Ab106114

H413 clone-1 cells were treated with different conditions as described above. Whole cell proteins were prepared by scraping the cells in cold PBS, extracted in SDS sample buffer, separated by SDS-PAGE using 5% to 12% gradient mini-gels, transferred to nitrocellulose membranes (Bio-Rad), and blocked overnight with 3% BSA (Sigma) in 0.1 mol·L^-1 Tris buffered salts solution pH 7.4 (TBS). The nitrocellulose membranes were then incubated with rabbit polyclonal antibodies to human occludin (ab31721), JAM-A (ab106114), claudin-1 (ab15098), claudin-4 (ab53156), ZO-1 (ab59720), claudin-15 (ab215354; all 1 μg·mL⁻¹, Abcam, Cambridge, UK), and β-actin (0.1 μg·mL⁻¹, GenTex, Zeeland, MI, USA) in 0.05% Tween20/TBS for 4 h. β-actin was used as a loading control. After the incubation, the membranes were washed three times and subsequently incubated with alkaline phosphatase (AP)-conjugated secondary antibody (goat-anti rabbit IgG, DAKO) diluted 1:1 500 in Tween20/TBS for 2 h. Bound antibodies were displayed with AP substrate (Bio-Rad) after the development of reactivity for proteins.

CrFK cells were grown overnight on glass coverslips and infected with FCV at a multiplicity of infection (MOI) of 5 at the indicated times, or at an MOI of 10 for 30 min at 4 °C as indicated. The cells were treated with cytoskeleton buffer for 5 min and permeabilized in 4% formaldehyde solution for 5 min at room temperature (RT). The samples were washed three times with phosphate buffer saline (PBS) for 5 min, blocked with 0.5% gelatin in PBS for 40 min at RT, washed three times with PBS for 5 min, and incubated with the anti-FCV (FCV1-43, Santa Cruz Biotechnology, CA, USA) that recognizes an epitope on the capsid protein or the anti-JAM-1 (ab106114, Abcam, Cambridge, UK), at 4 °C overnight. Samples were washed three times with cold PBS for 5 min and incubated with the appropriate secondary antibodies (Invitrogen, MA, USA) for 1 h at RT. The samples were washed three times with PBS and incubated with 1 mg/mL of 4′6′-diamidino-2-phenylindole (DAPI) for 2 min. The samples were washed six times with PBS and three times with distilled water. The samples were treated with VECTASHIELD liquid mounting media (Vector Laboratories A.C., CA, USA) and analyzed using a Zeiss LSM-700 confocal microscope.