Mouse anti tubulin

Neurospheres or cells adhered to PO-precoated coverslips were incubated in 4% paraformaldehyde for 10 min at room temperature and then washed with phosphate buffer saline (PBS, pH ~7.4) for three times. The samples were permeated with 0.5% Triton X-100 PBS for 30 min and blocked by 5% bovine serum albumin (BSA) for 2 h after washing three times. Thereafter, the samples were incubated in primary antibodies, goat anti-Nestin (1 : 100, Santa Cruz Biotechnology, CA, USA), rabbit anti-MAP-2 (1 : 100, Proteintech Group, Inc., Beijing, China), rabbit anti-glial fibrillary acidic protein (GFAP, 1 : 100, Abcam, Cambridge, UK), mouse anti-Olig2 (1 : 100, Milipore Corp., Billerica, MA, USA), rabbit anti-α-Smooth Muscle Actin (ACTA2) (1 : 100, Beyotime, Beijing, China), and mouse anti-Tubulin (1 : 100, Beyotime, Beijing, China) for 10–14 h at 4°C. After washing, relative fluorescence secondary antibodies were incubated at room temperature for 2 hours. Cell nuclei were counterstained with 4′-6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO) for 10 min at room temperature. Then, coverslips were mounted onto glass slides and the images were captured by a confocal microscope (Carl Zeiss, LSM780, Weimar, Germany) and examined using Zen 2011 software (Carl Zeiss, Weimar, Germany).

Total proteins were isolated from Drosophila tissues and the cultured cells with different treatments, and the protein concentration in the lysates was quantified using Bio-Rad protein assay reagent. Equal amounts of total protein were subjected to western blotting. The antibodies and dilutions used in the study were as follows: goat anti-Drosophila Fzr (1:1000; Santa Cruz), rabbit anti-Drosophila Myc (1:1000; Santa Cruz), goat anti-human Fzr (1:1000; Santa Cruz), mouse anti-human Myc (1:1000; Santa Cruz), mouse anti-Drosophila CycB (1:5000; DSHB), rabbit anti-human CycB (1:5000; Cell Signaling), mouse anti-H2B (1:10 000; Beyotime), rabbit anti-H2Bub (1:20 000; Cell Signaling), mouse anti-V5 (1:5000; Abcam), rabbit anti-Flag (1:5000; Sigma), mouse anti-Myc tag (1:5000; Sigma), rabbit anti-MCM6 (1:5000; Zoonbio Biotechnology), and mouse anti-tubulin (1:10 000; Beyotime). The following secondary antibodies were used, including HRP-conjugated goat anti-rabbit (1:10 000; Beyotime), goat anti-mouse (1:10 000; Beyotime), and donkey anti-goat (1:10 000; Beyotime).

Total proteins were isolated from the silkworm silk gland and then quantified by the Bradford assay (Sigma) using a microplate reader (BioTek) at the absorbance of 562 nm. Equal amounts of total protein were subjected for Western blotting. The antibodies and dilutions used in the study were as follows: rabbit anti-Fzr (1:1000, Zoonbio Biotechnology), rabbit anti-CycB (1:1000, Zoonbio Biotechnology), mouse anti-CycD (1:1000, Zoonbio Biotechnology), mouse anti-CycE (1:1000, Zoonbio Biotechnology), rabbit anti-pH3 (1:1000, Thermo fisher), mouse anti-H2B (1:10, 000; Beyotime), rabbit anti-H2Bub (1:20, 000; Cell Signaling), rabbit anti-Ub (1:1000, Proteintech), rabbit anti-RpS15A (1:5000, Abclonal), rabbit anti-RpS25 (1:5000, Abclonal), rabbit anti-GNS (1:1000, Sangon Biotech), rabbit anti-UBE2C (1:1000, Sangon Biotech) and mouse anti-Tubulin (1:10000, Beyotime).