R position 18. For PCR, primers 1 and 2 and primer mixes 3 and 4 were

R position 18. For PCR, primers 1 and 2 and primer mixes 3 and 4 were combined in the ratio of 24:16:30:30 to reflect their PD1-PDL1 inhibitor 1 approximate relative proportions in vertebrate genomes. The PCR reaction mixture contained the forward primers, the PolyT reverse primer, and LA Taq DNA polymerase (BD Bioscience Clontech). The double-stranded cDNAs were amplified for 16 cycles using the following thermal profile: 94uC for 15 seconds, 42uC for 30 seconds, 72uC for 6 minutes. The amplified double-stranded cDNA fragments were cloned into the pEGFP-cl vector (BD Bioscience Clontech) together with fragment 1 (YFP1 amino acids 1?58) of a yellow fluorescent protein variant (Addgene) and a 10-amino-acid linker Z-360 consisting of (Gly-Gly-Gly-Gly-Ser)2x [36]. First, the EGFP cDNA was replaced with the Kozak sequence by using NheI and XhoI enzymes. The PCR-amplified YFP1 cDNA was digested with XhoI and EcoRI to make cohesive 59- and 39 ends. The linker oligo DNA was synthesized to contain the cleaved EcoRI and BamHI overhangs at the 59- and 39 ends. The YFP1 cDNA with the linker DNA was cloned at the XhoI and BamHI sites. Amplified double-stranded cDNAs from a ureter were then cloned at the BamHI site using the In-Fusion cloning kit (BD Bioscience Clontech) to encode Cterminal fusion protein to YFP1. Subsequent bacterial transformation and amplification of the library was performed according to the protocols of the In-Fusion SMARTer cDNA library construction kit (BD Bioscience Clontech).Bait ConstructThe pcDNA3.1 vector (Life Technologies) was used to construct the bait by inserting ARL11 cDNA linked to the 10-amino-acid flexible linker consisting of (Gly-Gly-Gly-Gly-Ser)2x at NheI and XhoI sites, and YFP2 (amino acids 159?39) was inserted at the XhoI site to encode the C-terminal fusion YFP2.In-Frame cDNA LibraryTable 1. Sequences of forward primers used to construct in-frame cDNA library.Sequence Positionligation sequenceKozak sequence*_____59- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 39 primer 1 primer 2 primer mix 3 primer mix 4 combination 1 combination 2 combination 3 combination 4 combination 5 combination 6 combination 7 combination 8 combination 9 C G G A G G A A G C G G A T C C C C C G C C G C C A C C A T G G C G G A G G A A G C G G A T C C C C C G C C G C C G C C A T G G C G G A G G A A G C G G A T C C D D D H D D H D A A A G A T G H C G G A G G A A G C G G A T C C D D D H D D H D K G K W A T G H C G G A G G A A G C G G A T C C A A A C A A C A G G G A A T G C C G G A G G A A G C G G A T C C G A A C A A C A G G G A A T G C C G G A G G A A G C G G A T C C T A A C A A C A G G G A A T G C C G G A G G A A G C G G A T C C A G A C A A C A G G G A A T G C C G G A G G A A G C G G A T C C A T A C A A C A G G G A A T G C C G G A G G A A G C G G A T C C G G A C A A C A G G G A A T G C C G G A G G A A G C G G A T C C G T A C A A C A G G G A A T G C C G G A G G A A G C G G A T C C T G A C A A C A G G G A A T G C C G G A G G A A G C G G A T C C T T A C A A C A G G G A A T G C*D is an equal mixture of A, G and T, H is an equal mixture of A, C and T, K is an equal mixture of G and T, and W is an equal mixture of A and T. doi:10.1371/journal.pone.0052290.tScreening of the In-frame cDNA LibraryHEK-293T cells were transiently co-transfected with in-frame library 18325633 and ARL11-YFP2 plasmids using Lipofectamine 2000 reagent (Life Technologies). After 24 hours, the transfected cells were harvested, washed with