Ab6142

The brain tissues and collected cells were stored at − 80 °C before use and homogenized to determine protein expression using the western blotting protocol described in our previous study [19 ]. The protein concentration was measured using the BCA Protein Assay Kit (P0010; Beyotime, China). Electrophoresis was performed using 10% or 12.5% SDS-polyacrylamide gels, and then proteins were electrotransferred to polyvinylidene fluoride membranes (IPVH0010; Millipore, Germany). The membranes were blocked with BSA or 5% non-fat milk and incubated with primary antibodies β-tubulin III (1:1000, T2200; Sigma-Aldrich), GFAP (1:10,000, ab53554; Abcam, UK), nestin (1:1000, ab6142; Abcam), ATN1 (1:500, orb213859; Biorbyt, UK), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000, 60004-1-Ig; Proteintech, USA) overnight at 4 °C. The membranes were incubated with second antibodies the next day for 2 h at room temperature, and then photographed using a GE Amersham Imager 600. Number of tissues was 5 per group and number of cells was 3 per group in western blot experiments. Images were analyzed using Image-Pro Plus 6.0 software.

Mice were daily given an IP injection of 5-bromo-2′-deoxyuridine (BrdU; 50 mg/kg, Sigma-Aldrich, St. Louis, MO, USA) for 12 days, beginning after stereotaxic surgery [14 (link)]. R6/2 mice euthanasia performed at 6 weeks (early-HD stage) and 13 weeks of age (late-HD stage) by transcardial perfusion with cold 1X PBS, followed by 4% paraformaldehyde (PFA). Harvested brain tissues were cryosectioned 16-μm thick slices, and immunohistochemical staining was performed on four sections, representing a range of more than 128 μm. The tissue sections were stained with the following antibodies: cell proliferation marker; BrdU (1:200, abcam, ab6326) and Ki67 (1:400, Leica Biosystems, NCL-Ki67p); OCT4 tagging marker HA (1:400, CSF, 3724S); OCT4 (1:100, santacruz, sc-5279); neuron-specific class III β-tubulin (βIII-tubulin, 1:400, abcam, ab18207) and mature neuronal marker NeuN (1:400, Millipore, MAB377); glial fibrillary acidic protein (GFAP, 1:400, abcam, ab10062) and s100β (1:400, Sigma, S2532); Nestin (1:400, abcam, ab6142); neural/glial antigen 2 (NG2, 1:200, Millipore, ab5320) and dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32, 1:400, cell signaling technology, 2306). The stained sections were observed by confocal microscopy (LSM700, Zeiss, Gottingen, Germany) and analyzed using ZEN black and blue edition (Zeiss, Gottingen, Germany).

The antibodies used for western blotting (for human) were as follows: GFP (abcam, ab290, 1:2,000), α-tubulin (CST, 3873s, 1:2,000), GAPDH (CST, 2118s, 1:2,000), Flag (MBL, M185, 1:2,000), Myc (MBL, M047-3, 1:5,000), HA (MBL, M180-3, 1:5,000), Phospho-JNK (CST, 9255, 1:1,000), and WDR62 (bethyl, A310-550A, 1:1,000). For mouse, they were as follows: WDR62 (abcam, 1:1,000), WDR62 antibody generated by MBL company using antigen VGQGGNQPKAGPLRAGTC, Phospho-WDR62 1053T (present from Dominic) [52 (link)], and MEKK3 (CST, 5727, 1:1,000). The antibodies used for immunostaining were Sox2 (abcam, ab97959, 1:1,000), Pax6 (Covance, PRB-278P, 1:400), Tbr2 (Millipore, ab2283, 1:1,000), β-III Tubulin/Tuj1 (abcam, ab7751, 1:1,000), γ-Tubulin (abcam, ab11316, 1:1,000), α-Tubulin (CST, 3873, 1:2,000), Phosph-Histone 3 (P-H3) (abcam, ab10543, 1:1,000), Nestin (abcam, ab6142, 1:1,000), GFP (abcam, ab13970, 1:1,000), Ki67 (abcam, ab15580, 1:1,000), BrdU (abcam, ab6326, 1:500), Phospho-JNK (abcam, ab124956, 1:1,000), activated-caspase3 (abcam, ab13847, 1:1,000). Nuclei were stained with DAPI (4’,6-diamidino-2-phenylindole) (Invitrogen).

Mouse anti-TUJ1 1:1000 (MMS-435P-250, Covance), rabbit anti-CHAT 1:1000 (AB 143, Millipore), mouse anti-NFM 1:500 (MAB1621, Millipore), rabbit anti-ISLET1 1:1000 (20670, Abcam), rabbit anti-GFAP 1:1000 (AB5804, Millipore), rabbit anti-AQP4 1:100 (AB3594, Millipore), rabbit anti-EAAT2 1:100 (ab41621, Abcam), mouse anti-GS 1:100 (MAB302, Millipore), mouse anti-O4 1:1000 (MAB345, Millipore), rabbit anti-IBA1 1:500 (019-19741, Wakochemical), rat anti-CD11b 1:200 (550282, BD pharmingen), mouse anti-MAP2 1:500 (MAB3418, Millipore), mouse anti-TDP-43 1:300 (gift from Dr J-P Julien’s laboratory), mouse anti-NeuN 1:500 (MAB377, Millipore), mouse anti-CNPase 1:200 (NE1020, Millipore), mouse anti-Nestin 1:200 (AB6142, Abcam), mouse anti-SOX2 1:500 (MAB4343, Millipore), mouse anti-Vimentin 1:1000 (AB28028, Abcam), rabbit anti-OLIG2 1:500 (AB9610, Millipore), rat anti-CD31 1:500 (550274, BD Biosciences), rabbit anti-cleaved caspase 3 1:200 AB3623, Millipore), Alexa fluor 488 goat anti-rabbit (A-11008, Life Technologies), Alexa fluor 594 goat anti-mouse (A-11005, Life Technologies)

The antibodies specific for Rnd3, Notch1, cleaved Notch1, hairy and enhancer of split-1 (Hes1), p-His H3, and GAPDH; the SMARTpool siRNA for Rnd3 knockdown; and the Myc-Rnd3 plasmid for Rnd3 overexpression that were used in this study are described in our previous work [16 (link)]. Antibodies specific for Nestin (ab6142) and GFAP (ab4674) were both purchased from Abcam.

Intervened cells were fixed in 4% paraformaldehyde, penetrated with 0.1% Triton X-100, blocked with 5% bovine serum albumin (BSA) and incubated with primary antibodies at 4°C overnight. Afterwards, the cells were stained with fluorescein-labeled secondary antibodies and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) under dark conditions, sealed on the slides, and observed under a laser confocal microscope. The respective concentrations of primary antibodies were: Iba1 (wako, Lot: 019–19741, 1:1000), CD86 (BD, Lot: 553689, 1:200), iNOS (BD, Lot: 610328, 1:200), CD206 (Santa, sc-34577, 1:100), Arg1 (Santa, sc-18351, 1:100), Nestin (Abcam, ab6142, 1:200), SOX2 (Abcam, ab97597, 1:200), Tuj-1 (Abcam, ab78078, 1:200), Olig2 (Abcam, ab109186, 1:100), GFAP (Abcam, ab7260, 1:1000), MAP2 (Abcam, ab5392, 1:500), O4 (Sigma, O7139, 1:100), A minimum of four images was captured using a 20X objective on a Zeiss microscope (Zeiss AxioCam, Germany) and the positive cells were counted using Image J.