FIGURE 5.

Lipopolysaccharide (LPS)‐trigged translocation of mixed lineage kinase domain‐like pseudokinase (MLKL) in macrophages. (A) Bone marrow–derived macrophages (BMDMs) isolated from C57BL/6J mice were exposed to LPS for 24 h. Colocalization of MLKL with Alexa Fluor–labeled phalloidin, which stains cell surface–associated F‐actin, was examined by confocal microscopy. (B,D) P10 (10,000 g pellet) fraction was isolated from LPS‐treated RAW cells in response to 1 μM GW806742X or 5 μM GSK872. (C) P10 and plasma membrane fractions were isolated from LPS‐treated RAW cells. (B–D) Proteins were resolved by nonreducing plPAGE and probed with antibody to MLKL. Images are representative on n = 3 independent experiments. (E,F) BMDMs were exposed to LPS, as described in Figure 4. BMDMs were incubated with CellLight BacMam 2.0 dye for overnight to label early endosomes, late endosomes, and Golgi. (E) Colocalization of MLKL and (F) quantification with phagosomes, lysosomes, early endosomes, late endosomes, and Golgi was examined by confocal microscopy. All images were obtained using a 40× objective (Zoom 4). Representative images are shown. Single‐channel images are shown in Figure S4. Values represent means ± SEM. Values with different superscripts are significantly different from each other; n = 3–4 independent BMDM isolations, p < 0.05. IF, immunofluorescence; IB, immunoblotting.