The ipsilateral cortex and underlying white matter around the injury epicentre were dissected at 2 hpi, 6 hpi, 24 hpi, 7 dpi and sham mice. Brain lysates (30 µg) were prepared and resolved in 10% sodium dodecyl sulfate-polyacrylamide gels as previously described (Theus et al., 2014 (link)). The primary mouse anti-EphB3 (Novus Abnova, Littleton, CO, USA), anti-caspase-3 and cleaved caspase-3 (Cell Signaling, Danvers, MA, USA), anti-caspase-8 and cleaved caspase-8 (Cell Signaling), anti-caspase-9 and cleaved caspase-9 (Abcam, Cambridge, MA, USA) or anti-FHL-2 (Abcam) antibody were applied at 1:200 dilution in 5% bovine serum albumin solution overnight at 4°C. Protein expression was normalized to β-actin (Cell Signaling).
For IP, 150 µg of protein sample was incubated with 1.5 µg of anti-EphB3 IgG2a mouse antibody (Novus Abnova) or a negative mouse mAb IgG2a isotype control (Cell Signaling). The membrane was subsequently incubated with the primary antibody of interest (anti-EphB3, anti-caspase-3, -8 and -9 and anti-FHL-2).
For IP, 150 µg of protein sample was incubated with 1.5 µg of anti-EphB3 IgG2a mouse antibody (Novus Abnova) or a negative mouse mAb IgG2a isotype control (Cell Signaling). The membrane was subsequently incubated with the primary antibody of interest (anti-EphB3, anti-caspase-3, -8 and -9 and anti-FHL-2).
