Ab201687

Tissue samples were lysed in RIPA lysis buffer containing a protease inhibitor and a phosphatase inhibitor (Applygen Technologies Inc., Beijing, China). The protein concentrations of all samples were measured using the BCA Protein Assay Kit (Applygen Technologies Inc.). Protein samples (equal amounts per lane) were run on Criterion SDS–PAGE gels (Bio–Rad) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Darmstadt, Germany). The transfer membranes were blocked in blocking buffer (TBS with 0.1% Tween 20 (TBST) and 5% skim milk) for 1 h at RT followed by incubation with the following primary antibodies: anti-NG2 (1 : 1000, ab129051, Abcam), anti-Olig2 (1 : 500, MABN50, Millipore), anti-GST-pi (1 : 800, 312, MBL), anti-MBP (1 : 1000, GB12226, Servicebio), anti-GEF (1 : 1000, ab201687, Abcam), anti-Rac1 (1 : 500, ARC03, Cytoskelet), anti-phospho-Rac1 (1 : 1000, 2641, CST), anti-Cdc42 (1 : 1000, 2466S, CST), anti-MRCKα (1 : 1000, 81681S, CST), anti-PAK1 (1 : 1000, 2602, CST), anti-Tubulin (1 : 20,000, 66031-1, Proteintech), anti-GAPDH (1 : 20,000, 60004-1, Proteintech), and the corresponding HRP-conjugated anti-IgG antibodies. Finally, the protein bands were detected using a gel chemiluminescence imaging system and quantified by determining the grayscale values using ImageJ. All experiments were repeated three to four times.

For immunofluorescence analysis, paraffin-embedded brains were deparaffinized and rehydrated. After being permeabilized with 0.3% Triton X-100 for 30 min and blocked with 1% bovine serum albumin (BSA) for 1 h, brain sections were incubated with primary antibodies against MBP (1 : 200, 10458-1-AP, Proteintech), GST-pi (1 : 500, 312, MBL), NG2 (1 : 200, ab12905, Abcam), GEF (1 : 500, ab201687, Abcam), Rac1 (1 : 20, ARC03, Cytoskelet), and Cdc42 (1 : 500, ab187643, Abcam) overnight at 4°C. The corresponding secondary antibodies, conjugated with ALexa Fluor 488 or ALexa Fluor 594 (1 : 400, Abcam), were added, and the samples were incubated for 1 h at room temperature (RT). Nuclei were counterstained with DAPI. Brain images were collected by fluorescence microscopy. Data are presented in standardized form as the mean ± SD and reported as the number or percentage of positive elements.

Immunostaining was performed as described before [31 (link)]. Briefly, cells were grown on cover glasses and fixed for 15 min with 4% paraformaldehyde (PFA) solution at room temperature. Cells were washed twice with PBS and then permeabilized with 0.1% Triton-X100 in PBS for 10 min. Then, cells were incubated with a blocking buffer (PBS with 5% BSA). Primary antibody was applied at the specified dilution in blocking solution overnight at 4 °C. The following morning, cells were washed twice with PBS and incubated with the appropriate fluorescent secondary antibody (in blocking solution) for 30 min in the dark. Coverslips were then washed three times with PBS, mounted with DAPI, and examined under Olympus FV1000D microscope. The following antibodies were used for immunostaining: ARHGEF2 (ab201687, Abcam), CHGA (ab45179, Abcam), and AR (ab133273, Abcam).