Hbcag

Protein samples were subjected to SDS-PAGE gel electrophoresis and blotted with primary antibodies recognizing HA-tag, TAK1, β-actin, JNK1/2/3, ERK1/2, p38, NF-κB p65, phospho-JNK1/2, phospho-ERK1/2, phospho-p38, phospho-NF-κB (p65) (Cell Signaling Technology), FXRα (R&D Systems, Minneapolis, MN, USA), and HBcAg (Abcam, Cambridge, MA, USA). Protein bands were visualized using ECL Plus western blotting detection reagents (Amersham Biosciences, Buckinghamshire, UK) and an ImageQuant LAS 2000 mini system, as previously described11 (link).

The recombinant protein human serum albumin (HSA) (Cat# 10968-HNAY) and GST (Cat#11213-HNAE) were purchased from Sino Biological. HBsAg (Cat# ab91276), HBcAg (Cat# ab49013), and GST-HBeAg (Cat# ab91273) were purchased from Abcam. HBeAg was prepared in our laboratory as previously described [9 (link)].

Formalin-fixed biopsies were deparaffinized, rehydrated, and subjected to antigen retrieval using citrate pH 6.0 (for HbcAg), Tris-EDTA pH 9.0 (for CD4 and CD8), pepsin (for CD68), or commercial CC1 buffer Ventana (for PD-1 and PD-L1). Sections were then incubated with antibodies to the following: HBsAg (clone A10F1, 1:100, Cell Marque, Rocklin, CA), HbcAg (1:800, Abcam, Cambridge, UK), CD4 (clone 4B12, 1:200, Leica, Buffalo Grove, IL), CD8 (clone 4B11, 1:1000, Leica, Buffalo Grove, IL), CD68 (clone PG-M1, 1:300, Dako, Santa Clara, CA), PD-1 (clone NAT105, 1:200, Abcam, Cambridge, UK), PD-L1 (clone 28-8, 1:200, Abcam, Cambridge, UK). Expression of the markers was quantified using the Visiopharm software, by dividing the number of strongly positive cells by the total stained area. PD-L1 staining on hepatocytes was scored on a semi-quantitative scale: 1+ 25% positive, 2+ as intermediate, and 3+ diffuse staining.