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1 SUPPLEMENTARY INFORMATION doi:10.10/nature10195 NCBI gene: Tagged Subunit(s: HA-Vpx; FLAG-Cul4 HA-DCAF1 FLAG-Cul4 HA-FLAG-Vpx Mock Vpx (SIVmac 100 (a ; (b ; 0.05 DCAF1 DDB1 DDA1 Cul4A 1; ; ; ; ; ; ; ; ; ; ; SAMHD1 TUBB2C EEF1A1 PRKDC SLC25A1 RPN1 AIFM1 KPNB1 TCP1 MIOS ATP2A2 ATP5C1 CAD SLC25A12 ATP5A1 ATP1A1 WDR59 ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; Supplementary Table I. MudPIT identification of cellular proteins that specifically co-purify with Cul4 DCAF1 E complex in the presence of Vpx. Protein complexes were purified by two sequential immunoprecipitation via HA-tag and then via FLAG-tag each followed by competitive elution with peptide epitope and analyzed by MudPIT. Upper rows: (a spectral count and (bd NSAF values for subunits of CRL4 DCAF1 and Vpx are shown; Lower rows: proteins that co-purified with HA-FLAG-Vpx and with Vpx-CRL DCAF1 complex but not with CRL DCAF1 complex in the absence of Vpx are shown. (a spectral count number of mass spectrometry spectra matching peptides from the indicated protein (bd NSAF distributed normalized spectral abundance factor 1
2 RESEARCH SUPPLEMENTARY INFORMATION doi:10.10/nature10195 ;<=> " % &'(*+ 5@D : ( *+&'*45% -.? "%%@AB=%&' &'% &'/012 E<EF /012?+ &' 1 9 D %GHI<JFK=FE=F " &'@AB=% % -.? : 5@D /012 +?C?+ 1? %GHI<JFK=FE=F Supplementary Figure 1. Vpx-mediated relief of the inhibition of HIV-2 & SIVmac infection in MDM requires Vpx glutamine Q. MDM were infected with vpx-defective HIV-2 (HIV-2vpxGFP (a or SIVmac 29 (SIVmacvpx GFP (b single cycle reporter viruses alone or in combination with SIV VLP loaded with wild type (wt or QA -substituted (QA HIV-2 Rod (a or SIVmac 29 (b Vpx proteins. (c All SIV VLP were normalized for their p24 CA and Vpx contents by Western blotting. 2
3 %&'(%*+- doi:10.10/nature10195 RESEARCH SUPPLEMENTARY INFORMATION " " <BCBD%0 " <BCBD%E A" " :" <BCBD%= 4(52./012 /9:; <=0': /9:; <=0': /9:; <=0': Supplementary Figure 2. HIV-1GFP transduction efficiency of MDM following RNAi to SAMHD1 or DCAF1. MDM subjected to the sirna targeting DCAF1 SAMHD1 or non-targeting sirna (NT1/2 in a protocol described in Figure c were infected with SIV VLP loaded with SIVmac Vpx. Two days later cells were challenged with HIV-1GFP reporter virus. GFP-positive cells were quantified days later by flow cytometry. Results of three typical experiments with MDM from three donors are shown.
4 doi:10.10/nature10195 RESEARCH SUPPLEMENTARY INFORMATION./ < &'( "% *+- = ; % < ; 9 ; 9 % % < =< 9 < %= :; ; 9 ; 9 % % < >:9 < < %>: ==> % ; 9 ; 9 % % " < > 9< < 9%% 9<9 % ; 9 ; 9 % % < %> % < % 9:% ; 9 ; 9 % % < =>< =>9 < ;= ;9 ; 9 ; 9 % % % = 9 % = 9?@0ABCDEFG&HGIHG % = 9 % = 9 Supplementary Figure. Flow cytometry analysis of the effects of SAMHD1 depletion on HIV-1GFP transduction of MDM. Primary data for the experiment with MDM from Donor in Figure 4a are shown. 4
