Hrp conjugated secondary ab

All antibodies used for western blotting are listed in Supplementary Table 3. 0.8–1 × 10⁶ primary naive B cells were recovered for protein extraction, which was performed using RIPA lysis buffer (Pierce) followed by cycles of sonication performed using the Biorupter Sonicator (Diagenode). Protein concentration was analysed using the BCA Protein Assay kit (cat no. 23225 from Pierce) and read by the Model 680 Microplate reader (Bio-Rad). Proteins were separated by the NuPAGE SDS-PAGE Gel system (Thermo Scientific) and transferred onto Immobilon-P PVDF membranes (Sigma-Aldrich) according to standard procedure. Detections were performed with HRP-conjugated secondary Ab (Bio-Rad) and enhanced chemiluminescent (ECL Plus) reagent (Amersham) using the G:BOX Chemi imaging system (Syngene). GAPDH or β-ACTIN on the same membrane served as loading control. Uncropped original scans of immunoblots are provided in Supplementary Fig. 9.

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).

Cells were lysed in NP-40 lysis buffer containing 1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 7.4, and 5 mM MgCl₂. Protein concentrations in cell lysates were assayed in triplicate using the Pierce BCA protein assay kit (Pierce, Rockford, IL). Proteins were separated using SDS-PAGE, transferred to BA-85 nitrocellulose membrane (Whatman, Florham Park, NJ), and probed with the indicated primary Abs, followed by an HRP-conjugated secondary Ab (Bio-Rad). Equal loading of samples was verified using Ponceau S (Sigma-Aldrich) to stain the nitrocellulose membrane. Polypeptides were visualized using the Pierce Supersignal chemiluminescence reagents (Pierce, Rockford, IL) and in some cases quantitated with an Alpha Imager.

NuPAGE gels (Novex, Invitrogen, Carlsbad, CA, USA) at appropriate concentrations were used for proteins separation as described [40 (link),41 (link)]. Filters were probed with the following antibodies at reported dilution: mouse polyclonal anti-GLV-capsid protein_N-terminal (GLV-CP_NT) 1:2000; mouse polyclonal anti-GLV-capsid protein_C-terminal (GLV-CP_CT) 1:2000; mouse polyclonal anti-J17/10_B-ORF1 N-terminal (J_B-ORF1_NT) 1:2000; mouse anti-αTubulin (clone B-5-1-2, Sigma-Aldrich, Merck Life Science S.r.l., Milan, Italy), 1:10,000; rabbit polyclonal N14 (anti-g14-3-3) [40 (link)] 1:5000. Interaction was revealed by incubation with HRP-conjugated secondary Ab (Bio-Rad, Hercules, CA USA) at 1:2000–1:3000 dilution, followed by chemiluminescence (Millipore, Merck Life Science S.r.l., Milan, Italy).