A) Anti-Flag immunoblot of ArhGap24 with tags in the indicated positions. The transfections and expression analyses were performed in duplicates (a and b). B) Immunoblot analysis of Nadrin with tags in the indicated position. The top panel was probed with anti-Flag antibody. The immunoblot was then stripped of antibody and reprobed with anti-Nadrin (bottom panel). C) Quantitative analysis of protein levels for six different proteins with Triple-Flag epitope tags at the indicated positions. For each replicate, the band intensity, as detected by Flag M2 antibody, was normalized by the transfection efficiency as detected by fluorescent values of a co-transfecting plasmid pLP ECFP C1. The normalized band intensity was then average between the two replicates. Ratios of each tagged-protein relative to the protein expressed from the same ORF from the TOPO vector are shown above the bars. Abbreviations: SPLICE – Creator Splice Donor (V37-based) and Creator Splice 5' acceptor (V180) – splicing occurs at 5' end, 5' – standard Clontech system (donor – V7-based and acceptor – V179) – no splicing at either end, 3' – standard Clontech system (donor – V7-based and acceptor – V181) – splicing at 3' end to remove the CmR gene, 5' SA, Creator Splice Donor (V37-based) and 5' acceptor vector (V179) – no splicing at either end, TOPO – 5' tag non-recombination system, CNT – counts, RFU – relative fluorescent units.