Image analyzer

Astrocytes treated with andrographolide with and without chemical inhibitors were lysed in situ on tissue culture plates by adding boiling Laemmli sample buffer (Bio-Rad, Berkeley, CA, USA), heated further at 95 °C for 5 min, then allowed to cool before electrophoretic separation on 10 % polyacrylamide gels, transferred onto nitrocellulose membranes (ThermoFisher Scientific, Waltham, MA, USA), and blocked with 5 % non-fat milk in 10 mM PBS, pH7.4 with 0.1 % Tween® 20 (PBST) at room temperature for 1 h. Membranes were then washed and probed with primary antibody diluted in PBST with 5 % bovine serum albumin overnight at 4 °C. The primary antibodies used are listed in Table 1. Following primary antibody incubation, membranes were washed with PBST, then incubated with horse radish peroxidase (HRP)-conjugated secondary antibodies (goat anti-rabbit, goat anti-mouse, and donkey anti-goat, respectively, from Jackson ImmunoResearch, West Grove, PA, USA), and diluted at 1:5000 for 1 h at 25 °C. To detect the proportion of phosphorylated protein, membranes were first probed for phospho-proteins then stripped and reblotted for total proteins. Immunoblots were visualized using HRP substrate (Luminata™ Forte or Crescendo, Merck Millipore, Darmstadt, Germany) and quantified by image analyzer (UVItec Ltd., Cambridge, UK).