P mlkl ser358

Cultured cells were lysed at 4 °C in lysis buffer and protein concentrations determined by the bicinchoninic acid (BCA) method. Equal amounts of total cell lysates were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels (8-12% gradient) and transferred to PVDF membranes (Millipore, Bedford, MA, USA). Membranes were blocked for 2 h with Tris-buffered saline containing 0.1% Tween and 5% nonfat dry milk and incubated with primary antibodies overnight at 4 °C. After incubation with horseradish peroxidase-conjugated secondary antibodies (1:5000; Multi Sciences Biotech), immunoreactions were visualized with an ECL detection kit (Biological Industries, Beit HaEmek, Israel). Primary antibodies specific for the following proteins were used: RIPK1, p-RIPK1 (Ser166), BCR/ABL, p-BCR/ABL (Tyr177), and β-actin (Cell Signaling Technology, CST; Beverly, MA, USA); RIPK3, p-RIPK3 (Ser223), MLKL, and p-MLKL (Ser358) (Abcam, Cambridge, UK).

Total proteins from cultured cells were separated on SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were blocked in 5% nonfat milk for 1 h at room temperature, and then incubated overnight at 4 °C with primary antibodies against cleaved caspase-3, caspase-3, p-Jak2 tyr1007/1008, Jak2, p-p38, p38, PI3K, p-STAT3, STAT3, p-STAT6, STAT6, arginase-1, β-actin (Cell Signaling Technology, 1:1000), MLKL and p-MLKL ser358 (Abcam, 1:1000). Blots were subsequently washed and incubated with secondary antibodies for 1 h at room temperature. The immunoblots were visualized by chemiluminescence and quantified using ImageJ software.