Fig. 3

The interaction of SIRT5 and ANXA1 increases after OGD/R. A, B HEK293T cells were transfected with plasmids encoding Flag-SIRT5-WT or Flag-SIRT5-H158Y together with HA-ANXA1. The cell extracts were collected for IP with anti-HA (A) or anti-Flag beads (B), followed by IB analysis with the indicated antibodies and showing the interaction of ectopically expressed ANXA1 and SIRT5. C, D Primary cultured microglia were subjected to OGD/R. Co-IP analysis indicating the interaction of endogenous ANXA1 and SIRT5. E HEK293T cells were transfected with plasmids encoding HA-ANXA1-WT or HA-ANXA1-K166R together with Flag-SIRT5. The cell lysates were collected for IP with anti-HA beads, followed by IB analysis with the indicated antibodies, showing the interaction of ectopically expressed ANXA1 and SIRT5. F Primary cultured microglia were subjected to OGD/R. Immunofluorescence assays of ANXA (green) and SIRT5 (red) with colabelled DAPI (blue, nuclei). Scale bars, 20 μm. G Quantitative analysis of the colocalization was conducted by Pearson’s coefficient measurement using ImageJ software. Data are presented as the mean ± SEM from at least three independent experiments. **P < 0.01