Mouse β actin

Samples from the whole liver tissue lysates (50 μg protein/lane) were resolved on SDS-PAGE (Hoefer Mighty small SE245, Pharmacia Biotech, USA) and electrophoretically transferred (Trans-Blot Semi-dry, BioRad) onto PVDF membranes (Bio-Rad, CA, USA). Gal1 protein was detected with the above mentioned rabbit anti-Gal1 (1:5000). Mouse β-actin (1:200, Abcam, UK) was used as a control to monitor sample loading. The immunocomplexes were visualized using the anti-rabbit (1:300), or anti-mouse (1:200) antibodies described above for 45 min and the EZ-ECL detection system (Biological Industries, Beit Ha-Emek, Israel) or Amersham ECL Prime (RPN2232, GE Healthcare UK Ltd, England) for 5 min.

The microglia from the control and experimental groups (1×10⁷ cells/mL) were homogenized in the RIPA buffer (Beyotime, Shanghai, China). The protein concentrations were quantified using the BCA assay (Pierce, Rockford, IL) according to the manufacturer’s instructions. Then, 40 μg of whole cell protein lysates were boiled in 5μl sample buffer for 5 min followed by separation of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat dry milk in TRIS-buffered saline (TBS) for 1h and incubated overnight at 4^oC with primary antibodies, namely, rabbit anti-IRE1α (1:500, Abcam, Camb, UK), rabbit anti-PERK (1:500, Abcam, Camb, UK), and rabbit anti-ATF6 (1:500, Abcam, Camb, UK), and mouse β-actin (1:1000, Abcam, Camb, UK). Then, after washing thoroughly, we incubated the blots with HRP-conjugated secondary antibodies (1:1000, Sigma, St. Louis, MO, USA) at room temperature for 2 h. The blots were developed using the Enhanced Chemiluminescence (ECL) kit (Cell Signaling Technology, Boston, USA). The levels of IRE1α, PERK and ATF6 proteins relative to β-actin expression were quantified using the Image J software (NIH, Bethesda, USA).