Sc 20003

Total lysates (500 μg protein) were diluted in 200 μL IP buffer [1% Triton X-100, 150 mM NaCl, 20 mM Tris buffer (pH 7.5)] in the presence of proteases and phosphatases inhibitors and immunoprecipitated with anti-p53 (SC-126, Santa Cruz Biotechnology) for 18 h at 4 °C. Protein A/G plus agarose beads (SC-20003; Santa Cruz Biotechnology) were added for 2 h at 4 °C. Immunoprecipitates were electrophoresed and immunoblotted with antibodies against p53 or UGT2B15. Total lysates were loaded as input co-IP control for each sample.

Formalin fixed and paraffin embedded liver fibrosis tissue arrays were purchased from US Biomax (LV805a) (Rockville, MD). Tissue array patient information is shown in Supplementary Table 2. IHF was performed according to a previous report34 (link). Thin cryosections (4 μm) of liver tissue were fixed in acetone for immunohistofluorescence, stained with the indicated antibodies. Antibodies used in IHF were: anti-CUGBP1 (Santa Cruz Biotechnology, SC-20003, 1: 50), α-SMA (Santa Cruz Biotechnology, SC-32251, 1: 50), Reelin (Abcam, ab78540, 1: 50), IFN-γ (Abcam, ab133566, 1: 50), Cytoglobin (Abcam, ab57713, 1: 50), CUGBP1 conjugated to Alexa Fluor 488 (Abcam, ab129115, 1: 100), IFN-γ conjugated to PE-CF594 (BD Biosciences, 562303, 1: 50), goat anti-mouse IgG2a conjugated to Alexa Fluor 594 (Invitrogen, A-21135, 1: 500), goat anti-mouse IgG1 conjugated to Alexa Fluor 647 (Invitrogen, A-21240, 1: 500), goat anti-rabbit IgG conjugated to Alexa Fluor 594 (Invitrogen, R37117, 1: 500), goat anti-mouse IgG2b conjugated to Alexa Fluor 488 (Invitrogen, A-21141, 1: 500). The sections were then stained with DAPI and examined with a confocal laser scanning microscope (Leica, Wetzlar, Germany). Sirius Red staining was performed by Servicebio (Wuhan, China). The liver fibrosis stage was assessed by Ishak scale35 (link).