Figure 5.

Full-length ATM is expressed in an LCL from patient A-T213, and immunoprecipitated ATM from this LCL has kinase activity in vitro. (A) Western blot (top and second panel; OE=overexposed) showing expression of ATM in a normal LCL (lanes 1 and 2), absence of ATM in an LCL from a classical A-T patient (A-T183, lanes 3 and 4) and reduced expression of ATM in LCL from A-T213 (lanes 5 and 6). Whereas the ATM in patient A-T213 appears to be full length, the ATM in an LCL from patient A-T86 with the c.2T>C mutation (and also null mutation c.7665delinsGTGA;p.His2555Gln ins^*) is slightly truncated (lanes 7 and 8). It is likely that truncated ATM from c.2T>C in patient AT187 is obscured by the p.Arg3047X (c.9139C>T) mutant protein (lanes 9 and 10) and the truncated ATM from c.1A>G in patient A-T213 is obscured by the p.Gly2891Asp (c.8672G>A) mutant protein (lanes 5 and 6). Immunoprecipitated ATM was used to phosphorylate the p53_1–72 substrate in vitro (third and fourth panel) following activation of ATM by IR. The level of normal radiation-induced ATM kinase activity is shown in lane 2. There is absence of induced activity in cells from classical A-T patient A-T183 (lane 4) with no ATM protein. Lane 6 shows induced activity of ATM from the LCL of A-T213 but not from ATM of patient A-T86 with a c.2T>C mutation and a second truncating mutation (lane 8) or from patient A-T187 with a c.2T>C mutation and c.9139C>T (p.Arg3047X). (B) Histogram of relative levels of ATM in vitro kinase activity from the immunoprecipitates in Figure 5A.