5 RESEARCH SUPPLEMENTARY INFORMATION doi:10.10/nature10195 %&'( +'-. / &*&" " 0?>@:A 90 :;<=> "0 " 12415( " ;< 5; & " 0 / ; BC %&' D +'-. Supplementary Figure 4. Increased permissiveness to HIV-1GFP infection of SAMHD1-depleted MDM is not due to off target effects of sirna pool targeting SAMHD1. a. To exclude the possibility that the observed enhancement of macrophage transduction following RNAi to SAMHD1 is caused by off target effects of the sirna pool targeting SAMHD1 individual sirnas from the ON-TARGETplus SMARTpool SAMHD1 (J J and J were tested using the assay described in Figure 4. The enhanced MDM transduction by HIV-1GFP was observed with all three out of three sirnas to SAMHD1 that we tested (panel a. Thus it is unlikely that the enhanced MDM transduction by HIV-1GFP following RNAi to SAMHD1 is due to an off target effect of the sirna pool targeting SAMHD1. b. SAMHD1 protein levels in cells transfected with individual sirna duplexes targeting SAMHD1. U2OS cells stably expressing human FLAG- tagged SAMHD1 were transfected with the indicated sirnas targeting SAMHD1 (J (9 J (10 J (11 and J (12 the SMARTpool SAMHD1 (pool or non-targeting sirna pool (NT1 as described previously12. Detergent extracts were prepared from the cells days after initiation of RNAi and SAMHD1 was visualized by immunoblotting for the FLAG epitope. Mock not transfected U2OS cells. 5
6 doi:10.10/nature10195 RESEARCH SUPPLEMENTARY INFORMATION ;<= -./ "0( *+ % &'( % &'( % ;<= " """" 12% *4595: %&'"( " Supplementary Figure 5. Model for Vpx-mediated relief of the inhibition of HIV-1 infection in myeloid cells by SAMHD1. SAMHD1 interferes with HIV-1 infection at an early post-entry step preventing efficient viral cdna synthesis probably by interfering with the reverse transcription process itself. Vpx relieves SAMHD1- imposed inhibition by binding to SAMHD1 and loading the protein onto CRL4 DCAF1 E ubiquitin ligase complex via its DCAF1 substrate receptor subunit. Vpx targets specifically DCAF1-DDB1 complex that is not associated with the inhibitory COP9 Signalosome (CSN or displaces CSN upon binding to DCAF1 20. CRL4 DCAF1 E poly-ubiquitylates SAMHD1 and directs it for degradation by the proteasome.
7 RESEARCH SUPPLEMENTARY INFORMATION " % " &'( % &' * ( ( * *2 */ ( 01 2 doi:10.10/nature10195.*2 Supplementary Figure. SAMHD1 protein levels in myeloid cells and established cell lines. Detergent extracts prepared from monocyte-derived macrophages and dendritic cells (MDM & MDDC as well as HEK 29T cells Jurkat T cells and monocytic THP-1 cells were normalized for their protein concentrations resolved by SDS-PAGE and immunoblotted for endogenous SAMHD1. Extract from HEK 29T cells transiently overexpressing FLAG- epitope tagged SAMHD1 (HEK 29T + HFA.SAMHD1 provided positive control.
8 RESEARCH SUPPLEMENTARY INFORMATION doi:10.10/nature10195 " E 9 D C ABC AB 45" _-./0/12 4:;<=->?@ 9 ABCD *% ( ABCE *( *+ ' & % % & % & % & >?@FG1/HIJII KLF%MN 4:;<=->?@ E 9 D ABC ABC 4:;<=->?@ 9 *' ( 45" _-./0/12 ABCD ABCE +*+ *& ' & % % & % & % & >?@FG1/HIJII N@- 45" _-./0/12 4:;<=->?@ E 9 D ABC ABC 4:;<=->?@ 9 ABCD *& ( ABCE *+ *M+ ' & % % & % & % & >?@FG1/HIJII Supplementary Figure. SAMHD1 does not restrict SIVmacGFP infection in HEK 29T and THP-1 cells. Monocyte-derived macrophages (MDM monocytic THP-1 cells and epithelial HEK 29T cells were infected with SIVmac-GFP reporter viruses containing wild type (vpx+ or inactivated (vpx vpx open reading frames. Three days after infection the cells were harvested for flow cytometry quantification of GFP-positive productively infected cells and for immunoblot analysis of SAMHD1 levels in the infected cells. Mock non-infected control cells. It is evident that SIVmac-GFP transduction rates are not affected by endogenous SAMHD1 levels in HEK 29T and THP-1 cells. Differences between transduction efficiencies of HEK 29T and THP-1 cells reflect differences in infection multiplicities.
