Supplementary data 1. Prmers and probes used in Taqman real-time PCR.
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1 Supplementary data 1. Prmers and probes used in Taqman real-time PCR. PIG-T Forward Primer: gatctgcctcacgtgcactgt Reverse Primer: aggttcgggtgaggagattgt Probe: 6FAMTGG CCG TGT GCT ATG GCT CCT TCTAMRA PIG-U Forward Primer: agccctccagccagagttg Reverse Primer: acttgtgaccctggactcgaa Probe: 6FAMCAG GCG AGT GCT TGG GCA GAA GATAMRA GPAA1 Forward Primer: ccgggctgggacagaga Reverse Primer: cagacactcatttatttccccagaa Probe: 6FAMTCC CCA AGG ACC CCA TTC TGC CTAMRA β-actin Forward Primer: tcacccacactgtgcccatctacga Reverse Primer: tcggtgaggatcttcatgaggta Probe: VICATG CCC TCC CCC ATG CCA TCCTAMRA
2 Supplementary data 2. The Oligonucleotides sequences used for synthesizing the PIG-T, PIG-U and GPAA1 RNAi. PIG-T Si1A (sense): TCGAGACACTGTCACTGATGTGGATTTCAAGAGAATCCACATCAGTGACAGTGTCTTTT Si1B(antisense): CTAGAAAAGACACTGTCACTGATGTGGATTCTCTTGAAATCCACATCAGTGACAGTGTC Si2A(sense): TCGAATGCACGCTGTACTAGCATCTTTCAAGAGAAGATGCTAGTACAGCGTGCATTTTTT Si2B(antisense): CTAGAAAAAATGCACGCTGTACTAGCATCTTCTCTTGAAAGATGCTAGTACAGCGTGCAT Si3A(sense): TCGAACCTCAACATCCAGCTCAAGTTTCAAGAGAACTTGAGCTGGATGTTGAGGTTTTTT Si3B(antisense): CTAGAAAAAACCTCAACATCCAGCTCAAGTTCTCTTGAAACTTGAGCTGGATGTTGAGGT PIG-U Si1A(sense): TCGAGTCTACCTGTGCCATCAACAATTCAAGAGATTGTTGATGGCACAGGTAGACTTTTT Si1B(antisense): CTAGAAAAAGTCTACCTGTGCCATCAACAATCTCTTGAATTGTTGATGGCACAGGTAGAC Si2A(sense): TCGACCATCTCTACAGATTCCTGAGTTCAAGAGACTCAGGAATCTGTAGAGATGGTTTTT Si2B(antisense): CTAGAAAAACCATCTCTACAGATTCCTGAGTCTCTTGAACTCAGGAATCTGTAGAGATGG GPAA1 Si1A ( sense): TCGACCTTGACCTGCTCAATCTCTTTTCAAGAGAAAGAGATTGAGCAGGTCAAGGTTTTT Si1B ( antisense) CTAGAAAAACCTTGACCTGCTCAATCTCTTTCTCTTGAAAAGAGATTGAGCAGGTCAAGG Si2A ( sense) TCGATAGCTTCCGCCAGTACAAGTATTCAAGAGATACTTGTACTGGCGGAAGCTATTTTT Si2B ( antisense) CTAGAAAAATAGCTTCCGCCAGTACAAGTATCTCTTGAATACTTGTACTGGCGGAAGCTA
3 Supplementary data 3. RT-PCRs were performed to compare the expression pattern of PIG-U, PIG-T and GPAA1 gene in MCF12A, MCF10A, HMEC and normal breast tissue. We found, in most cases, the expression level of these three GPI transamidase subunits are the same in MCF10A and MCF12A, but lower in HMEC and normal breast tissue. MCF12A MCF10A HMEC Normal -PIG-U -GAPDH
4 Supplementarydata 4. Summary of gain of three genes copy number in 69 cases of breast tumors. PIG-U 21 3 PIG-T GPAA1
5 Supplementary data 5. Subcellular localization of PIG-T and GPAA1 in cells and stable clone selection in NIH3T3 cells. A: Western blotting analysis shows PIG-T-HA and GPAA1-HA expression in 293 cells. B: PIG-T and GPAA1 are located in the ER by immunofluorescence detection. C: Western blotting in two independent NIH3T3 clones show stable high expression of PIG-T-HA and GPAA1-HA fusion proteins. β-actin was used as protein loading control. D: Differential expression of PIG-T-HA and GPAA1-HA fusion proteins in NIH3T3 clones was detected by immunofluorescence. A. B. Gene Nucleus Merged 75KD- Vector GPAA1 PIG-T -HA -β-actin Gene Nucleus Merged C. Vector Clone5 Clone13 D. Clone 5 Clone 13 -β-actin Clone 6 Clone 2 Vector Clone6 Clone2 -β-actin
