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. 2021 Oct 27;12:6207. doi: 10.1038/s41467-021-26240-9

Fig. 6. Doxorubicin treatment but not cGAMP signaling promotes nuclear cGAS localization.

Fig. 6

a Immunofluorescence of HA-cGAS and γH2AX in MEF cells mock treated or exposed to doxorubicin (2 μM for 6 h), scale bar = 10 μm. Representative images from three independent biological replicates are shown. b Immunoblots for endogenous cGAS, STING, and TBK1 in the cytoplasmic and nuclear fractions of THP1 cells after mock treatment (−) or treatment with doxorubicin (+) (2 μM for 6 h). Tubulin and TBP served as cytoplasmic and nuclear loading controls, respectively. Bands of interest from representative immunoblots from three independent experiments are shown. c Immunoblots for endogenous cGAS in the cytoplasmic and nuclear fractions of THP1 cells after mock treatment (−) or treatment with cGAMP (+) for 16 h. Tubulin and TBP served as cytoplasmic and nuclear loading controls, respectively. Bands of interest from representative immunoblots from three independent experiments are shown. d Immunoblots for phosphorylated and total endogenous TBK1 in the cytoplasmic and nuclear fractions of THP1 cells after mock treatment (−) or treatment with cGAMP (+) for 16 h. Tubulin and TBP served as cytosolic and nuclear loading controls, respectively. Bands of interest from representative immunoblots from three independent experiments are shown. e Immunoblots for DDR signaling proteins H2AX (γH2AX), phosphorylated CHK2 (pCHK2), and endogenous cGAS in WT and cGAS−/− THP1 cells after treating with mock (−) or cGAMP (+) for 16 h. Total H2AX (H2AX) and tubulin serve as internal controls. Bands of interest from representative immunoblots from three independent experiments are shown. f Immunoblot (IB) for γH2AX, ATM, and HA-cGAS of anti-HA or anti-IgG immunoprecipitates (IP) from HA-cGAS-reconstituted cGAS−/− immortalized MEF’s in the presence (+) or absence (−) of doxorubicin (2 μM for 6 h). γH2AX but not ATM was enriched in the cGAS immunoprecipitate. Bands of interest from representative immunoblots from three independent experiments are shown.