SUPPLEMENTAL FIGURE LEGENDS
SUPPLEMENTAL FIGURE LEGENDS Supplemental Figure S1: Endogenous interaction between RNF2 and H2AX: Whole cell extracts from 293T were subjected to immunoprecipitation with anti-rnf2 or anti-γ-h2ax antibodies
Supplementary Materials
Supplementary Materials Supplementary Figure S1 Regulation of Ubl4A stability by its assembly partner A, The translation rate of Ubl4A is not affected in the absence of Bag6. Control, Bag6 and Ubl4A CRISPR
Vpx rescue of HIV-1 from the antiviral state in mature dendritic cells is independent of the intracellular deoxynucleotide concentration
University of Massachusetts Medical School escholarship@umms University of Massachusetts Medical School Faculty Publications 2-1-2014 Vpx rescue of HIV-1 from the antiviral state in mature dendritic cells
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Supplemental Information. NRF2 Is a Major Target of ARF. in p53-independent Tumor Suppression
Molecular Cell, Volume 68 Supplemental Information NRF2 Is a Major Target of ARF in p53-independent Tumor Suppression Delin Chen, Omid Tavana, Bo Chu, Luke Erber, Yue Chen, Richard Baer, and Wei Gu Figure
(B D) Three views of the final refined 2Fo-Fc electron density map of the Vpr (red)-ung2 (green) interacting region, contoured at 1.4σ.
Supplementary Figure 1 Overall structure of the DDB1 DCAF1 Vpr UNG2 complex. (A) The final refined 2Fo-Fc electron density map, contoured at 1.4σ of Vpr, illustrating well-defined side chains. (B D) Three
RAW264.7 cells stably expressing control shrna (Con) or GSK3b-specific shrna (sh-
1 a b Supplementary Figure 1. Effects of GSK3b knockdown on poly I:C-induced cytokine production. RAW264.7 cells stably expressing control shrna (Con) or GSK3b-specific shrna (sh- GSK3b) were stimulated
293T cells were transfected with indicated expression vectors and the whole-cell extracts were subjected
SUPPLEMENTARY INFORMATION Supplementary Figure 1. Formation of a complex between Slo1 and CRL4A CRBN E3 ligase. (a) HEK 293T cells were transfected with indicated expression vectors and the whole-cell
Supplementary Figure 1.TRIM33 binds β-catenin in the nucleus. a & b, Co-IP of endogenous TRIM33 with β-catenin in HT-29 cells (a) and HEK 293T cells
Supplementary Figure 1.TRIM33 binds β-catenin in the nucleus. a & b, Co-IP of endogenous TRIM33 with β-catenin in HT-29 cells (a) and HEK 293T cells (b). TRIM33 was immunoprecipitated, and the amount of
Appendix. Table of Contents
Appendix Table of Contents Appendix Figures Figure S1: Gp78 is not required for the degradation of mcherry-cl1 in Hela Cells. Figure S2: Indel formation in the MARCH6 sgrna targeted HeLa clones. Figure
T H E J O U R N A L O F C E L L B I O L O G Y
Supplemental material Chairoungdua et al., http://www.jcb.org/cgi/content/full/jcb.201002049/dc1 T H E J O U R N A L O F C E L L B I O L O G Y Figure S1. Expression of CD9 and CD82 inhibits Wnt/ -catenin
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SUPPLEMENTARY INFORMATION doi:1.138/nature9814 a A SHARPIN FL B SHARPIN ΔNZF C SHARPIN T38L, F39V b His-SHARPIN FL -1xUb -2xUb -4xUb α-his c Linear 4xUb -SHARPIN FL -SHARPIN TF_LV -SHARPINΔNZF -SHARPIN
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sirna pool: Control Tetherin -HA-GFP HA-Tetherin -Tubulin Supplementary Figure S1. Knockdown of HA-tagged tetherin expression by tetherin specific sirnas. HeLa cells were cotransfected with plasmids expressing
Tel: ; Fax: ;
Tel.: +98 216 696 9291; Fax: +98 216 696 9291; E-mail: mrasadeghi@pasteur.ac.ir Tel: +98 916 113 7679; Fax: +98 613 333 6380; E-mail: abakhshi_e@ajums.ac.ir A Soluble Chromatin-bound MOI 0 1 5 0 1 5 HDAC2
supplementary information
DOI: 10.1038/ncb1875 Figure S1 (a) The 79 surgical specimens from NSCLC patients were analysed by immunohistochemistry with an anti-p53 antibody and control serum (data not shown). The normal bronchi served
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doi:10.1038/nature11700 Figure 1: RIP3 as a potential Sirt2 interacting protein. Transfected Flag-tagged Sirt2 was immunoprecipitated from cells and eluted from the Sepharose beads using Flag peptide.
