Figure 2

Absolute reverse-transcription quantification of ATM transcripts and ATM western blot. (A) Calibration curves for the three RT-qPCR assays shown in Fig. 1E. Y-axis reports the Ct values obtained from quantitative real time analysis; on the X-axis, the number of plasmid copies used (data in Supplemental Table 3). For full-length ATM, we used a plasmid containing the entire gene coding region (pMAT); for ATMdexa1 we generated a specific plasmid as described in Supplemental Fig. 1. (B) Absolute PCR quantification, in number of copies, of the full-length ATM and ATMdexa1 transcripts in three A–T cell lines and three controls, treated with solvent (EtOH) or dexamethasone. We did not detect ATMdexa1 in any case (Supplemental Table 4 in dataset 1). (C) Western blot analysis of the ATM protein in control (CTR1) and A-T46 fibroblasts; cells were treated with vehicle (EtOH), 300 μM of t-butyl hydroperoxide (TBH) or dexamethasone at increasing concentrations (tenfold increase from 0.1 to 100 µM). Cell lysates were analyzed by western blotting using Memcode as reference (left panel) or the ATM 2C1 antibody, specific for the C-terminal portion of the ATM protein (aa. 2577–3056). No bands beyond ATM were visible on the gel. Histograms show the quantitative analysis of ATM in CTR1 and A-T46.