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. 2004 Apr 20;101(18):6905–6910. doi: 10.1073/pnas.0400099101

Fig. 1.

Fig. 1.

Suppression of hPNK gene expression in the human A549 cell line. (A) Upper Western blot shows expression of hPNK, p53, and actin in A549 control, vector only (pSup), and three G418 resistant clones: C-ter1–3 cells. Lower Western blot shows the stability of down-regulation of hPNK in the C-ter3 cells over a period of 12 weeks. XRCC1 expression was used as a control. (B) Immunofluorescence of hPNK in A549 control and C-ter3 cells. (C) hPNK DNA kinase activity in fractionated cell extracts isolated from A549 cells, vector-only controls (pSup), and several G418-resistant clones. Confirmation that fraction 6 obtained from C-ter3 cells lacks hPNK is shown in the Western blot. (D) hPNK 3′-phosphatase activity in crude cell extracts of wild-type A549 and C-ter3 cells. The duplex substrate consisting of three oligonucleotides, a 45-mer complementary to a 24-mer, and a 5′-labeled 21-mer bearing a 3′-phosphate (p21p) to mimic a single-strand nick has been fully described (13). The lanes marked p21p and p21 indicate the starting material and a marker for the dephosphorylated product, respectively.