Figure 8.

Sig-1R activates and interacts with Rac1 to regulate macrophage efferocytosis. (A) Macrophages were incubated with dead cells for 30 min before processed for immunostaining. Representative images of Rac1 (Red), Sig-1R (Cyan) and CD68 (Green) triple staining were shown from at least three repeated experiments with similar results. Scale bar: 20 μm. (B) Macrophages were pre-treated with BD1047 (10 μM) for 30 min before stimulation with PRE084 (10 μM) for 30 min. The cells were then incubated with dead cells for additional 60 min before collection for further assays. Co-immunoprecipitation (Co-IP) analysis for Rac1 and Sig-1R interaction was shown. (C) HEK293T cells were co-transfected with Myc-Rac1 and Sig-1R plasmids. the interaction between Rac1 and Sig-1R was monitored by co-immunoprecipitation (Co-IP) with anti-Myc beads, followed by SDS-PAGE separation and detected by respective antibody. (D) Single mutation of Rac1 plasmid was prepared as described in Method section, HEK293T cells were transfected with Flag-Sig-1R and wild type Myc-Rac1 or its single mutation, as indicated. After Co-IP with anti-Myc beads, the IP and input samples were separated by SDS-PAGE and probed with indicated antibodies.