The complement component 2(C2) p.Glu318Asp mutation model was built on the crystal structure reported by Milder et AZD3965 al. (PDB ID: 2I6Q).14 It is not feasible to model the mutation on transmembrane protein 2 (TMEM2) at present, as very little is known of the structure of TMEM2 or its homologous proteins. TMEM2 p.Ser1254Asn was found to be associated with CHB, but with no indications of immunological function of the wildtype protein. We therefore performed expression studies. Immunohistochemistry

was performed on formalin-fixed and paraffin-embedded healthy liver tissues from 12 individuals, with polyclonal rabbit antihuman TMEM2 antibody (Aviva Systems Biology, San Diego, CA). The sections were incubated with the first antibody at 1:40-1:160 dilution at 4°C overnight. The second, peroxidase-labeled goat antirabbit/mouse antibody (Dako K5007, Carpinteria, CA) was applied to the sections for 30 minutes at 37°C and the sections were developed with Diaminobenzidine (DAB) solution. The staining was replicated in healthy liver tissues from another six subjects using rabbit polyclonal

antibody to human TMEM2 from a different company (Jin Tiancheng, Beijing, China). Negative controls were performed with phosphate-buffered saline (PBS) replacing the first antibody preparation. Real-time PCR was performed in liver tissues from three CHB patients and normal GDC-0199 liver tissues from three subjects who underwent surgical ablation of hemangioma Succinyl-CoA in the liver. The latter had normal liver function (normal ALT, aspartate aminotransferase [AST], and total bilirubin) and were negative for HBsAg and

HBeAg. Total RNA was extracted. Real-time PCR for TMEM2 was performed using the primers 5′-GGAGATATGCTCCGTCTGACC-3′ and 5′-CATCTGACTTGCCATACAAGGT-3′ and 5′-CCA TCTTCCAGGAGCGAGA-3′ and 5′-TGGTTCACA CCCATGACGAA-3′ for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Real-time PCR was also performed with the same primers in two cell lines (1) HepG2.2.15 containing the complete HBV genome and capable of stable HBV expression and replication in the culture system15 and (2) a non-HBV-containing HepG2 cell line (ATCC, Manassas, VA). The two cell lines were maintained in the exponential growth phase in Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies, Carlsbad, CA), supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 0.1% (w/v) streptomycin. The mean and standard error (SE) were calculated from three independent experiments. Western blotting was also performed on the two cell lines. After lysis of the harvested HepG2 and HepG2.2.15 cells and gel electrophoresis, the rabbit antihuman TMEM2 antibody (Aviva Systems Biology) and mouse antihuman GAPDH monoclonal antibody (Kang Chen Biotech, Shanghai, China) were applied for detection of the proteins. Apart from meeting all the criteria described in the “candidate selection” section above, TMEM2 p.