Elevated Levels of DNA Strand Breaks Induced by a Base Analog in the Human Cell Line with the P32T ITPA Variant

Figure 4

Specificity of polyclonal antibodies against ITPA in Western blot. Western blots were performed as described in Section 2. Antibodies against ITPA react largely with ITPA (a band closer to 20 kDa marker, see compared with lane where pure ITPA was loaded, and predicted molecular mass was 21.4 kDa) and, less intense, with an unrelated protein running at 30 kDa. The level of ITPA, but not of this non-specific protein, is decreased almost 10-fold in the soluble fraction of the P32T cell line. In the control reprobing experiment the amount of GAPDH was the same in all three cell lines (not shown). The position of the immunoreactive band was shifted up, closer to the 30 kDa marker in the lane with the 6 His-tagged pure ITPA-P32T (predicted molecular mass 23.6 kDa [47]). We also analyzed the extracts of 293 cell lines transfected with expressing ITPA-EGFP fusion protein (51.4 kDa) and have found that the major band was detected at a position corresponding to 50 kD.