mut Human PD-1 promoter SBE-D 5 -GTCTG- -1.2kb SBE-P -CAGAC- -1.kb
Supplementary Figures
Inhibition of Pulmonary Anti Bacterial Defense by IFN γ During Recovery from Influenza Infection By Keer Sun and Dennis W. Metzger Supplementary Figures d a Ly6G Percentage survival f 1 75 5 1 25 1 5 1
% of live splenocytes. STAT5 deletion. (open shapes) % ROSA + % floxed
Supp. Figure 1. a 14 1 1 8 6 spleen cells (x1 6 ) 16 % of live splenocytes 5 4 3 1 % of live splenocytes 8 6 4 b 1 1 c % of CD11c + splenocytes (closed shapes) 8 6 4 8 6 4 % ROSA + (open shapes) % floxed
Page 39 of 44. 8h LTA & AT h PepG & AT h LTA
Page 39 of 44 Fig. S1 A: B: C: D: 8h LTA 8h LTA & AT7519 E: F: 8h PepG G: 8h PepG & AT7519 Fig. S1. AT7519 overrides the survival effects of lipoteichoic acid (LTA) and peptidoglycan (PepG). (A) Human
NK cell flow cytometric assay In vivo DC viability and migration assay
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D CD8 T cell number (x10 6 )
IFNγ Supplemental Figure 1. CD T cell number (x1 6 ) 18 15 1 9 6 3 CD CD T cells CD6L C CD5 CD T cells CD6L D CD8 T cell number (x1 6 ) 1 8 6 E CD CD8 T cells CD6L F Log(1)CFU/g Feces 1 8 6 p
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1 Supplementary Data Figure legends Supplementary Figure 1. IL-12 serum levels and frequency of subsets in FL patients. (A) IL-12 serum levels measured by multiplex ELISA (Luminex) in FL patients before
a surface permeabilized
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Supplementary Figures
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Appendix Figure S1 A B C D E F G H
ppendix Figure S1 C D E F G H ppendix Figure S1. RT and chemotherapy alter PD-L1 expression in PDC cells. Flow cytometric analysis of PD-L1 expression in () KPC and () Pan02 cells following treatment with
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Supplementary Figures
Supplementary Figures Supplementary Figure 1 Characterization of stable expression of GlucB and sshbira in the CT26 cell line (a) Live cell imaging of stable CT26 cells expressing green fluorescent protein
Supplementary Figure 1. Normal T lymphocyte populations in Dapk -/- mice. (a) Normal thymic development in Dapk -/- mice. Thymocytes from WT and Dapk
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Supplementary Figure 1: Co-localization of reconstituted L-PTC and dendritic cells
a CD11c Na + K + ATPase Na + K + ATPase CD11c x-y CD11c Na + K + ATPase Na + K + ATPase CD11c x-z c b x-y view BoNT NAPs CD11c BoNT CD11c NAPs BoNT NAPs CD11c 90 x-z view Apical Basolateral Supplementary
Supplementary Materials for
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Tbk1-TKO! DN cells (%)! 15! 10!
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Supplementary Figure 1
Supplementary Figure 1 YAP negatively regulates IFN- signaling. (a) Immunoblot analysis of Yap knockdown efficiency with sh-yap (#1 to #4 independent constructs) in Raw264.7 cells. (b) IFN- -Luc and PRDs
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Supplementary Figure 1 a γ-h2ax MDC1 RNF8 FK2 BRCA1 U2OS Cells sgrna-1 ** 60 sgrna 40 20 0 % positive Cells (>5 foci per cell) b ** 80 sgrna sgrna γ-h2ax MDC1 γ-h2ax RNF8 FK2 MDC1 BRCA1 RNF8 FK2 BRCA1
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Pearson r = P (one-tailed) = n = 9
8F4-Specific Lysis, % 1 UPN1 UPN3 8 UPN7 6 Pearson r =.69 UPN2 UPN5 P (one-tailed) =.192 4 UPN8 n = 9 2 UPN9 UPN4 UPN6 5 1 15 2 25 8 8F4, % Max MFI Supplementary Figure S1. AML samples UPN1-UPN9 show variable
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Supplementary Figure 1 Gata1 expression in heamatopoietic stem and progenitor populations. (a) Unsupervised clustering according to 100 top variable genes across single pre-gm cells. The two main cell
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Canberra, Australia). CD11c-DTR-OVA-GFP (B6.CD11c-OVA), B6.luc + and. Cancer Research Center, Germany). B6 or BALB/c.FoxP3-DTR-GFP mice were
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Supplementary Figure 1
Supplementary Figure 1 Identification of IFN-γ-producing CD8 + and CD4 + T cells with naive phenotype by alternative gating and sample-processing strategies. a. Contour 5% probability plots show definition
Pathologic Stage. Lymph node Stage
ASC ASC a c Patient ID BMI Age Gleason score Non-obese PBMC 1 22.1 81 6 (3+3) PBMC 2 21.9 6 6 (3+3) PBMC 3 22 84 8 (4+4) PBMC 4 24.6 68 7 (3+4) PBMC 24. 6 (3+3) PBMC 6 24.7 73 7 (3+4) PBMC 7 23. 67 7 (3+4)
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DOI: 1.138/ncb222 / b. WB anti- WB anti- ulin Mitotic index (%) 14 1 6 2 T (h) 32 48-1 1 2 3 4 6-1 4 16 22 28 3 33 e. 6 4 2 Time (min) 1-6- 11-1 > 1 % cells Figure S1 depletion leads to mitotic defects
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Supplementary Figure 1 Supplementary Figure 1 SNARE Probes for FRET/2pFLIM Analysis Used in the Present Study. mturquoise (mtq) and Venus (Ven) are in blue and yellow, respectively. The soluble N-ethylmaleimide-sensitive
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doi: 10.1038/nature05732 SUPPLEMENTARY INFORMATION Supplemental Data Supplement Figure Legends Figure S1. RIG-I 2CARD undergo robust ubiquitination a, (top) At 48 h posttransfection with a GST, GST-RIG-I-2CARD
ILC1 and ILC3 isolation and culture Following cell sorting, we confirmed that the recovered cells belonged to the ILC1, ILC2 and
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a c e doi:10.1038/nature10407 b d f Supplementary Figure 1. SERCA2a complex analysis. (a) Two-dimensional SDS-PAGE gels of SERCA2a complexes. A silver-stained SDSPAGE gel is shown, which reveals a 12 kda
Nature Protocols: doi: /nprot Supplementary Figure 1
Supplementary Figure 1 Traditional electronic gating strategy for analysing cell death based on A5-FITC and 7-AAD. a, Flow cytometry analysis showing the traditional two-stage electronic gating strategy
Supplementary Materials
Supplementary Materials Figure S1. MTT Cell viability assay. To measure the cytotoxic potential of the oxidative treatment, the MTT [3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide] assay
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Rescue of mutant rhodopsin traffic by metformin-induced AMPK activation accelerates photoreceptor degeneration Athanasiou et al
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