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. 2022 Jan 28;13:791522. doi: 10.3389/fimmu.2022.791522

Figure 1.

Figure 1

ATM and ATM kinase activity/signalling in cells from ataxia telangiectasia patients 1–6. Assay showing the presence or absence of ATM in cells from each patient and whether this was associated with some activity/signalling ability. Cells were either irradiated (IR) with 2-Gy x-rays or not irradiated. The left-hand section show the results of the three classical patients, the middle section the results of variant patient 2, and the right-hand section the results of variants 4 and 5. Left-hand section, lanes 1 & 2—positive control. (A) Cells from a normal individual showing presence of ATM protein (B) and normal ATM activity/signalling showing phosphorylation of the targets SMC1 ser966 (C), KAP-1 ser 824 (E), Nbn ser343 (G), and CREB ser121 (I). There is a strong signal for each of these in lane 2 after activation. The total levels of SMC1, KAP-1, Nbn, and CREB are also shown in (D, F, H, J), respectively. Lanes 3 and 4—negative control. Cell lysate from a classical A-T patient with mutations ATM, c.1355delC; p.(Thr452AsnfsTer21) and c.3802delG; p.(Val1268Ter). Both are frameshift mutations leading to instability and loss of the ATM from both alleles. There is no differential phosphorylation of these targets in this patient’s cells inane 4—consistent with the patient having A-T; there is no ATM (B). Lanes 5 and 6 show cell lysates from patient 6, lanes 7 and 8 lysates from patient 3, and lanes 9 and 10 lysates from patient 1. Just as with the negative control (lane 4), lysates from these three patients show absence of ATM protein (B) and absence of a differentially increased phosphorylation induced by irradiation, as indicated by absence of phosphorylation of SMC1 ser966, KAP-1 ser824, Nbn ser343, and CREB ser121 in lanes 6, 8, and 10, respectively. Middle section: lanes 11 and 12 show lysates from the same normal control as in lanes 1 and 2. Lanes 13 and 14 are cell lysates from the same classical A-T patient as in lanes 3 and 4. Lanes 15 and 16 are lysates from variant patient 2. (B) confirms that the lysate shows a low level of ATM protein. In contrast with the negative control (lane 14), this lysate shows a clear low level of ATM kinase activity/signalling as indicated by the presence of some moderate phosphorylation of SMC1 ser966, KAP-1 ser824, Nbn ser343, or CREB ser121 in lane 16. Right-hand section, lanes 17 and 18 and 19 and 20 are lysates from variant patients 4 and 5. The positive and negative controls have been removed but were the same as in lanes 1–4 and 11–14. (B) confirms that the variant lysates show a low level of ATM protein. In contrast to the negative controls in lanes 4 and 14, these lysates also show a clear low level of ATM that has some retained kinase activity/signalling as indicated by the presence of moderate phosphorylation of SMC1 ser966, KAP-1 ser824, and Nbn ser343 in particular.