Platelet derived growth factor receptor pdgfr

Cells were washed with phosphate-buffered saline (PBS) and then fixed in 4% paraformaldehyde (Merck) for 10 minutes. Cells were then washed with PBS three times for 3 minutes per wash, and subsequently incubated with a blocking solution (10% normal donkey serum, Jackson ImmunoResearch) for 1 hour at room temperature (RT). Cells were incubated with primary antibodies for 1 hour at RT, washed three times with PBS, incubated with secondary antibody for 30 minutes at RT, and finally washed three more times and mounted with a mounting solution (Vector Laboratories). Cells were analyzed using an Olympus BX51 fluorescence microscope (Olympus) and an LSM 700 confocal microscope (ZEISS). The primary antibodies used in this study were: Tuj1 (1:2000, Abcam), MAP2 (1:500, Abcam), Nestin (1:1000, Millipore), GFAP (1:1000, Abcam), NG2 (1:200, Millipore), Olig2 (1:200, Abcam), platelet-derived growth factor receptor (PDGFR; 1:500, Santa Cruz) and Ki67 (1:250, Abcam).

SAHA (Purity ≥99%) was purchased from Selleck Chemicals (Houston, TX). Matrigel and the anti-Semaphorin-4D (Sema-4D) antibody were obtained from BD Biosciences (San Jose, CA). Trypan blue was purchased from Beyotime Biotechnology (Shanghai, China). Annexin V-FITC apoptosis detection kit was purchased from Biotech Co., Ltd (Nanjing, China). RNase-free DNase I was from Qiagen (Hilder, Germany). RevertAid™ First Strand cDNA Synthesis Kit was purchased from Fermentas Life Sciences (Chicago, IL). Taq™ DNA Polymerase was from TaKaRa Biotechnology Co., Ltd (Dalian, China). Propidium iodide (PI), monoclonal antibody against β-actin and gelatin were obtained from Sigma (St. Louis, Mo). The anti-cyclin-D1 antibody was obtained from ABGENT (Suzhou, China). Anti-epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) antibodies were purchased from Santa Cruz Biotech (Santa Cruz, CA). Akt, p-Akt (Ser 473), p70S6 kinase (S6K1), p-S6K1 (Thr 389), S6, p-S6 (Ser 235/236), mTOR, p-mTOR (Thr 289), Ulk1, p-Gsk-3β, Ulk1, Erk1/2 and p-Erk1/2 antibodies were purchased from Cell Signaling Tech (Beverly, MA). Primers were synthesized by GENEWIZ, Inc. (Suzhou, China).