A. List of selected proteins with high SILAC (H/L) ratios identified in mass
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1 Supplementary material Figure S1. Interaction between UBL5 and FANCI A. List of selected proteins with high SILAC (H/L) ratios identified in mass spectrometry (MS)-based analysis of UBL5-interacting proteins, as described previously 1. HeLa cells transfected with Strep-HA-UBL5 plasmid or empty vector were cultured in heavy (H) or light (L) SILAC medium, respectively. Cell lysates were incubated with Strep-Tactin Sepharose. Bound complexes were washed extensively and analyzed by MS. B. Whole cell extracts () of U2OS cells transfected with cdna encoding GFP-FANCI were subjected to GFP immunoprecipitation followed by immunoblotting with SART1 and GFP antibodies. C. U2OS cells transfected with cdna encoding Strep-HA-tagged UBL5 were treated or not with 1 µm mitomycin C (MMC) for 18 h. Whole cell extracts () were subjected to Strep-Tactin pull-down followed by immunoblotting with FANCI and HA antibodies. *, non-specific band.
2 A Protein symbolsilac ratio (H/L) SART *UBL MFAP WBP NPW PRPF38A 3.70 FANCI 3.09 EFTUD SNRPD PRPF SNRPD Oka et al., Figure S1 B IP: GFP + + GFP-FANCI SART1 GFP-FANCI C Pull-down: Strep U2OS Strep-HA-UBL5 MMC -Ub Strep-HA-UBL5 -Ub Strep-HA-UBL5
3 Figure S2. UBL5 is required for the integrity of the FA pathway A. Extracts of HeLa cells transfected with UBL5 sirnas were analyzed by immunoblotting with indicated antibodies. B. Extracts of HeLa cells transfected with non-targeting (CTRL) or FANCI sirnas were analyzed by immunoblotting with indicated antibodies. C. U2OS cells were transfected with non-targeting control (CTRL) or UBL5 sirna, subsequently transfected with a construct encoding HA-FANCI and then treated with 0.2 µm MMC for 24 h. Cells were fixed and immunostained with HA antibody. Representative images are shown. Arrows indicate cells transiently expressing HA-FANCI. Scale bar, 10 µm. For quantification, 200 cells were counted in each experiment. Mean values (±SD) from three independent experiments are shown. D. Extracts of U2OS cells transfected with indicated sirnas were analyzed by immunoblotting. See also Fig. 2F.
4 Oka et al., Figure S2 A siubl5(#1) siubl5(#2) FANCC B 48 h 72 h sifanci(utr) sifanci(#1) sifanci(utr) sifanci(#1) Time after sirna FANCE UBL5 β-tubulin UBL5 β-tubulin C siubl5 HA-FANCI DAPI Cells with HA-FANCI foci (%) siubl5 D siwapl siubl5(#1)+siwapl siubl5(#2)+siwapl WAPL UBL5 β-actin sifanci sifancd2 SMC1 FANCD2 SMC1
5 Figure S3. UBL5 has a direct role uncoupled from its pre-mrna splicing involvement in promoting the FA pathway A. HeLa cells were transfected with non-targeting control (CTRL), UBL5 or SART1 sirnas. Total mrnas were extracted and levels of FANCI mrna were measured by reverse transcription-qpcr, normalized to the expression of β-actin. B. U2OS cells stably expressing GFP-tagged FANCI and transfected with nontargeting control (CTRL) or UBL5 sirnas were harvested following addition of MG132. Cell lysates were immunoblotted with GFP, UBL5 and β-tubulin antibodies. C. Whole cell extracts () of HeLa cells transfected with indicated Strep-HA- UBL5 constructs were subjected to Strep-Tactin pull-down followed by immunoblotting with FANCI and HA antibodies. D. U2OS cells stably expressing sirna-resistant (si R ) forms of Strep-HA-UBL5 WT or D64A mutant were induced or not with doxycycline, transfected with non-targeting control (CTRL) or UBL5 sirnas and treated with MMC for 24 h. Cell extracts were analyzed by immunoblotting with indicated antibodies.
6 Oka et al., Figure S3 A Relative FANCI mrna level siubl5 sisart1 B U2OS/GFP-FANCI DMSO MG siubl5 GFP-FANCI UBL5 β-tubulin +Strep-HA-UBL5 +Strep-HA-UBL5 WT E3A D8A K12A/K13A R15L D21A D22A D26A R38L WT E61A D64A C Pull-down: Strep WB: HA WB: HA D U2OS/Strep-HA-UBL5(si R )-WT U2OS/Strep-HA-UBL5(si R )-D64A MMC DOX siubl5 -Ub FANCD2-Ub FANCD2 Fold increase of FANCD2-Ub Strep- HA-UBL5 UBL5 β-tubulin
7 Figure S4. Binding of FANCI fragments to FANCD2 Whole cell extracts () of HEK293T cells transfected with indicated constructs were subjected to GFP immunoprecipitation followed by immunoblotting with FANCD2 and GFP antibodies.
