Anti fhl2

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).

The cells were collected from the cultures, placed in the RIPA lysis buffer on ice (BestBio, Shanghai, China). The whole proteins were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride membranes (PVDF; Millipore Corporation, Billerica, MA, USA). The PVDF membrane was incubated with 5% defatted milk powder at room temperature for 1 h, then incubation with the following specific primary antibodies at 4℃ overnight: anti-FHL2 (Abcam, Cambridge, MA, USA), anti- MYOD1 (Abcam), anti-MyoG (Abcam), anti-MYH3 (Abcam), anti-ATG5 (Cell Signaling, Danvers, MA, USA), anti-ATG7 (Cell Signaling), and anti-β-Actin (Abcam). The secondary antibodies HRP-labeled mouse and rabbit IgG (Cell Signaling) were added at room temperature for 1h. Following each step, the membranes were washed five times with PBS-T for 3 min. The proteins were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ, USA) with a Kodak imager (Eastman Kodak, Rochester, NY, USA). Quantification of protein blots was performed using the Quantity One 1-D software (version 4.4.0) (Bio-Rad, Hercules, CA, USA) on images acquired from an EU-88 image scanner (GE Healthcare, King of Prussia, PA, USA).