Mouse anti β actin

Rabbit polyclonal anti-HBc antibody is from DAKO (B0586). For the detection of capsids, mouse monoclonal anti-HBc C1 (ab8637) was purchased from Abcam, and mouse monoclonal anti-HBc 2A7 was kindly provided by Prof. Quan Yuan (Xiamen University, China). For the immunoprecipitation of HBV virions, rabbit anti-HBs (NB100-62652, Novus, Centennial, CO, USA) and rabbit anti-preS1 antibodies (kindly provided by Prof. Shuping Tong, Brown University, USA) were used. For immunofluorescence staining, commercially available rabbit polyclonal anti-Alix (12422-1-AP, Proteintech, Chicago, IL, USA), and rabbit polyclonal anti-HBc (0282, Long Island Antibody, Shanghai, China) were used. Other commercially available antibodies are as follows: mouse anti-β-tubulin (M20005, Abmart, Shanghai, China), mouse anti-β-actin (T40104, Abmart, Shanghai, China), and rabbit anti-Flag (14793S, Cell Signaling Technology, Boston, MA, USA).

Protein solutions from three mice in each group were denatured with 5 × loading buffer at 95 °C for 10 min and stored at − 20 °C. Twenty to forty micrograms of total protein sample were separated by SDS-polyacrylamide gel electrophoresis using a Bio–Rad protein assay and transferred onto polyvinylidene fluoride (PVDF) membranes. Membranes were blocked in Tris-buffered saline/0.1% Tween buffer (TBST; 25 mM Tris–Cl, 125 mM NaCl, 0.1% Tween-20) with 5% skim milk powder at room temperature for 2 h and incubated with primary antibodies at 4 °C overnight: mouse anti-GLUT5 (Santa Cruz, sc-271055, 1:300), mouse anti-GLUT3 (Abcam, ab150299, 1:1000), mouse anti-ADAM10 (Santa Cruz, sc-28358, 1:500), mouse anti-SYK (Santa Cruz, sc-1240, 1:300), rabbit anti-phospho-SYK (Tyr525/526) (CST, 2710, 1:300), mouse anti-β-tubulin and mouse anti-β-actin (Abmart, 1:1000). The corresponding secondary antibody was incubated at room temperature for 2 h (1:4000). The results were analyzed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, USA).