6 Supplementary data 6. Representive pictures of soft agar colonies in vector transfected cells, and cell clones stably expressing PIG-T-HA and GPAA1-HA fusion proteins. Clone1 Clone7 -control Clone5 Clone13 Clone6 Clone2
7 Supplementary data 7. RNAi decreases expression of PIG-U in MCF7cells. The top panels show protein levels by western blotting and the bottom panels depict the colony formation assay. Error bars shown are derived from three independent experiments. Control Si PIGU1 Si PIGU2 -PIG-U -β-actin Number of Colonies
8 Supplemetary data 8. The physical interaction of PIG-T, GPAA1 and Paxillin in human breast cancer cells. A. Colocalization of PIG-T, GPAA1 with paxilin in breast cancer MDA231, MDA436 cells. I shows merged images. II ( green signals) shows PIG-T (top panel) and GPAA1 (low panel) images. III (Red signal) is Paxillin image. IV (Blue signal) is nucleus staining. B. Immunoprecipitation (IP) assay. Left two penals show cell lysis immunoprecipitated with control Flag antibody. Right two panels show cell lysis immunoprecipitated with paxillin antibody. Top panel shows western blotting with anti serum in MDA231 cells. Low panel shows western blotting with anti serum in MDA436 cells. A. I II III IV -MDA231 I II III IV -MDA436 B. In-put IP FLAG In-put IP Paxillin IB:PIG-T IB: GPAA1
9 Supplementary data 9. Breast cancer cell line invasion assay using Matrigel invasion chamber. MDA231 and MDA436 show much stronger invasive ability than MDA157 and MCF7 cells (p <0.006 and p <0.02). 0.5x10 4 cells were plated in each well of 24 well matrigel invasion chamber. Invasive cells were counted in ten different fields in three independent experiments after 22 hrs. 120 Invasive cell number /field MDA157 MCF7 MDA231 MDA436
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Figure S1. Transformed human epithelial cells showed up-regulated p63 but down-regulated p53. (a) Heavy particle radiation promotes human bronchial cell transformation. The immortalized cells without IR
Supplementary Information
Supplementary Information MLL histone methylases regulate expression of HDLR- in presence of estrogen and control plasma cholesterol in vivo Khairul I. Ansari 1, Sahba Kasiri 1, Imran Hussain 1, Samara
CRISPR RNA-guided activation of endogenous human genes
CRISPR RNA-guided activation of endogenous human genes Morgan L Maeder, Samantha J Linder, Vincent M Cascio, Yanfang Fu, Quan H Ho, J Keith Joung Supplementary Figure 1 Comparison of VEGF activation induced
supplementary information
DOI: 10.1038/ncb2116 Figure S1 CDK phosphorylation of EZH2 in cells. (a) Comparison of candidate CDK phosphorylation sites on EZH2 with known CDK substrates by multiple sequence alignments. (b) CDK1 and
Supplementary Methods Quantitative RT-PCR. For mrna, total RNA was prepared using TRIzol reagent (Invitrogen) and genomic DNA was eliminated with TURB
Supplementary Methods Quantitative RT-PCR. For mrna, total RNA was prepared using TRIzol reagent (Invitrogen) and genomic DNA was eliminated with TURBO DNA-free Kit (Ambion). One µg of total RNA was reverse
Supplementary Table 1. The Q-PCR primer sequence is summarized in the following table.
