Anti cleaved casp3

For Western Blot, anti- α-SMA (1:1000, 113200) was obtained from Calbiochem (San Diego, CA, USA), anti-p-IκBα (1:1000, 2859) and anti-IκBα (1:1000, 9242) from Cell Signaling Technology (Danvers, MA) and anti-GAPDH (1:5000, MA1-22670) from Thermo Fisher Scientific (Waltham, MA). For immunofluorescence staining, anti-SPC (1:500, proSP-C) was purchased from Merck Millipore (Burlington, MA, USA), anti-ki67 (1:200, SP6) from Novus Biologicals (Littleton, CO, USA), anti-CD31 (1:100, D8V9E), anti-PDGFRα (1:500, D1E1E) and anti-cleaved-Casp-3 (1:200, Asp175) from Cell Signaling, anti-PDGFRα from Santa Cruz (1:50, C-20), anti-F4/80 (1:100, ab6640) and anti-Ly6G (1:50, ab2557) antibody from abcam (Cambridge, UK), all secondary antibodies from Thermo Fisher Scientific. For anti-cleaved-Casp-3 signal stain boost IHC detection reagent (Cell Signaling) was used for improved signal detection. All antibodies used in this manuscript can be found in SciCrunch database. Animal-free recombinant TRAIL (315-19-50UG) was obtained from PeproTech (Hamburg, Germany).

The total proteins were extracted using a protein Lysis buffer RIPA containing 1 mM PMSF (Solarbio; Beijing, China). The protein levels were normalized by probing the same blots with a β-actin antibody. The extracts were boiled with an SDS loading buffer and separated by SDS-PAGE. The membrane was incubated overnight at 4 °C with primary antibodies specific to anti-PDCD4 (CoWin Biosciences, Beijing, China), anti-β-actin (CoWin Biosciences, Beijing, China), anti-cleaved-CASP3 (Cell Signaling Technology Inc., Shanghai, China), anti-cleaved PARP (Cell Signaling Technology Inc., USA). Anti-immune rabbit IgG-HRP (Sungene Biotech, Tianjin, China) served as a secondary antibody. The intensity of each band was scanned and quantified using the image J software.

To detect levels of proteins involved in inflammation and regeneration after brain ischemia, mice stroke affected lateral portions of the hemisphere were isolated and snap-frozen in liquid nitrogen and stored at −80 °C. Proteins were isolated by mechanical homogenisation in SDS-urea-β-mercaptoethanol solution containing proteinase inhibitor on ice. Electrophoresis and transfer on the PVDF-membrane was done using BioRad Mini-PROTEAN3 System (BioRad, Hercules, CA, USA).
Primary antibodies were β3-Tubulin (5568 S, Cell Signalling Technology, Beverly, MA, USA) as a loading control, DLG4 (PSD95) (3450, Cell Signalling Technology Beverly, MA, USA), GAP43 (AB5220, Merck, Kenilworth, NJ, USA), synaptophysin clone SY38 (MAB5258, Merck, Kenilworth, NJ, USA), and anti-cleaved CASP3 (Cell Signaling, Danvers, MA, USA). Secondary antibodies were goat anti-mouse, and goat anti-rabbit (32430 and A27011, Thermo Fisher Scientific, Waltham, MA, USA). The chemiluminiscent signal achieved by Western Lightning – EXL Enhanced Chemiluminiscence Substrate (Perkin Elmer, Waltham, MA, USA) was imaged by ChemiDoc MP System (BioRad, Hercules, CA, USA) and analysed using Image Lab Software (BioRad, Hercules, CA, USA).