28 Purified immunoglobulin G (IgG) from rat anti-CLDN1 serum were obtained by MAbTrap kit (GE Healthcare). To analyze cross-reactivity of antibodies with other members of the CLDN family, 293T cells were transfected to express AcGFP tagged CLDN1, 4, 6, 7, 9, 11, 15, and 17 or chimeric CLDN1/7 (described by Evans et al.9) and 48 hours later stained with rat anti-CLDN1 antibodies and Alexa-633

coupled anti-rat immunoglobulin (Invitrogen). Polyclonal rat anti–SR-BI or CD81 antibodies were obtained by genetic immunization as described.26 R-phycoerythrin–conjugated and Cy5-conjugated anti-rat IgG were obtained from Jackson ImmunoResearch Laboratories, mouse IgG was obtained from Caltag, and mouse anti-CD81 (JS-81) was obtained from BD Biosciences. Living Huh7.5.1 cells were incubated with preimmune or anti-CLDN1 serum (1/50) and a Cy5-conjugated anti-rat secondary antibody (1/300). Polarized selleck chemicals Caco-2 cells, as described by Mee et al.,23 were fixed in 3% paraformaldehyde, permeabilized with

saponin, and stained with polyclonal anti-CLDN1 (1/50) or control serum. Following staining, cells were fixed, mounted, and observed using a Leica TCS SP2 CLSM (for Huh7.5.1) or a Zeiss Cell Axio Observer Z1 microscope (for Caco-2). To determine Carfilzomib the functionality of TJs and whether they restrict the paracellular diffusion of solutes from the bile-canalicular (BC) lumen to the basolateral medium (barrier function), HepG2 cells were treated with either control (PBS), rat anti-CLDN1, rat control serum, or interferon-γ and incubated with 5 mM 5-chloromethylfluorescein diacetate

(CMFDA) (Invitrogen) at 37°C for 10 minutes to allow internalization and translocation to BC lumen by MRP2. After washing with PBS, the capacity of BC lumens to retain CMFDA was analyzed as described.18 Cell culture–derived HCV (HCVcc) (Luc-Jc1 or Jc1) were generated as described.6, 26, 29 For infection experiments, Huh7.5.1 cells were preincubated in the presence or absence of antibodies for 1 hour at 37°C and infected at 37°C for 4 hours with HCVcc. Forty-eight hours later HCV infection was analyzed in cell lysates by quantification of luciferase activity or viral RNA.6, 26, 29, 30 Kinetic studies in the presence of antibodies or inhibitors were performed as described.6, 26, 29, 30 Infection of 293T/CLDN1 or Huh7.5.1 cells with murine leukemia virus–based HCV pseudoparticles Progesterone (HCVpp) in kinetic assays was performed as described.5, 6 Primary hepatocytes were infected with HIV-based HCVpp expressing envelope glycoproteins of strains HCV-J (genotype 1b), JFH-1 (genotype 2a), UKN3A.1.28 (genotype 3a), and UKN4.21.16 (genotype 4). One day following hepatocyte isolation and plating, hepatocytes were washed with PBS and preincubated with rat anti-CLDN1 or control serum (1/50) for 1 hour at 37°C in William’s E medium. Then, HCVpp were added for 3 hours at 37°C. Following infection, the supernatant was removed and replaced by fresh William’s E medium.