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SUPPLEMENTARY INFORMATION doi:10.1038/nature11429 S1a 6 7 8 9 Nlrc4 allele S1b Nlrc4 +/+ Nlrc4 +/F Nlrc4 F/F 9 Targeting construct 422 bp 273 bp FRT-neo-gb-PGK-FRT 3x.STOP S1c Nlrc4 +/+ Nlrc4 F/F casp1
SUPPLEMENTARY FIGURES
SUPPLEMENTARY FIGURES Supplementary Figure 1. (A) Left, western blot analysis of ISGylated proteins in Jurkat T cells treated with 1000U ml -1 IFN for 16h (IFN) or left untreated (CONT); right, western
Nature Immunology doi: /ni.3268
Supplementary Figure 1 Loss of Mst1 and Mst2 increases susceptibility to bacterial sepsis. (a) H&E staining of colon and kidney sections from wild type and Mst1 -/- Mst2 fl/fl Vav-Cre mice. Scale bar,
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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
Supplementary information
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Supplementary Discussion The cell cycle machinery and the DNA damage response network are highly interconnected and co-regulated in assuring faithful duplication and partition of genetic materials into
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Supplementary Information
Supplementary Information mediates STAT3 activation at retromer-positive structures to promote colitis and colitis-associated carcinogenesis Zhang et al. a b d e g h Rel. Luc. Act. Rel. mrna Rel. mrna
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Supplementary Figure 1. MAT IIα is Acetylated at Lysine 81.
IP: Flag a Mascot PTM Modified Mass Error Position Gene Names Score Score Sequence m/z [ppm] 81 MAT2A;AMS2;MATA2 35.6 137.28 _AAVDYQK(ac)VVR_ 595.83-2.28 b Pre-immu After-immu Flag- WT K81R WT K81R / Flag
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SUPPLEMENTARY INFORMATION doi:10.1038/nature13418 Supplementary Results: USP30 opposes autophagic flux In HEK-293 cells, USP30 overexpression increased basal LC3-II levels, dependent on enzymatic activity,
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Supplemental information contains 7 movies and 4 supplemental Figures
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DOI: 10.1038/ncb3076 Supplementary Figure 1 btrcp targets Cep68 for degradation during mitosis. a) Cep68 immunofluorescence in interphase and metaphase. U-2OS cells were transfected with control sirna
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FIG S1 Examination of eif4b expression after virus infection. (A) A549 cells
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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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doi:10.1038/nature11095 Supplementary Table 1. Summary of the binding between Angptls and various Igdomain containing receptors as determined by flow cytometry analysis. The results were summarized from
T H E J O U R N A L O F C E L L B I O L O G Y
Supplemental material Jewell et al., http://www.jcb.org/cgi/content/full/jcb.201007176/dc1 T H E J O U R N A L O F C E L L B I O L O G Y Figure S1. IR Munc18c association is independent of IRS-1. (A and
Type of file: PDF Title of file for HTML: Supplementary Information Description: Supplementary Figures
Type of file: PDF Title of file for HTML: Supplementary Information Description: Supplementary Figures Type of file: MOV Title of file for HTML: Supplementary Movie 1 Description: NLRP3 is moving along
Reviewers' comments: Reviewer #1 (Remarks to the Author):
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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
Supplementary Figure S1 Supplementary Figure S2
Supplementary Figure S A) The blots shown in Figure B were qualified by using Gel-Pro analyzer software (Rockville, MD, USA). The ratio of LC3II/LC3I to actin was then calculated. The data are represented
Supplementary Fig. 1. GPRC5A post-transcriptionally down-regulates EGFR expression. (a) Plot of the changes in steady state mrna levels versus
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Supplementary Materials for
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MO reports PR3 dephosphorylates TZ Xian-o Lv et al xpanded View igures igure V1. PR3 dephosphorylates and inactivates YP/TZ., Overexpression of tight junction proteins Pals1 () or LIN7 () has no effect
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Supplementary Table 1. Metabolic parameters in GFP and OGT-treated mice
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doi:10.1038/nature12652 Supplementary Figure 1. PRDM16 interacts with endogenous EHMT1 in brown adipocytes. Immunoprecipitation of PRDM16 complex by flag antibody (M2) followed by Western blot analysis
Supplement Figure S1. Real Time PCR analysis of mrna levels of C/EBPα and PU.1 in wild type (WT) and NQO1-null (NQO1-/-) mice.
