For transfection, HEK 293T cells (5x10^5) were seeded in 6-well plate for 24 h and transfected with indicated plasmids (pcDNA3.1(+), pcDNA3.1(+)-FLAG-CAPG, pcDNA3.1(+)-FLAG-CGNL1, pcDNA3.1(+)-FLAG-EPS8L2, pcDNA3.1(+)-FLAG-PDXK, pcDNA3.1(+)- FLAG-TPD52) by using jetOPTIMUS. 48 h after transfection, cells were fixed with 4% PFA (CGNL1, EPS8L2 and PDXK) or pre-cooled ethanol (-20°C) (CAPG and TPD52) 15 min and then washed twice with PBS and permeabilized with 0.3% Triton X-100, followed by blocking with 3% BSA for 1 h. Cells were incubated at 4°C with the following primary antibodies in PBS/1% BSA: mouse anti-FLAG (1:200, cat. #F3165, sigma), CAPG (1:100, cat. #10194-1-AP, Proteintech), rabbit anti-CGNL1 (1:100, cat. #18031-1-AP, Proteintech), rabbit anti-EPS8L2 (1:100, cat. #20461-1- AP, Proteintech), rabbit anti-PDXK (1:50, cat. #15309-1-AP, Proteintech) and rabbit anti-TPD52 (1:50, cat#A10254; Abclonal). Excess antibody was removed by rinsing in PBS for 10 min and 3 times. Cells were then incubated at room temperature for 1 h with the following secondary fluorescently labeled antibodies: Alexa Fluor 488 donkey anti-rabbit IgG (1:500, Jackson ImmunoResearch) and Alexa Fluor 594 goat anti-mouse IgG (1:500, Jackson ImmunoResearch). Slides were mounted in Anti-fade Reagent with DAPI (cat. #S2110, Solarbio) and imaged with laser scanning confocal microscope (Leica, stellaris 5).

The blood-brain barrier (BBB) is primarily manifested by a variety of physiological properties of brain endothelial cells (ECs), but the molecular foundation for these properties remains incompletely clear. Here, we generate a comprehensive molecular atlas of adult brain ECs using acutely purified mouse ECs and integrated multi-omics. Using RNA sequencing (RNA-seq) and proteomics, we identify the transcripts and proteins selectively enriched in brain ECs and demonstrate that they are partially correlated. Using single-cell RNA-seq, we dissect the molecular basis of functional heterogeneity of brain ECs. Using integrative epigenomics and transcriptomics, we determine that TCF/LEF, SOX, and ETS families are top-ranked transcription factors regulating the BBB. We then validate the identified brain-EC-enriched proteins and transcription factors in normal mouse and human brain tissue and assess their expression changes in mice with Alzheimer's disease. Overall, we present a valuable resource with broad implications for regulation of the BBB and treatment of neurological disorders.