Extended Data Fig. 4: BLK-mediated phosphorylation of cGAS at Y215 regulates the nuclear translocation of cGAS in response to DNA damage.

a, Alignment of primary sequences of H. sapiens cGAS and its homologues in 22 species: H. sapiens (Hsa), Pan troglodytes (Ptr), Pan paniscus (Pps), Gorilla gorilla (Ggo), Pongo abelii (Pon), Macaca mulatta (Mmu), Ailuropoda melanoleuca (Aml), Canis familiaris (Cfa), Felis catus (Fca), Sus scrofa (Ssc), Bos taurus (Bta), Mus musculus (Mms), Rattus norvegicus (Rno), Monodelphis domestica (Mdo), Sarcophilus harrisii (Shr), Meleagris gallopavo (Mgp), Gallus gallus (Gga), Taeniopygia guttata (Tgu), Anolis carolinensis (Acs), Xenopus tropicalis (Xtr), Oryzias latipes (Ola) and Danio rerio (Dre). Consensus sequences (similarity score, >0.7) are listed at the bottom of the column; conserved amino acids are highlighted in yellow, and absolutely conserved amino acids are highlighted in red. The filled triangle indicates the conserved Y215. The primary sequence numbers of H. sapiens cGAS are labelled at the top of the line. b–e, Immunofluorescence assay results showing the localization of HA–cGAS and HA–cGAS(Y215E) (anti-HA, green) in PC-9 cells exposed to camptothecin (1 μM) for 4 h (b) or to H₂O₂ (10 mM) for 30 min (d). Data represent n = 3 independent experiments. Quantitative data for b and d are depicted in c and e, respectively. Data are expressed as mean ± s.e.m. of n = 3 independent experiments. f, Immunoblot results of anti-HA immunoprecipitates from HEK293T cells transfected with HA–cGAS or the HA–cGAS(Y215A) mutant in the absence or presence of pervanadate for 30 min, followed by cell collection. Data represent n = 3 independent experiments. g, Immunoblot of lysates from PC-9 cells that had been stimulated with camptothecin (1 μM) for the indicated times. Data represent n = 3 independent experiments. h, i, Results of immunofluorescence analysis showing localization of HA–cGAS (anti-HA, green) in PC-9 HA–cGAS cells transfected with control shRNA (sh-Ctrl) or with shRNA targeting BLK (sh-BLK) and then exposed to camptothecin (1 μM) for 4 h (h). Nuclei were stained with DAPI (blue). Data represent n = 3 independent experiments. Quantitative data are shown in i. At least 100 transfected cells were counted in each experiment. Data are expressed as mean ± s.e.m. of n = 3 independent experiments. j, Immunoblot results of lysates from PC-9 HA–cGAS cells that had been transfected with either control shRNA or with shRNA targeting BLK and then exposed to camptothecin (1 μM) for 4 h. Data represent 3 independent experiments. Student’s t tests (unpaired and two-tailed) were used for statistical analysis (c, e, i). Scale bar, 5 μm. For gel source data, see Supplementary Fig. 1.