Notch1

Total protein was isolated from the ipsilateral hemisphere samples, and the amount of protein was measured using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, USA). The procedure of Western blotting was performed as previously described [62 (link)]. The primary antibodies were as follows: CD63 (1:1000), Tsg101 (1:1000), CD206 (1:1000) (Abcam, Shanghai, China); caspase-3 (1:1000), cleaved caspase-3 (1:1000), ARG (1:200), Notch1 (1:500), and GAPDH (1:1000) (BD Biosciences, San Jose, USA). Proteins were determined using enhanced chemiluminescence (MilliporeSigma, Burlington, USA) and photographed using a Molecular Imager VersaDoc 4000 system (Bio-Rad, Hercules, USA).

Single-cell suspensions were obtained from isolated tissue samples. Cells were washed, counted and stained with the following murine phycoerythrin, fluorescein isothiocyanate, allophycocyanin or cyanine 7 (Cy7)—conjugated antibodies: CD4, CD8, CD44, CD25, CD30, CD117, CD3, Vα2 TCR, Notch1 (BD Biosciences, Oxford, UK). After staining (30 min at 4 °C, 1:1,000), cells were washed and analysed using fluorescence-activated cell sorter (FACS) Canto or Fortessa flow cytometers (BD Biosciences). For intracellular stain, the cells were fixed in 0.01% formaldehyde for 10 min, permeabilized in 0.5% Tween 20 v/v in PBS (15 min in the dark at RT), cells were washed in PBS 0.1% Triton and re-suspended in 100 μl of 0.1% Triton in PBS, cells were stained and analysed as previously described. Alternatively, cell populations were sorted using a FACS Aria machine as detailed in ref. 11 (link).

DAPTA was purchased from Tocris Bioscience (Bristol, UK). Fluorescein isothiocyanate, PE/Cyanine7, Allophycocyanin, Alexa Fluor^® 488, PE/Dazzle™ 594, Alexa Fluor^® 647, and phycoerythrin-labeled CD40, NF-κB p65, IκBα, Notch1, Notch3, GM-CSF, iNOS, RORγT, MCP-1, TNF-α, lysis, and permeabilization solution, and fixation buffers were purchased from BD Biosciences and BioLegend (San Diego, CA, USA). SYBR Green and cDNA kits were purchased from Applied Biosystems (Foster City, CA, USA). Primers were purchased from GenScript (Piscataway, NJ, USA). TRizol was purchased from Invitrogen (Carlsbad, CA, USA).