PBS, and sorted to col.R position 18. For PCR, primers 1 and 2 and primer mixes 3 and 4 were combined in the ratio of 24:16:30:30 to reflect their approximate relative proportions in vertebrate genomes. The PCR reaction mixture contained the forward primers, the PolyT reverse primer, and LA Taq DNA polymerase (BD Bioscience Clontech). The double-stranded cDNAs were amplified for 16 cycles using the following thermal profile: 94uC for 15 seconds, 42uC for 30 seconds, 72uC for 6 minutes. The amplified double-stranded cDNA fragments were cloned into the pEGFP-cl vector (BD Bioscience Clontech) together with fragment 1 (YFP1 amino acids 1?58) of a yellow fluorescent protein variant (Addgene) and a 10-amino-acid linker consisting of (Gly-Gly-Gly-Gly-Ser)2x [36]. First, the EGFP cDNA was replaced with the Kozak sequence by using NheI and XhoI enzymes. The PCR-amplified YFP1 cDNA was digested with XhoI and EcoRI to make cohesive 59- and 39 ends. The linker oligo DNA was synthesized to contain the cleaved EcoRI and BamHI overhangs at the 59- and 39 ends. The YFP1 cDNA with the linker DNA was cloned at the XhoI and BamHI sites. Amplified double-stranded cDNAs from a ureter were then cloned at the BamHI site using the In-Fusion cloning kit (BD Bioscience Clontech) to encode Cterminal fusion protein to YFP1. Subsequent bacterial transformation and amplification of the library was performed according to the protocols of the In-Fusion SMARTer cDNA library construction kit (BD Bioscience Clontech).Bait ConstructThe pcDNA3.1 vector (Life Technologies) was used to construct the bait by inserting ARL11 cDNA linked to the 10-amino-acid flexible linker consisting of (Gly-Gly-Gly-Gly-Ser)2x at NheI and XhoI sites, and YFP2 (amino acids 159?39) was inserted at the XhoI site to encode the C-terminal fusion YFP2.In-Frame cDNA LibraryTable 1. Sequences of forward primers used to construct in-frame cDNA library.Sequence Positionligation sequenceKozak sequence*_____59- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 39 primer 1 primer 2 primer mix 3 primer mix 4 combination 1 combination 2 combination 3 combination 4 combination 5 combination 6 combination 7 combination 8 combination 9 C G G A G G A A G C G G A T C C C C C G C C G C C A C C A T G G C G G A G G A A G C G G A T C C C C C G C C G C C G C C A T G G C G G A G G A A G C G G A T C C D D D H D D H D A A A G A T G H C G G A G G A A G C G G A T C C D D D H D D H D K G K W A T G H C G G A G G A A G C G G A T C C A A A C A A C A G G G A A T G C C G G A G G A A G C G G A T C C G A A C A A C A G G G A A T G C C G G A G G A A G C G G A T C C T A A C A A C A G G G A A T G C C G G A G G A A G C G G A T C C A G A C A A C A G G G A A T G C C G G A G G A A G C G G A T C C A T A C A A C A G G G A A T G C C G G A G G A A G C G G A T C C G G A C A A C A G G G A A T G C C G G A G G A A G C G G A T C C G T A C A A C A G G G A A T G C C G G A G G A A G C G G A T C C T G A C A A C A G G G A A T G C C G G A G G A A G C G G A T C C T T A C A A C A G G G A A T G C*D is an equal mixture of A, G and T, H is an equal mixture of A, C and T, K is an equal mixture of G and T, and W is an equal mixture of A and T. doi:10.1371/journal.pone.0052290.tScreening of the In-frame cDNA LibraryHEK-293T cells were transiently co-transfected with in-frame library 18325633 and ARL11-YFP2 plasmids using Lipofectamine 2000 reagent (Life Technologies). After 24 hours, the transfected cells were harvested, washed with PBS, and sorted to col.

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