Fig 1: Identification of RAD17 as an SMG7-binding protein. (a) Verification of SMG7-binding proteins identified from complex purification. The total cell lysates and a-Flag/HA immunoprecipitated materials from H1299 and H1299-FH-SMG7 cells were assayed by western blot analysis using a-RAD17, a-UPF1, a-SMG5, and a-HA (SMG7) antibodies. (b) Schematic presentation of the RAD17 domain structure containing P-loop, Walker B, Sensor 1, Sensor 2 and C-terminal highlighted SQ motifs. (c) As in (a), the total cell lysates and a-Flag immunoprecipitates from H1299 and H1299-FH-SMG7 cells were assayed by western blot analysis using a-RAD17, a-RAD17-pS645, a-SMG7, a-RFC2 and a-RFC3 antibodies. (d) H1299 cells were transfected with SMG7- and RAD17-expressing plasmid DNA, and the cell extracts and a-Flag immunoprecipitated materials were analyzed by western blot with a-RAD17 and a-HA (SMG7) antibodies. (e) The total cell extracts and a-Flag immunoprecipitated materials from H1299 cells transfected with empty vector or FH-RAD17-expression plasmid DNA were examined by western blot analysis using a-SMG7 and a-RAD17 antibodies. (f–g) SMG7 direct binding to RAD17 in vitro. GST, full-length GST-RAD17 (f), or truncated GST-RAD17 fragment (g) fusion proteins were used in pulldown assays with purified FH-SMG7 proteins. FH-SMG7 was detected by western blot analysis using a-SMG7 antibody, and GST proteins visualized by Ponceau S staining in (f) and Supplementary Fig. 2d.