Mca4739

Total protein was extracted from cultured cells or frozen clamped liver tissue that had been stored at −80 °C. The whole tissues were homogenized in Triton lysis buffer (TLB) supplemented with protease inhibitors (Complete Mini 11 836 153 001; Roche, Indianapolis, IN, USA). Protein concentration was measured with a Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Equal amounts of total protein were fractionated by polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Darmstadt, Germany). Primary antibodies against α-SMA (A5228; Sigma Aldrich, St. Louis, MO, USA) and GAPDH (MCA4739; AbDSerotec, Oxford, UK) were used in this experiment. The membranes were developed by chemiluminescence (ATTO Corporation, Tokyo, Japan). The blots obtained from three independent experiments were scanned and a region of interest (ROI) around the band of interest was defined. The band intensities were calculated by using CS analyzer 4 program (ATTO Corporation.)

Total protein was extracted from frozen liver tissues and cultured cells. Samples were homogenized in Triton lysis buffer (TLB) supplemented with protease inhibitors (Roche). Equal amount of total protein were fractionated by SDS-PAGE on 15% tris-tricine gel as described by Hermann [25 (link)] and transferred to polyvinylidene difluoride (PVDF) membranes. Other detailed procedures were conducted as previously described [5 (link)]. Primary antibodies against Tβ4 (A9520, Immunodiagnostik AG, Bensheim, Germany) and GAPDH (MCA4739, AbDSerotec, Raleigh, NC) were used in this experiment. Membranes were developed by chemiluminescence (ATTO Corporation). The blots obtained from 3 independent experiments were scanned and an region of interest (ROI) around the band of interest was defined. Band intensities were calculated by using CS Analyzer Version 2.0 (ATTO Corporation).

Tissue samples were homogenized in NP-40 lysis buffer using a tissue grind pestle (Kimble/Chase) or with a bead ruptor 12 (Omni International) to obtain protein lysates. These were separated by SDS–PAGE, transferred to polyvinylidene difluoride membrane and analysed by immunoblotting. Membranes were probed with the following antibodies: anti-p-AKT (#4060), anti-AKT (#4685), anti-p-GSK-3 (#8566), anti-GSK-3 (#5676), anti-p-ERK (#4377) and anti-ERK (#4695; Cell Signaling), anti-Caspase-8 (Enzo #ALX-804-447), anti-RIPK3 mouse (IMGENEX #IMG-5523), anti-RIPK3 human (#ab56164), anti-phospho-MLKL human (#ab187091) and mouse (#ab196436; Abcam) and anti-GAPDH (ABD Serotec #MCA4739). All primary antibodies were used at the dilution 1:2,000. As secondary antibodies, anti-rabbit-horseradish peroxidase (HRP; #NA934V) and anti-mouse-HRP (#NA931V; Amersham) and anti-rat-HRP (Santa Cruz #sa2956) were used. All secondary antibodies were used at the dilution 1:5,000. Unedited scans of all western blot images are shown in Supplementary Fig. 11.