ATRX overexpression plasmids contain IS10 insertions. (A) Schematic map of the insertions found in IS10-GFP-ATRX and IS10-ATRX-YFP. The exon structure (alternate white and gray bars) of ATRX cDNA isoform 2 (top) and some relevant domains from the protein sequence (bottom) are shown. The insertion sites are denoted by black bars and their positions in the cDNA and the protein are described in the right box. Black arrows show the direction of the IS10 elements found. The stop codons introduced by IS10 in the protein sequence are indicated with asterisks. The position of two of the primer pairs used for analyzing ATRX sequence is shown (white and gray arrows). The NLS within ATRX cDNA is indicated with a black bar. (B) PCR analysis of IS10-GFP-ATRX, IS10-ATRX-YFP and IF-GFP-ATRX using the primers shown in (A). Both amplicons of IF-GFP-ATRX have the expected size (1,449 bp for amplicon I and 1,935 for amplicon II) while amplicon I shows an insertion in IS10-ATRX-YFP and amplicon II shows an insertion in IS10-GFP-ATRX (both have additional 1,336 bp). (C) Representative EcoRI digestion patterns of the IF-GFP-ATRX plasmid grown in two different bacteria strains. The Stbl4-derived plasmid has the expected pattern while the plasmid derived from DH5α shows an insertion in the 2,400bp fragment (shift from a 2,468 bp fragment to ~3,800 bp).