Cell lysis buffer

PHH were plated onto 6-well plates at a density of 1.5 million cells per well. The cells were transduced with GFP- or HBx-expressing lentiviruses (MOI of 3) 24 h after plating. At 72 h posttransduction, cell lysates were generated by adding 250 μl 1× cell lysis buffer (Cell Signaling) supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific) and then scraping the wells using a cell scraper. The lysates were sonicated for 30 s and centrifuged for 1 min at 21,130 × g. The supernatants were collected, and 65 μl of each was set aside for Western blotting. Anti-DDB1 antibody (clone 5428) (Cell Signaling) was added to the remainder of the supernatant at a 1:250 dilution, and the samples were mixed gently with rocking overnight at 4°C. Protein A/G magnetic beads (Thermo Fisher Scientific) were washed according to the manufacturer’s protocol, added to the lysate, and then incubated for 1 h at room temperature. The magnetic beads were washed three times using 1× cell lysis buffer (Cell Signaling) and then once with deionized water. Proteins bound to the beads were eluted by boiling in SDS-PAGE buffer for 10 min. The eluted proteins were subsequently electrophoresed for 1.5 h at 150 V on a NuPage 4-to-12% gradient gel in 1× MES buffer (Thermo Fisher Scientific) and Western blotted as described above. HBx was detected using an anti-Myc antibody.

The PathScan RTK signaling antibody array kit (Cell Signaling Technology) is a slide-based antibody array product founded upon the sandwich immunoassay principle. Briefly, 1 × 10e6 CD34⁺ HPCs were infected at a multiplicity of infection (MOI) of 1,000 with adenovirus vector expressing GFP (Ad-GFP) or UL7 (Ad-UL7), sorted after 24 h for viable CD34⁺ HPCs, washed in phosphate-buffered saline (PBS), lysed in 1× cell lysis buffer (Cell Signaling Technology), and processed according to the manufacturer’s protocol. The PathScan phospho-Flt3 (panTyr) is a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) (Cell Signaling Technology). Briefly, 8 × 10e5 cells were plated in 24-well flat-bottom plate and grown overnight at 37°C with 5% CO₂. The cells were starved in 0.5% FBS for 24 h before treatment with Flt-3 ligand (Flt-3L) or purified UL7 (pUL7) (0.05 to 10 nM) for 10 min at 37°C with 5% CO₂. The cells were washed in PBS, lysed in cell lysis buffer (Cell Signaling Technology), and quantified by Pierce BCA protein assay kit (ThermoFisher). Lysates (20 µg) were processed according to the manufacturer’s protocol.

Immunoprecipitations were performed after HCT116 cells were lysed with cell lysis buffer (Cell Signaling Technology, Danvers, MA) and then clarified by centrifugation at 13,000 rpm for 10 min to remove cell debris. Antibodies (indicated in Table SI) were added and incubated for the indicated times, and antibodies were then precipitated with protein G magnetic beads. Samples with biotinylated Omomyc were precipitated with streptavidin magnetic beads (Thermo-Fisher, Carlsbad, CA). Magnetic beads were then washed 5 times with cell lysis buffer and radioimmunoprecipitation assay (RIPA) buffer (Cell Signaling Technology, Danvers, MA). Beads were resuspended in 1× sample buffer (Thermo-Fisher, Carlsbad, CA). Samples were then subjected to Western blotting by running samples on a 4 to 20% polyacrylamide gel (Thermo-Fisher, Carlsbad, CA) and then transferring them to a polyvinylidene difluoride (PVDF) membrane using an I-blot apparatus (Thermo-Fisher, Carlsbad, CA). Membranes were then probed with the appropriate primary antibody, followed by a secondary antibody, either goat anti-rabbit antibody–horseradish peroxidase (HRP) or goat anti-mouse antibody–HRP (Cell Signaling Technology, Danvers, MA). Blots were developed with ECL developing solution (GE Bioscience, Westborough, MA) and exposed to X-ray film (GE Bioscience, Westborough, MA).