Restore plus

Cells were trypsinized for histone extraction as per the Abcam protocol. Additionally, cells were lysed using RIPA buffer for whole-cell lysates. Protein concentration was assessed (BioRad, 5000116). A total of 10 µg total histones and 40 µg whole-cell lysates were loaded on gels. Membranes were incubated overnight at 4 °C with primary antibody in 5% BSA in TBST. Primary antibodies used were: H3K27me3 (Cell signaling, 9733S), total histone H3 (Cell Signaling, 9715), EZH2 (Cell signaling, 5246), KDM6A (Atlas Antibodies, HPA002111), ALDH2 (Abcam, ab108306), and CK5 (Covance, PRB 160-P). Membranes were incubated with HRP-conjugated secondary rabbit antibody (GE Life Sciences, NA934V). The secondary antibody was detected using Luminata Crescendo Western HRP Substrate (WBLUC0500). The membranes were stripped with Restore Plus (Thermo Scientific, 46428) and re-probed with GAPDH (Abcam, ab9485) or Total H3.

Protein extracts were prepared by cell lysis in boiling 1% SDS buffer (18 (link)). Protein extracts (and Co-IP samples) were then mixed with loading buffer and denatured (10 minutes, 95°C–100°C), before separation by SDS-PAGE and transfer to nitrocellulose membranes (Bio-Rad). Membranes were blocked [5% milk or 3% BSA in Tris-buffered saline with 0.1% Tween 20 (TBST)], incubated with primary antibodies (Table S5), washed with TBST, incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Southern Biotech or Thermo Fisher Scientific, 1:10,000–1:50,000), and then washed again with TBST. Membranes were developed using HRP substrate [ECL Prime (GE Healthcare) or SuperSignal West Pico PLUS (Thermo Fisher Scientific)]. Membranes were stripped with Restore Plus (Thermo Fisher Scientific) and blocked before the detection of additional targets. Band intensities were quantified using Image Quant TL (GE Healthcare). Expression levels of bacterial and host proteins were normalized to the expression of the Chlamydia protein Slc1 and host β-actin, respectively.

Protein lysates from frozen liver tissue of subject ID#11 and from two controls without acute liver failure were prepared with tissue homogenization in RIPA buffer with protease inhibitors, sonication, and centrifugation as described previously [16 (link)]. Protein concentration was determined using a Bradford assay (Bio-Rad, Hercules, CA). Protein samples (80 μg/well) were resolved on a 4–12% Bis-Tris gel (Life Technologies, Grand Island, NY) and transferred to a 0.2 μm nitrocellulose membrane (Bio-Rad). Membranes were blocked with 10% Blotting Grade Blocker in TBST (Bio-Rad) for 1 hour at 25°C, incubated with primary antibody (Ab) overnight at 4°C, washed 3x in TBST, and incubated with HRP-conjugated secondary Ab (1:10,000 dilution) for 2 hours at 25°C. Blots were developed with enhanced chemiluminescence (ECL; Pierce Biotechnology, Rockford, IL) and read on a low-light digital camera (LAS-1000; Fujifilm Medical Systems USA, Stamford, CT). The membrane was incubated for 1 hour at 25°C with RestorePLUS (Thermo Scientific, Rockford, IL) and reprobed using anti-GAPDH as a loading control with the above technique. Primary Abs were purchased from Abcam (Cambridge, MA): rabbit anti-human p53R2 (#130321, 1:500 dilution, NP_001165948.1), mouse anti-GAPDH (#9484, 1:2000 dilution). Secondary Ab included: HRP-conjugated anti-rabbit IgG and anti-mouse IgG (Promega, Madison, WI).