Figure 4

Protein expression levels in the Creator and Creator Splice system. The indicated constructs were transiently transfected into HEK293T cells. After 48 hours, the cells were lysed and the lysate subjected to immunoblot analysis. A) WWP2 and BulI fused to Double-Myc epitope tags were detected by Myc antibody 9E10. B) ArhGEF9 and ArhGEF1 fused to Triple-Flag tags were detected by Flag antibody M2. Abbreviations: 5' SA – Vector created by recombining a Creator Splice donor vector, (splice acceptor at 5' end – V37-based), with a 5' tag acceptor vector lacking a splice donor/intron (V179). No splicing occurs in these transcripts, 5' – Vector created by recombining a donor lacking a 5' splice acceptor (V7 or V308-based) and a 5' tag acceptor vector lacking a splice donor/intron (V516- Double Myc, V179 – Triple Flag). No splicing occurs in these transcripts, SPLICE – Expression vector created by recombining a Creator Splice donor (5' splice acceptor – V37-based) and a Creator Splice 5' tag acceptor vector containing a splice donor/intron (V517 – Double Myc, V180 – Triple Flag). Resulting transcripts undergo splicing at the 5' end to remove the loxP sequence and juxtapose the tag onto the ORF, 3' – Vector created by recombining a donor lacking a 5' splice acceptor (V7 or V308-based) with a 3' tag acceptor vector lacking a splice donor/intron (V518 – Double Myc, V181 – Triple Flag). Splicing only occurs at the 3' end to remove the CmR gene (The donor contains a splice donor preceding the CmR ORF and the acceptor vector contains a splice acceptor preceding the tag), 3' SA – Vector created by recombining a Creator Splice donor vector containing a 5' splice acceptor (V37-based) and a 3' tag acceptor vector lacking a splice donor/intron (V181 -Triple Flag). Splicing only occurs at the 3' end to remove the CmR gene (same as 3' tag) and TOPO – 5' tag, non-Creator system.