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. 2023 Feb 17;77(3):902–919. doi: 10.1002/hep.32612

FIGURE 4.

FIGURE 4

Expression and phosphorylation of mixed lineage kinase domain‐like pseudokinase (MLKL) in lipopolysaccharide (LPS)‐treated macrophages. (A) Expression of messenger RNA (mRNA) for receptor interacting protein kinase (RIP) 1‐RIP3‐MLKL‐axis in bone marrow–derived macrophages (BMDMs) after challenge with 10 ng/ml LPS for 24 h. (B) MLKL protein in lysates of LPS‐treated RAW cells over 24 h was assessed by western blot and normalized to HSC70. (C–E) Protein expression of phospho‐MLKL (pMLKL), MLKL, RIP3, phospho‐STAT1 (pSTAT1), and signal transducer and activator of transcription 1 (STAT1) in (C) BMDM lysates, (D) RAW cell lysates, and (E) RAW cell cytosolic and nuclear fractions after LPS challenge assessed by western blot and normalized to HSC70 or GAPDH. (D,E) RAW cells were pretreated with or without 25 μM Fludarabine (Fludara) for 2 h prior to challenge with LPS. (E) Lamin B1 and GAPDH were employed as loading controls for nuclear and cytoplasmic fractions, respectively. (F) RAW cells were pretreated with or without 5 μM GSK872 for 2 h prior to challenge with LPS and phosphorylation of MLKL assessed by western blot. Values represent means ± SEM. Values with different superscripts are significantly different from each other; n = 3–6, p < 0.05.