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. 2022 Jan 21;50(3):1501–1516. doi: 10.1093/nar/gkac009

Figure 2.

Figure 2.

TOPORS is phosphorylated at Thr515 in response to IR. (A) Schematic diagrams of the TOPORS protein domains with putative ATM phosphorylation sites indicated. (B) Determination of IR-induced phosphorylation sites in TOPORS by mass spectrometry. A peptide containing Thr515 phosphorylation is shown. HeLa cells transfected with control siRNA or ATM siRNA were treated with or without 5 Gy of IR (C) or 2 mM HU (D). Whole cell lysates were then subjected to immunoprecipitation using an anti-TOPORS antibody followed by immunoblotting using indicated antibodies. (E) HeLa cells transfected with control siRNA or ATR siRNA were treated with or without 2 mM HU. Whole cell lysates were then subjected to immunoprecipitation using an anti-TOPORS antibody followed by immunoblotting using indicated antibodies. (F) HEK293T cells transfected with control GFP vector, GFP-TOPORS-WT or GFP-TOPORS-T515A were treated with IR (5 Gy). Cell lysates were immunoprecipitated with anti-GFP antibody and subjected to immunoblot analysis with anti-phospho-threonine antibody. Asterisk indicates degradation products of GFP-TOPORS. (G) HA-RAD51-expressing HEK293T cells transfected with control GFP vector, GFP-TOPORS-WT or GFP-TOPORS-T515A were treated with 5 Gy of IR and subjected to immunoprecipitation and immunoblots as indicated. Asterisk indicates degradation products of GFP-TOPORS.