Mouse anti human monoclonal antibody

Western blotting was performed as we previously described (Duan et al., 2012b (link)). Briefly, ARPE-19 cells were infected by HSV-1 F strain at MOIs of 1 and 0.1, and then stimulated by LPS as described above. At 24 h p.i., cells were harvested and lysed with lysate buffer (20 nM tris-HCL). The samples were freeze-thawed 3 times and then centrifuged at 15777✕g for 30 min at 4 °C to remove cellular debris. The protein content in the supernatant was determined by the bicinchoninic acid method using bovine serum albumin (BSA) as the standard. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted on polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Billerica, USA). The membranes were incubated with 1 μg/mL of mouse anti-human monoclonal antibodies (Abcam, Cambridge, UK) that recognize HSV-1 infected cell protein (ICP4) or 0.2 μg/mL of mouse anti-human β-actin antibodies (Kang- Chen, Shanghai, China) respectively at 4 °C overnight. Then, they were exposed to secondary goat anti-mouse IgG antibodies (Zhongshan Goldenbridge, Beijing, China) for 1 h. The protein bands were visualized with a kit of chemiluminescence Phototype (R)- Horseradish peroxidase (HRP) Western Blot Detection System (Cell Signaling Technology, Inc., Danvers, MA, USA) and exposed by Kodak Imaging Station 4000MM (Kodak, Rochester, NY, USA).