mau A B C Qsepharose051229manual001:1_UV@01,SHFT Qsepharose051229manual001:1_Conc Qsepharose051229manual001:1_Fractions Qsepharose051229manual001:1_Inject Manual run 3:1_UV@01,SHFT Manual run 3:1_Fractions Qsepharose051229manual001:1_Logbook Manual run 3:1_Logbook mau %B 400 S-300 SDS cut 2000 1500 0.8 300 0.6 80 72 kda 60 1000 0.4 200 40 34 kda 500 0 123456 789 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 Waste 0.2 100 0.0 20 25 kda 0 100 200 300 400 500 600 min con 29 37 39 41 43 45 47 49 0 1 2 34 5 6 7 8 9 101112131415161718192021222324252627282930313233343536 0 0 50 100 150 200 250 300 min 17 kda 72 kda 10 kda 34 kda 25 kda 15 kda 10 kda Silver Staining of rllc1 (15% Acrylamide gel)
Immunized with rllc1 Spleen Cell Fusion HPRT- Myeloma 43 kda 34 kda 26 kda Hybridoma Cells HAT selection 17 kda 11 kda ELISA screening Sample: Ad-LLC1 H1703 - : uninfected + : infected Western blotting
A549 A549 LLC1 DAPI Merge vector LLC1 WI26 WI26 LLC1 DAPI Merge vector LLC1
Lung Salpinx Skin
Ad-LacZ Ad-LLC1 Con 10 20 50 100 10 20 50 100 (pfu/cell) H1588 PARP-48 hr Beta-Actin PARP, 48 hr Beta actin H1703 Caspase 3 Caspase 9 (47/37 kda)
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 O.D. 0.2 M O I 0.8 M O I 3.2 M O I 0.6 0.5 0.4 0.3 0.2 0.1 O.D. G F P L L C 1-G F P 0 G F P L L C - G F P G F P L L C - G F P G F P L L C - G F P 0 1703 1588 1833 1703 1588 1833 3.2 MOI c o lo n y n u mb e r ( 800 700 600 500 400 300 200 100 0 V e cto r L L C 1 co lo n y n u m b er (n ) 200 180 160 140 120 100 80 60 40 5mm< = 4mm 3mm 1~2mm 300 H1833 20 0 v e c to r L L C 1 H1299 c o lo n y n u m b e r 250 200 150 100 50 0 v e c to r L L C 1 H520 4mm 3mm 1~2mm c o lo n y n u m b e r 300 250 200 150 100 50 0 v e c to r LLC 1 A549 4mm 3mm 1~2mm
300 c o lo n y n u m b e rb 250 200 150 100 50 5 m m 4 m m 3 m m 1 ~ 2 m m 0 v e c to r L L C 1 Vector LLC1 Negative Control
#! + - #1 : yeast transformant of C20orf85 bait + : Positive control of protein-protein interaction - : Negative control of protein-protein interaction Master plate lacz expression SD-L.W. Filter assay ADE2 expression URA3 expression SD-L.W.A SD-L.W.U. The PBN204 yeast strain has lacz, URA3 and ADE2 as reporters. μ
lacz expression URA3 expression ADE2 expression Filter assay SD-LWU SD-LWA Master plate With bait With vector With bait + With vector - - : Negative control of protein-protein interaction + : Positive control of protein-protein interaction Prey plasmids were prepared by conventional E. coli transformation with yeast DNAs from 15 Ura + lacz + Ade + colonies. SD-LW Number of Colonies Library A Library B Library C 1 st 2 nd Reporter I (URA3) 64 45 23 18 Reporter II (lacz) 22 29 1 2 Reporter III (ADE2) 15 17 1 1 Real Positives 0 0 0 0
Genomic DNA Targeting vector BamHI/XhoI cut BamHI RI KpnI XhoI RI XhoI RI 1 2 3 4 probe 4.3kb RI RI RI RI RI neo DT Targeted locus BamHI/XhoI cut 1 3.1 1 4.8 1.3 BamHI RI KpnI RI XhoI RI 8.6kb neo 1 2 3 Targeting vector Digested by EcoRI 4.8kb 3.1kb 1.3kb 1kb
SAGE (Serial Analysis of Gene Expression) cdna synthesis Concatamer NlaIII digestion Linker Ligation BsmFI digestion and gap filling Sequencing and counting Tag frequency Ditag formation PCR amplification and Nla III digestion Log(Relative LLC-1 expression) 2 1 0-1 -2-3 -4-5 22 285 277 46 57 142 293 217 16 246 61 198 357 24 151 128 2 1 0-1 -2-3 -4-5 H1703 H358 A549 H1299 Hop92 H226 H2170 H520 H522
13 adenocarcinoma, 9 squamous carcinoma: no expression 1 squamous and 1 adeno-carcinoma: weak and diffuse staining GATGAGCAAATGGGCCTAGAGGTGGACAAAGGGCCCAGAC*GTGAGCAAAGGATGGCAGCAAGCCC*GG ACCCAAGC*GAC*GC*GGC*GAAAGCACAGC*GACCCCACCCTC*GCCAGGTGCAGAGCCATTTCC*GCCTC C*GAGTCCTGC*GTTGCCTGGCAACC*GGTCTCCCAGGAGAC*GC*GAGAC*GCTGAGCCCAGGGCCAGAG CTTGC*GGGGCGCACAGAGCCCCCAGGCCTCATGGCGCAGAAACCTCTCAGCACCGCGGCGGCTGAACGC A Promoter GCCCGGACCCAAGCGACGCGGCGAAAGCACAGCGACCCCACCCTCG Unmethyl GTTTGGATTTAAGTGATGTGGTGAAAGTATAGTGATTTTATTTTTG Methylat GTTCGGATTTAAGCGACGCGGCGAAAGTATAGCGATTTTATTTTCG B H358 m CpG CpG H23 H1299 H522 Hop92