Supplementary Table 1. The Q-PCR primer sequence is summarized in the following table. Name Sequence (5-3 ) Application Flag-u ggactacaaggacgacgatgac Shared upstream primer for all the amplifications of
Supplementary Information. Construction of Lasso Peptide Fusion Proteins
Supplementary Information Construction of Lasso Peptide Fusion Proteins Chuhan Zong 1, Mikhail O. Maksimov 2, A. James Link 2,3 * Departments of 1 Chemistry, 2 Chemical and Biological Engineering, and
Supplemental material
Supplemental material Diversity of O-antigen repeat-unit structures can account for the substantial sequence variation of Wzx translocases Yaoqin Hong and Peter R. Reeves School of Molecular Bioscience,
Supplementary Figure 1. IFN-γ induces TRC dormancy. a, IFN-γ induced dormancy
Supplementary Figure 1. IFN-γ induces TRC dormancy. a, IFN-γ induced dormancy of various tumor type TRCs, including H22 (murine hepatocarcinoma) and CT26 (murine colon cancer). Bar, 50 µm. b, B16 cells
Materials Protein synthesis kit. This kit consists of 24 amino acids, 24 transfer RNAs, four messenger RNAs and one ribosome (see below).
Protein Synthesis Instructions The purpose of today s lab is to: Understand how a cell manufactures proteins from amino acids, using information stored in the genetic code. Assemble models of four very
Gene Forward Primer Reverse Primer GAPDH ATCATCCCTGCCTCTACTGG GTCAGGTCCACCACTGACAC SSB1 AACTTCAGTGAGCCAAACCC GTTCTCAGAGGCTGGAGAGG
Supplemental Data EXPERIMENTAL PROCEDURES Plasmids and Antibodies- Full length cdna of INT11 or INT12 were cloned into ps- Flag-SBP vector respectively. Anti-RNA pol II (RPB1) was purchased from Santa
Cat. # Product Size DS130 DynaExpress TA PCR Cloning Kit (ptakn-2) 20 reactions Box 1 (-20 ) ptakn-2 Vector, linearized 20 µl (50 ng/µl) 1
Product Name: Kit Component TA PCR Cloning Kit (ptakn-2) Cat. # Product Size DS130 TA PCR Cloning Kit (ptakn-2) 20 reactions Box 1 (-20 ) ptakn-2 Vector, linearized 20 µl (50 ng/µl) 1 2 Ligation Buffer
SUPPLEMENTARY INFORMATION
doi:1.138/nature11496 Cl. 8 Cl. E93 Rag1 -/- 3H9 + BM Rag1 -/- BM CD CD c-kit c-kit c-kit wt Spleen c-kit B22 B22 IgM IgM IgM Supplementary Figure 1. FACS analysis of single-cell-derived pre-b cell clones.
Supplementary Figure 1. Reintroduction of HDAC1 in HDAC1-/- ES cells reverts the
SUPPLEMENTARY FIGURE LEGENDS Supplementary Figure 1. Reintroduction of HDAC1 in HDAC1-/- ES cells reverts the phenotype of HDAC1-/- teratomas. 3x10 6 HDAC1 reintroduced (HDAC1-/-re) and empty vector infected
Isolation, culture, and transfection of primary mammary epithelial organoids
Supplementary Experimental Procedures Isolation, culture, and transfection of primary mammary epithelial organoids Primary mammary epithelial organoids were prepared from 8-week-old CD1 mice (Charles River)
Supplementary Figure 1 A) MMC provokes a rapid replication arrest. We used a strain with a dnaats allele to
Supplementary Figure 1 A) MMC provokes a rapid replication arrest. We used a strain with a dnaats allele to block the initiation of replication when placed at 40 C. The ori/ter ratio was monitored by qpcr.
An evolutionarily conserved negative feedback mechanism in the hippo pathway reflects functional difference between LATS1 and LATS2
/, Supplementary Advance Publications Materials 2015 2016 An evolutionarily conserved negative feedback mechanism in the hippo pathway reflects functional difference between LATS1 and LATS2 Supplementary
Expression of Recombinant Proteins
Expression of Recombinant Proteins Uses of Cloned Genes sequencing reagents (eg, probes) protein production insufficient natural quantities modify/mutagenesis library screening Expression Vector Features
Supplementary Figure 1. Quantitative RT-PCR experimental validation of CRISPR/Cas9 and sgrnas expression in HEK293A transfected cells.
Supplementary Figure 1. Quantitative RT-PCR experimental validation of CRISPR/Cas9 and sgrnas expression in HEK293A transfected cells. HEK293A cells were transfected with the indicated combinations of
evaluated with UAS CLB eliciting UAS CIT -N Libraries increase in the
Supplementary Figures Supplementary Figure 1: Promoter scaffold library assemblies. Many ensembless of libraries were evaluated in this work. As a legend, the box outline color in top half of the figure