Fig. 4: The K150 site is crucial for STING ubiquitination in microglia.

a Western blot showing STING ubiquitination levels in primary microglia. b Western blot showing STING ubiquitination levels in mouse hippocampus. c Mass spectrometry identification of STING ubiquitination site K150. d Western blot analysis of STING ubiquitination levels in primary microglia transfected with mutant plasmids for each of the seven identified ubiquitination sites. Western blot (e) and quantification (f) of K150 in hippocampal of mice (n = 6). g Western blot showing STING ubiquitination levels in hippocampus 21 days after K150R⁺ adeno-associated virus (AAV) injection. Representative 3D reconstructed imaging (h) and quantitative analysis (i) of microglia engulfing synapses (n = 6). Green, Iba-1; Red, PSD95; Magenta, CD68. Scale bar, 5 μm. Western blot (j) and quantification (k) of STING in hippocampus of mice received K150R⁺-AAV (n = 6). Western blot (l) and quantification (m) of C1q, PSD95 and SYN in hippocampal synaptic proteins of mice treated with K150R⁺-AAV (n = 6). n, o Representative transmission electron microscopy (TEM) images of synapse and mitochondria in the CA1 region of mice treated with K150R⁺-AAV (n = 6), ×15, Scale bar = 500 nm. ×20, Scale bar = 2.0 μm. Representative images (p) and statistical analysis (q) of the Morris water maze (n = 12). Representative images (r) and statistical analysis (s) of the Y-maze tests (n = 12). Representative images (t) and statistical analysis (u) of the open field tests (n = 12). v Statistical analysis of the novel object recognition tests (n = 12). Behavioral tests data are shown as mean ± SEM, the others are shown as mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001. ns, not significant. Statistical tests: two-sided Student’s unpaired t-tests (f, i, k, m, o, q, s, u) and multiple unpaired t-tests (v). AZ active zone, SC synaptic cleft, SV synaptic vesicles, PSD postsynaptic density. Source data and exact P values are provided as a Source Data file.