Bca protein quantitative kit

The total protein was extracted from harvested cells, and the concentration was measured with the BCA protein quantitative kit (Dingguo, Beijing) according to the manufacturer’s protocol. Thirty micrograms of total proteins were separated by 10% SDS-PAGE gels and transferred onto PVDF membranes (Millipore, Bedford, MA, USA). After blocking with 5% nonfat milk for 2 h at room temperature, membranes were incubated with primary and secondary (1:5000, MultiSciences, Hangzhou, China) antibodies [30 (link)]. The primary antibodies included Iba-1 (1:200, sc-32725, Santa Cruz, CA, USA), CD11b (1:200, sc-6612, Santa Cruz, USA), Arg1 (1:200, sc-271430, Santa Cruz, USA), BDNF (1:1000, Sangon, Shanghai, China), TrkB (1:1000, #46035, CST, Boston, Massachusetts, USA), P75 (1:400, sc-271708, Santa Cruz, USA), P-AKT (1:1000, #40605, CST, USA), AKT (1:1000, #46855, CST, USA), PI3K (1:200, sc-365404, Santa Cruz, USA), and β-Actin (1:400, sc-8432, Santa Cruz, USA). The proteins on the membranes were detected by enhanced chemiluminescence (ECL) (Millipore Corp., USA). The bands were analyzed by a chemiluminescence system (Tanon 5200, Shanghai, China). All target proteins were quantified by normalizing them to β-Actin reprobed on the same membrane and calculated as a percentage of the control group.

Approximately 40 mg of brain tissue lysed in RIPA buffer including protease inhibitors were homogenized and centrifuged at 12,000 rpm for 5 min at 4 °C. The concentration of protein was detected by a BCA Protein Quantitative Kit (Dingguo Changsheng Biotechnology, Beijing, China). Equivalent amounts of protein samples (40 ng) were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (PVDF). The antibodies used were as follows: APP 1:1000 (Abcam, ab126732, Cambridge, UK), SAA 1:1000 (Abcam, ab199030, Cambridge, UK), CYP27A1 1:1000 (Abcam, ab126785, Cambridge, UK), CYP7B1 1:1000 (ABclonal, A17872, Wuhan, China), CYP46A1 1:2000 (Abcam, ab244241, Cambridge, UK), RORγt 1:2000 (Abcam, ab207082, Cambridge, UK), Foxp3 1:1000 (Abcam, ab215206, Cambridge, UK), IL-17A 1:3000 (Abcam, ab189377, Cambridge, UK), GM-CSF 1:1000 (Proteintech, 17762-1-AP, Chicago, IL, USA), MIP-3α 1:1000 (Abcam, ab106151, Cambridge, UK), IL-10 1:1000 (Abcam, ab189392, Cambridge, UK), IFN-λ2 0.1 µg/mL (R and D, AF4635, Minneapolis, MN, USA). The protein density was measured by Image System Fusion FX (Vilber Lourmat, Paris, France) and GAPDH was used as the reference for standardization.

Protein expression of TrkB and P75 was measured by western blot in the amygdala. The total protein was extracted from the amygdala and the concentration was measured with the BCA protein quantitative kit (Dingguo, Beijing) according to the manufacturer’s protocol. 30 μg total protein were separated by 10% SDS PAGE gels and transferred onto PVDF membranes (Millipore, USA). After blocking with 5% non-fat milk for 2 h at room temperature, membranes were incubated with primary and secondary (MultiSciences, 1:5000) antibodies. The primary antibodies include TrkB (1:1000, ab187041, Abcom, UAS), P75 (1:400, sc-271708, Santa Cruz, USA) and β-Actin (1:400, sc-8432, Santa Cruz, USA). The proteins on the membranes were detected by enhanced chemiluminescence (ECL) (Millipore Corp., USA). The bands were analyzed by chemiluminescence system (Tanon 5200, Shanghai, China). All target proteins were quantified by normalizing them to β-Actin re-probed on the same membrane and calculated as a percentage of the control group.