8 Oka et al., Figure S4 FL 293T F1 F2 F3 F4 GFP-FANCI FANCD2-Ub FANCD2 97 IP: GFP WB: GFP kda FANCD2-Ub FANCD WB: GFP kda
9 Figure S5. Loss of UBL5 binding impairs the stability and activation of FANCI, while FANCD2 knockdown does not affect FANCI stability. A. Extracts of HeLa cells transfected with non-targeting control (CTRL), FANCI or FANCD2 sirnas for 48 or 72 h were analyzed by immunoblotting with antibodies to FANCI, FANCD2 or ORC2 (loading control). B. HeLa cells transfected with non-targeting control or FANCD2 sirna and subsequently transfected with GFP-tagged FANCI WT or UBL5 were treated with cycloheximide (CHX) for 3 h. Cell extracts were analyzed by immunoblotting with indicated antibodies. C. U2OS cells stably expressing HA-tagged FANCI WT or UBL5 were treated or not with 30 µm ML323 for 6 h. Cell extracts were analyzed by immunoblotting with HA and SMC1 antibodies.
10 Oka et al., Figure S5 A 48 h 72 h Time after sirna sifanci sifancd2 sifanci sifancd2 B WT +CHX UBL5 + + GFP-FANCI sifancd2 GFP-FANCI FANCD2 FANCD2 β-actin ORC2 C U2OS/HA-FANCI WT UBL5 + + ML323 HA-FANCI-Ub HA-FANCI SMC1
11 Figure S6. UBL5 promotes FANCI stability by facilitating its homodimerization A. Extracts of HEK293T cells co-transfected with GFP-FANCI and HA-FANCI constructs as indicated were subjected to HA immunoprecipitation followed by immunoblotting with GFP and HA antibodies. B. Schematic diagram of enforced homodimerization of FANCI by means of the DmrB system. C. HEK293T cells transfected with plasmids encoding DmrB-GFP-FANCI WT or UBL5 were treated with 100 nm AP20187 (B/B Homodimerizer) or left untreated, and then treated with cycloheximide (CHX). Cell extracts were analyzed by immunoblotting with GFP and β-actin antibodies.
12 Oka et al., Figure S6 A HEK293T GFP-FANCI HA-FANCI B DmrB GFP FANCI IP: HA GFP-FANCI HA-FANCI DmrB GFP FANCI B/B Homodimerizer GFP-FANCI HA-FANCI DmrB DmrB GFP GFP FANCI FANCI C WT UBL5 DmrB-GFP-FANCI + HEK293T CHX AP20187 DmrB-GFP-FANCI β-actin
13 Supplementary reference 1. Oka, Y. et al. UBL5 is essential for pre-mrna splicing and sister chromatid cohesion in human cells. EMBO Rep 15, (2014). 1
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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
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TABLE OF CONTENTS SUPPLEMENTARY LEGENDS... 2 11 MOVIE S1... 2 FIGURE S1 LEGEND... 3 FIGURE S2 LEGEND... 4 FIGURE S3 LEGEND... 5 FIGURE S4 LEGEND... 6 FIGURE S5 LEGEND... 7 FIGURE S6 LEGEND... 8 FIGURE
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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
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p = formed with HCI-001 p = Relative # of blood vessels that formed with HCI-002 Control Bevacizumab + 17AAG Bevacizumab 17AAG
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DOI:.38/ncb2822 a MTC02 FAO cells EEA1 b +/+ MEFs /DAPI -/- MEFs /DAPI -/- MEFs //DAPI c HEK 293 cells WCE N M C P AKT TBC1D7 Lamin A/C EEA1 VDAC d HeLa cells WCE N M C P AKT Lamin A/C EEA1 VDAC Figure
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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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ACC ELOVL MCAD. CPT1α 1.5 *** 0.5. Reverbα *** *** 0.5. Fasted. Refed
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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
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Supplementary Table 1. Cell sphingolipids and S1P bound to endogenous TRAF2. Sphingolipid Cell pmol/mg TRAF2 immunoprecipitate pmol/mg Sphingomyelin 4200 ± 250 Not detected Monohexosylceramide 311 ± 18
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
Supplementary Figure 1
A B D Relative TAp73 mrna p73 Supplementary Figure 1 25 2 15 1 5 p63 _-tub. MDA-468 HCC1143 HCC38 SUM149 MDA-468 HCC1143 HCC38 SUM149 HCC-1937 MDA-MB-468 ΔNp63_ TAp73_ TAp73β E C Relative ΔNp63 mrna TAp73