competes with 20S proteasome for binding with C/EBP leading to its stabilization and Relative mrna levels Supplement Figure S1. Real Time PCR analysis of mrna levels of C/EBPα and PU.1 in wild type (WT)
Nature Structural & Molecular Biology: doi: /nsmb.3218
Supplementary Figure 1 Endogenous EGFR trafficking and responses depend on biased ligands. (a) Lysates from HeLa cells stimulated for 2 min. with increasing concentration of ligands were immunoblotted
Supplementary Figure 1. Normal T lymphocyte populations in Dapk -/- mice. (a) Normal thymic development in Dapk -/- mice. Thymocytes from WT and Dapk
Supplementary Figure 1. Normal T lymphocyte populations in Dapk -/- mice. (a) Normal thymic development in Dapk -/- mice. Thymocytes from WT and Dapk -/- mice were stained for expression of CD4 and CD8.
effects on organ development. a-f, Eye and wing discs with clones of ε j2b10 show no
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Author s Choice 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
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supplementary information
DOI: 10.1038/ncb2153 Figure S1 Ectopic expression of HAUSP up-regulates REST protein. (a) Immunoblotting showed that ectopic expression of HAUSP increased REST protein levels in ENStemA NPCs. (b) Immunofluorescent
Nature Immunology: doi: /ni.3631
Supplementary Figure 1 SPT analyses of Zap70 at the T cell plasma membrane. (a) Total internal reflection fluorescent (TIRF) excitation at 64-68 degrees limits single molecule detection to 100-150 nm above
Supplementary Figure 1
Supplementary Figure 1 Supplementary Figure 1. Neither the activation nor suppression of the MAPK pathway affects the ASK1/Vif interaction. (a, b) HEK293 cells were cotransfected with plasmids encoding
Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1. Differential expression of mirnas from the pri-mir-17-92a locus.
Supplementary Figure 1 Differential expression of mirnas from the pri-mir-17-92a locus. (a) The mir-17-92a expression unit in the third intron of the host mir-17hg transcript. (b,c) Impact of knockdown
Supplementary Figure 1. PAQR3 knockdown inhibits SREBP-2 processing in CHO-7 cells CHO-7 cells were transfected with control sirna or a sirna
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A. List of selected proteins with high SILAC (H/L) ratios identified in mass
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SUPPLEMENTARY INFORMATION. Supp. Fig. 1. Autoimmunity. Tolerance APC APC. T cell. T cell. doi: /nature06253 ICOS ICOS TCR CD28 TCR CD28
Supp. Fig. 1 a APC b APC ICOS ICOS TCR CD28 mir P TCR CD28 P T cell Tolerance Roquin WT SG Icos mrna T cell Autoimmunity Roquin M199R SG Icos mrna www.nature.com/nature 1 Supp. Fig. 2 CD4 + CD44 low CD4
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T H E J O U R N A L O F C E L L B I O L O G Y Supplemental material Krenn et al., http://www.jcb.org/cgi/content/full/jcb.201110013/dc1 Figure S1. Levels of expressed proteins and demonstration that C-terminal
Vif Proteins of Human and Simian Immunodeficiency Viruses Require Cellular CBFβ to Degrade APOBEC3 Restriction Factors
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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
Trim29 gene-targeting strategy. (a) Genotyping of wildtype mice (+/+), Trim29 heterozygous mice (+/ ) and homozygous mice ( / ).
Supplementary Figure 1 Trim29 gene-targeting strategy. (a) Genotyping of wildtype mice (+/+), Trim29 heterozygous mice (+/ ) and homozygous mice ( / ). (b) Immunoblot analysis of TRIM29 in lung primary
supplementary information
DOI: 1.138/ncb1 Control Atg7 / NAC 1 1 1 1 (mm) Control Atg7 / NAC 1 1 1 1 (mm) Lamin B Gstm1 Figure S1 Neither the translocation of into the nucleus nor the induction of antioxidant proteins in autophagydeficient
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Supplementary Figure 1. The mir-182 binding site of SMAD7 3 UTR and the mutated sequence. 1 Supplementary Figure 2. Expression of mir-182 and SMAD7 in various cell lines. (A) Basal levels of mir-182 expression
Supplementary figures
Supplementary figures Supplementary Figure 1. B cells stimulated with pokeweed mitogen display normal mitotic figures but not cells infected with B95-8. The figures show cells stimulated with pokeweed
Nature Immunology doi: /ni Supplementary Figure 1. Raf-1 inhibition does not affect TLR4-induced type I IFN responses.
Supplementary Figure 1 Raf-1 inhibition does not affect TLR4-induced type I IFN responses. Real-time PCR analyses of IFNB, ISG15, TRIM5, TRIM22 and APOBEC3G mrna in modcs 6 h after stimulation with TLR4
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