Rabbit anti p erk

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).