Hc201 01

The protein level of cleaved caspase-3 in mouse brain was measured by Western blotting. In brief, the brain samples collected at 9 dpi were homogenized on ice in lysis buffer containing a complete protease and phosphatase inhibitor cocktail (23,227, Thermo Scientific, USA). After being centrifuged, the samples were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (ISEQ00010, Millipore, USA). The membranes were blocked with TBS buffer containing 10% skim milk and incubated with primary antibodies to cleaved caspase-3 (9664s, CST, diluted 1: 1000) or to β-actin (HC201-01, TransGen, China, diluted 1: 1000) overnight at 4 ​°C. As the secondary antibody, goat anti-rabbit IgG or donkey anti-mouse IgG (926–68071 or 925–32212, LI-COR, USA, diluted 1: 5000) was added and the protein band was visualized by using an infrared laser imager 767 (Odyssey® CLX, USA).

Cells were lysed in RIPA buffer (50 mM Tris-HCl at pH 7.4, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.25% sodium deoxycholate, and 1 mM dithiothreitol) supplemented with protease inhibitors (Roche, Basel, Switzerland) and incubated for 30 min on ice. After centrifugation at 18,800 g for 10 min, the protein concentration in the supernatant was determined using the BCA method. Equal amounts of protein were separated by sodium dodecyl sulfate (SDS)-sulfate-polyacrylamide gel electrophoresis in Tris/glycine/SDS running buffer. The separated proteins were transferred onto a polyvinylidene fluoride membrane in Tris-glycine transfer buffer that was blocked with 3% Tris-buffered saline with Tween 20 (BSA-TBST) for 1 h at room temperature. The membranes were probed with primary RanGAP1 antibody (ab92360, Abcam) at a 1:500 dilution and HBG2 (ab137096, Abcam), GATA1 (ab181544, Abcam), and β-actin antibodies (HC201-01, TransGen Biotech, Beijing, China) at a 1:1,000 dilution in 3% BSA-TBST at 4°C overnight. The membranes were subsequently rinsed with TBST three times for 10 min each and incubated with a secondary antibody for 1 h at room temperature. Finally, the membranes were rinsed three times with TBST solution and imaged using an Image Quant ECL machine (Tanon 5200, Tanon, Shanghai, China).