Non fat blocking grade milk

Cells were washed with phosphate buffered saline (PBS) and incubated in 100 μl of RIPA cell lysis buffer (Santa Cruz Biotechnology, Inc, Santa Cruz, CA) for 20 minutes. They were then snap-frozen in liquid nitrogen, thawed, and centrifuged at 125g for 10 minutes. The supernatant was collected, and assayed for protein concentration using the spectrophotometric Bradford protein Assay (Bio-Rad). Approximately 30 μg of proteins were electrophoresed in 10% SDS-PAGE gel, and transferred on a nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% non-fat blocking grade milk (Bio-Rad) and probed with a 1:1000 dilution of primary antibody overnight. The following antibodies were used: G-CSF receptor (Abcam), JAK-2 (Abcam), phospho-JAK2 (Abcam), PI3K (Cell Signaling Technology, Danvers, MA), phospho-PI3K (Cell Signaling Technology), Akt (Cell Signaling Technology), phospho-Akt (Cell Signaling Technology), PDE3B (Abcam), and actin (Santa Cruz Biotechnology). After washing three times, the membranes were probed with a 1:1000 dilution of secondary antibodies (Santa Cruz Biotechnology) for 1 hour at room temperature. The protein bands were visualized with ECL Plus, Chemiluminescence (GE Healthcare and Life Sciences, Piscataway, NJ). The densities were analyzed using Image J Software (Version 1.43u; National Institutes of Health, Bethesda, MD) and normalized to actin.

Western blotting was performed as previously described (Gamdzyk et al., 2018 (link)). A total of 30 μg protein, from ipsilateral hemisphere, was loaded and separated by 8%-12% SDS-PAGE gel and then transferred onto nitrocellulose membranes (0.45 μm). The membranes were blocked with 5% non-fat blocking grade milk (Bio-Rad, USA), and incubated overnight at 4°C with primary antibodies (anti-FPR2, 1:500, Abcam, USA; anti-NOX2, 1:500, Abcam, USA; anti-Rac1, 1:2000, Abcam, USA; IL-1β, 1:1000, Abcam, USA; TNF-α, 1:500, Abcam, USA; β-actin, 1:2000, Santa Cruz Biotechnology, USA). The next day, membranes were probed with anti-rabbit (or anti-mouse) secondary antibodies (1:3000 Santa Cruz Biotechnology, USA) for 2 h at room temperature. Finally, the bands were developed with ECL Plus kit (Amersham Bioscience, Arlington Heights, IL, USA) and exposed onto an X-ray film. For quantification, gray values were measured and normalized with the control band by Image J software (National Institutes of Health, USA).