Mouse anti β tubulin

The hippocampus and cortex of mice were extracted and lysed by RIPA buffer (Beyotime, P0013B, China) containing 1X halt™ protease and phosphatase inhibitor single-use cocktail (Invitrogen, 78,443). Afterward, Pierce™ BCA Protein Assay Kit (Invitrogen, 23,225) was used for quantitation. Samples were separated on SDS-PAGE gel and transferred to PVDF membranes (Millipore). Then we blocked the membranes with 5% milk for one hour at room temperature and incubated with primary antibodies. The primary antibodies we used include mouse-anti-β-Tubulin (1:5000, Proteintech, 66,240–1-Ig), rabbit-anti-PDGFRβ (1:1000, Abcam, ab32570), rabbit-anti-ZO1 (1:1000, ThermoFisher, 61–7300), rabbit-anti-Occludin (1:1000, ThermoFisher, 71–1500), rabbit-anti-Claudin-5 (1:1000, Abcam, ab131259), mouse-anti-tau5(1:5000, Invitrogen, AHB0042), and Phospho-Tau Family Antibody Sampler Kit (CST, 96628 T). Specific secondary HRP-linked antibodies (1:2000, Cell Signaling Technology, 7074S or 7076S) were subsequently incubated for an hour at room temperature.

The cells were harvested and lysis in RIPA buffer supplemented with 1 mM PMSF, protease inhibitor and phosphatase inhibitor cocktail. The cell lysates were quantified using BCA protein assay kit and the Western Blot assays were carried out as previously described (Li et al., 2014 (link)). The following primary antibodies were used: mouse anti-GAPDH (1:4000, TransGen Biotech), mouse anti-β-Tubulin (1:4000, Proteintech Group, IL, United States), rabbit anti-COX-2 (1:1000, Proteintech), rabbit anti-iNOS (1:1000, Abcam, Cambridge, United Kingdom), mouse anti-TLR4 (1:2000, Proteintech), rabbit anti-SF3A1 (1:2000, Proteintech), rabbit anti-NF-κB p65 (1:500, Cell Signaling Technology), rabbit anti-phospho-NF-κB p65 (1:1000, Cell Signaling Technology), rabbit anti-IκBα (1:1000, Proteintech), rabbit anti-phospho-IκBα (1:1000, Bioworld Technology, Bloomington, United States). The protein bands were quantified by ImageJ Software, and normalized to GAPDH protein levels.