Anti gas6

For western blotting, corpus callosum tissue of mice was quickly resected on ice and ultrasonically homogenised in RIPA buffer supplemented with 1 mM PMSF and protease inhibitor cocktail. Equal amounts of protein samples (∼20 μg) from each group were loaded in SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were incubated with the following primary antibodies: antimyelin basic protein (MBP) (1 : 400, Merck Millipore), anti-MerTK (1 : 1000, R&D Systems), anti-Axl (1 : 1000, R&D Systems), anti-Tyro3 (1 : 1000, R&D Systems), anti-Gas6 (1 : 1000, R&D Systems), and anti-β-actin (1 : 10000, ProteinTech). Protein expression was detected using an enhanced chemiluminescence detection system (Tanon) via incubation with peroxidase-conjugated secondary antibodies. Images were obtained using an ImageQuant LAS4000 mini image analyser (GE Healthcare).

Antibodies used were as follows: anti-Tyro3 (Santa Cruz, Dallas, TX, sc-1095, R&D Systems, Minneapolis, MN, AF759), anti-Axl (Santa Cruz sc-1097, R&D Systems AF854), anti-Mer (R&D Systems AF591), anti-HA (Covance, Princeton, NJ, MMS-101P, Roche, Mannheim, Germany 11 867 423 001), anti-Gas6 (R&D Systems AF986), anti-Akt (Cell Signaling, Beverly, MA, 4691), anti-Gapdh (Millipore, Billerica, MA, MAB374), anti-cleaved caspase 3 (Cell Signaling 9661), and anti-phosphotyrosine (Millipore 05–321). Human Pros1 was from Enzyme Research Laboratories, South Bend, IN, and Tet-Express from Clontech, Mountain View, CA.